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Methionine393 is an axial ligand of the heme b558 component of the cytochrome bd ubiquinol oxidase from Escherichia coli
Abstract
The cytochrome bd oxidase is one of two terminal oxidases in the aerobic respiratory chain of Escherichia coli. The complex is composed of two subunits (I and II) and three heme prosthetic groups (heme b(558), heme b(595), and a chlorin, called heme d). Both subunits are located within the bacterial cytoplasmic membrane, and each has multiple putative transmembrane helices. Heme b(558) is a six-coordinate, low-spin heme component of the oxidase which has been shown to be contained within subunit I and has been implicated in the oxidation of the substrate, ubiquinol-8, in the cytoplasmic membrane. Previous site-directed mutagenesis studies identified His186, predicted to be near the periplasmic side of transmembrane helix D of subunit I, as one of the axial ligands of heme b(558). Since mutagenesis of none of the other histidines in subunit I perturbs heme b(558), it was concluded that this heme cannot have bis(histidine) ligation. In this work, the properties of 14 mutants are reported, including substitutions for each of 10 methionine residues within subunit I. Among this set of mutants, only the replacement of M393 perturbs heme b(558). Replacement of M393 by leucine results in the conversion of heme b(558) to a high-spin state. Surprisingly, the M393L mutation does not eliminate enzymatic activity, and the mutant oxidase has sufficient turnover to support aerobic growth of the cells. The addition of imidazole to the purified M393L oxidase converts heme b(558) back to a low-spin configuration. The data strongly suggest that the sixth axial ligand of heme b(558) is methionine-393, and that this heme, therefore, has histidine-methionine ligation. The results are consistent with recent cryogenic near-infrared magnetic circular dichroism spectra that also indicate histidine-methionine ligation of heme b(558).
... These cytochromes bd consist of two different subunits, I and II, which carry three hemes, b LS (previously called "b 558 ") 3 , b HS (previously called "b 595 "), and d. The low-spin hexacoordinate heme b LS located within subunit I near a quinol-binding site has His186/ Met393 axial ligation [31,32] and mediates electron transfer from quinol to hemes b HS and d [33]. The high-spin pentacoordinate heme b HS has His19 of subunit I as the axial ligand. ...
... [64,65]. The strain lacks the bo 3 -type oxidase and overexpresses cytochrome bd [32]. ...
... Hence, the broadening is not sufficient to account for the difference. The low-spin hexacoordinate heme b LS in cytochrome bd from E. coli is known to have a His-Met axial ligation [31,32]. Indeed, the line shape of its MCD vibronic structure is very similar to that of cytochrome c which is a typical hemoprotein with His-Met coordinated heme (Fig. 5B). ...
Cytochromes bd are terminal oxidases in the respiratory chains of many prokaryotic organisms. They reduce O₂ to 2H₂O at the expense of electrons extracted from quinol. The oxidases can be divided into two subfamilies, L and S, based on the presence of either a long or a short hydrophilic connection between transmembrane helices 6 and 7 in subunit I designated as 'Q-loop'. The L-subfamily members, e.g. the enzyme from Escherichia coli, are relatively well-studied and were shown to generate proton-motive force. The S-subfamily comprises the majority of cytochromes bd including the enzyme from Geobacillus thermodenitrificans but is very poor studied. We compared the properties of cytochromes bd from G. thermodenitrificans and E. coli at room temperature using a combination of absorption, CD and MCD spectroscopy. The G. thermodenitrificans enzyme does contain the high-spin heme b(HS) ("b(595)") despite the fact that its characteristic Q(00)-band ("α"-band) at 595nm is not seen in the absorption spectra; stoichiometry of hemes b(LS), b(HS) and d per the enzyme complex is suggested to be 1:1:1. At 1mM CO, 20-25% of ferrous heme b(HS) in the G. thermodenitrificans oxidase binds the ligand, while in case of the E. coli enzyme such a reaction is minor. In the G. thermodenitrificans oxidase, the excitonic interaction between ferrous hemes b(HS) and d decreased as compared to that in the E. coli bd. The latter may suggest that the two enzymes differ in the distance between heme d and heme b(HS) and/or in the angle between their porphyrin planes.
... These peaks are characteristic for the heme b (560 nm, 532 nm, 594 nm) and the heme d (628 nm) prosthetic groups in cytochrome bd oxidase (Junemann et al., 1995). To prove that cytochrome bd oxidase indeed corresponds to the DsbB activating factor, we purified cytochrome bd oxidase to >95% purity from an overproducing strain of E. coli (GO105/pTK1) according to Kaysser et al., 1995. GO105/pTK1 contains a plasmid overproducing cytochrome bd oxidase and a null mutation in the genes for cytochrome bo oxidase. ...
... GO103/pJRhisA and GO105/pTK1 were generous gifts from Dr. Robert Gennis, University of Illinois. These strains overexpress cytochrome bo and cytochrome bd terminal oxidase, respectively, and were grown essentially as described before (Kaysser et al., 1995; Rumbley et al., 1997). ...
... The cytochrome bo terminal oxidase was purified from GO103/pJRhisA over Ni-NTA according to a published procedure (Rumbley et al., 1997). For the purification of cytochrome bd oxidase from GO105/PTK1 we followed the protocol described by (Kaysser et al., 1995), except that n-Dodecyl-β-D-maltoside was used as a detergent. ...
Many secreted proteins require the correct formation of one or more disulfide bonds for the proper folding into their native 3-D structure. DsbA catalyzes the formation of disulfide bonds in the periplasm of E. coli. DsbA's active site disulfide bond is reoxidized by the inner membrane protein DsbB. Electrons flow from DsbA via DsbB to ubiquinone and further on to terminal cytochrome oxidase complexes, which finally transfer electrons to molecular oxygen. While growing anaerobically, E. coli replaces ubiquinones by menaquinones, which then act as mobile electron carriers between anaerobic respiratory complexes in the inner membrane. DsbB directly interacts with menaquinones suggesting a mechanism whereby DsbB drives disulfide bond formation under anaerobic conditions. The ability of DsbB to utilize alternative electron acceptors such as ubiquinones and menaquinones ensures efficient disulfide bond formation over a wide variety of growth conditions. DsbA is a strong but non-specific oxidant leading to the complete but often incorrect oxidation of proteins. A second pathway ensures the isomerization of incorrect disulfide bonds. This pathway consists of two disulfide isomerases, DsbC and DsbG, and the inner membrane protein DsbD. DsbD is directly involved in the reduction of DsbC and DsbG by providing a link to the reducing power of the cytosol. It is puzzling how this reducing pathway can coexist with the oxidizing DsbA-DsbB system without going through futile cycles of mutual oxidation and reduction. A number of dsbC mutants were obtained which rescue a dsbA null mutant. These mutant proteins failed to dimerize and were reoxidized by DsbB in vivo and in vitro. Accordingly, DsbC seems to be protected from DsbB mediated oxidation only when present as a dimer. This is an important molecular barrier that allows the coexistence of an isomerization and oxidation pathway in the periplasm, thus securing the proper formation of disulfide bonds. Many secreted proteins require the correct formation of one or more disulfide bonds for the proper folding into their native 3-D structure. DsbA catalyzes the formation of disulfide bonds in the periplasm of E. coli. DsbA's active site disulfide bond is reoxidized by the inner membrane protein DsbB. Electrons flow from DsbA via DsbB to ubiquinone and further on to terminal cytochrome oxidase complexes, which finally transfer electrons to molecular oxygen. While growing anaerobically, E. coli replaces ubiquinones by menaquinones, which then act as mobile electron carriers between anaerobic respiratory complexes in the inner membrane. DsbB directly interacts with menaquinones suggesting a mechanism whereby DsbB drives disulfide bond formation under anaerobic conditions. The ability of DsbB to utilize alternative electron acceptors such as ubiquinones and menaquinones ensures efficient disulfide bond formation over a wide variety of growth conditions. DsbA is a strong but non-specific oxidant leading to the complete but often incorrect oxidation of proteins. A second pathway ensures the isomerization of incorrect disulfide bonds. This pathway consists of two disulfide isomerases, DsbC and DsbG, and the inner membrane protein DsbD. DsbD is directly involved in the reduction of DsbC and DsbG by providing a link to the reducing power of the cytosol. It is puzzling how this reducing pathway can coexist with the oxidizing DsbA-DsbB system without going through futile cycles of mutual oxidation and reduction. A number of dsbC mutants were obtained which rescue a dsbA null mutant. These mutant proteins failed to dimerize and were reoxidized by DsbB in vivo and in vitro. Accordingly, DsbC seems to be protected from DsbB mediated oxidation only when present as a dimer. This is an important molecular barrier that allows the coexistence of an isomerization and oxidation pathway in the periplasm, thus securing the proper formation of disulfide bonds.
... The low-spin hexacoordinate heme b 558 seems to be directly involved in the quinol oxidation, and the two quinol protons are released into the periplasm. His 186 and Met 393 of subunit I were identified as its axial ligands [31,32]. The high-spin heme d is the core of the enzyme where O 2 is bound, activated and reduced into H 2 O. ...
... The strain of Escherichia coli GO105 devoid of cytochrome bo 3 and cytochrome bd oxidases and harboring plasmid pTK1 with the genes encoding cytochrome bd was used for overexpressing cytochrome bd [32]. ...
Cytochrome bd is a tri-heme (b558, b595, d) respiratory oxygen reductase that is found in many bacteria including pathogenic species. It couples the electron transfer from quinol to O2 with generation of an electrochemical proton gradient. We examined photolysis and subsequent recombination of CO with isolated cytochrome bd from Escherichia coli in one-electron reduced (MV) and fully reduced (R) states by microsecond time-resolved absorption spectroscopy at 532-nm excitation. Both Soret and visible band regions were examined. CO photodissociation from MV enzyme possibly causes fast (τ<1.5 µs) electron transfer from heme d to heme b595 in a small fraction of the protein, not reported earlier. Then the electron migrates to heme b558 (τ∼16 µs). It returns from the b-hemes to heme d with τ∼180 µs. Unlike cytochrome bd in the R state, in MV enzyme the apparent contribution of absorbance changes associated with CO dissociation from heme d is small, if any. Photodissociation of CO from heme d in MV enzyme is suggested to be accompanied by the binding of an internal ligand (L) at the opposite side of the heme. CO recombines with heme d (τ∼16 µs) yielding a transient hexacoordinate state (CO-Fe2+-L). Then the ligand slowly (τ∼30 ms) dissociates from heme d. Recombination of CO with a reduced heme b in a fraction of the MV sample may also contribute to the 30-ms phase. In R enzyme, CO recombines to heme d (τ∼20 µs), some heme b558 (τ∼0.2-3 ms), and finally migrates from heme d to heme b595 (τ∼24 ms) in ∼5% of the enzyme population. Data are consistent with the recent nanosecond study of Rappaport et al. conducted on the membranes at 640-nm excitation but limited to the Soret band. The additional phases were revealed due to differences in excitation and other experimental conditions.
... The maximum and minimum of the γ-band in the " reduced minus oxidized " difference absorption spectrum are 429.5 and 413 nm, respectively (Table 3) [225]. Heme b 558 is low-spin hexacoordinate [37], and amino acid residues His186 ** and Met393 of subunit I (E. coli cytochrome bd-I) have been identified as its axial ligands226227228. The location of heme b 558 is predicted to be near the periplasmic surface [67,229]. ...
... Several polar or ionizable residues that are highly conserved in the bd-family have been postulated to be a part of this putative proton channel. The residues that are totally conserved within the entire bd-family include His19 (the heme b 595 axial ligand [230]), His186 and Met393 (the heme b 558 axial ligands226227228), Lys252 and Glu257 (involved in QH 2 binding [66]), Arg448 (unknown function), and Glu99, Glu107, and Ser140 (proposed to be components of a proton channel [48,67] and important for heme binding in the heme d/heme b 595 di-heme site [213,214]). Slightly less conserved (95–99%) are Glu445 (required for charge compensation of the b 595 /d O 2 reducing site upon its full reduction by two electrons [41]), Asn148 (plausible component of a proton channel), and Arg9 (unknown function) [213]. ...
Cytochrome bd is a respiratory quinol: O₂ oxidoreductase found in many prokaryotes, including a number of pathogens. The main bioenergetic function of the enzyme is the production of a proton motive force by the vectorial charge transfer of protons. The sequences of cytochromes bd are not homologous to those of the other respiratory oxygen reductases, i.e., the heme-copper oxygen reductases or alternative oxidases (AOX). Generally, cytochromes bd are noteworthy for their high affinity for O₂ and resistance to inhibition by cyanide. In E. coli, for example, cytochrome bd (specifically, cytochrome bd-I) is expressed under O₂-limited conditions. Among the members of the bd-family are the so-called cyanide-insensitive quinol oxidases (CIO) which often have a low content of the eponymous heme d but, instead, have heme b in place of heme d in at least a majority of the enzyme population. However, at this point, no sequence motif has been identified to distinguish cytochrome bd (with a stoichiometric complement of heme d) from an enzyme designated as CIO. Members of the bd-family can be subdivided into those which contain either a long or a short hydrophilic connection between transmembrane helices 6 and 7 in subunit I, designated as the Q-loop. However, it is not clear whether there is a functional consequence of this difference. This review summarizes current knowledge on the physiological functions, genetics, structural and catalytic properties of cytochromes bd. Included in this review are descriptions of the intermediates of the catalytic cycle, the proposed site for the reduction of O₂, evidence for a proton channel connecting this active site to the bacterial cytoplasm, and the molecular mechanism by which a membrane potential is generated.
... E. coli strain GO105 (cyd AB::kan, cyo, recA), which lacks both the cytochrome bo 3 and cytochrome bd quinol oxidases [15], was used as the host strain for overexpressing the wild type cytochrome bd from plasmid pTK1 [15] which was introduced into the strain. ...
... E. coli strain GO105 (cyd AB::kan, cyo, recA), which lacks both the cytochrome bo 3 and cytochrome bd quinol oxidases [15], was used as the host strain for overexpressing the wild type cytochrome bd from plasmid pTK1 [15] which was introduced into the strain. ...
Cytochrome bd catalyzes the two-electron oxidation of either ubiquinol or menaquinol and the four-electron reduction of O(2) to H(2)O. In the current work, the rates of reduction of the fully oxidized and oxoferryl forms of the enzyme by the 2-electron donor ubiquinol-1 and single electron donor N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD) have been examined by stopped-flow techniques. Reduction of the all-ferric form of the enzyme is 1000-fold slower than required for a step in the catalytic cycle, whereas the observed rates of reduction of the oxoferryl and singly-reduced forms of the cytochrome are consistent with the catalytic turnover. The data support models of the catalytic cycle which do not include the fully oxidized form of the enzyme as an intermediate.
... Heme b 558 is low-spin, six-coordinate and is located entirely within subunit I [39]. It has been shown that this low-spin heme has histidine/methionine ligation, with H186 in subunit I (I-H186) [40] and I-M393 [41] as the two axial ligands. ...
... R391 in subunit I (E. coli numbering) is the only highly conserved, positively charged residue within the membrane. It is located on the opposite face of the putative transmembrane helix VII from M393 (Fig. 6B), which is the axial ligand for heme b 558 [41]. The proximity to heme b 558 along with the sequence location of R391 at the end of the Q-loop suggests that it could participate in quinol binding or electron transfer from quinol. ...
Numerous sequences of the cytochrome bd quinol oxidase (cytochrome bd) have recently become available for analysis. The analysis has revealed a small number of conserved residues, a new topology for subunit I and a phylogenetic tree involving extensive horizontal gene transfer. There are 20 conserved residues in subunit I and two in subunit II. Algorithms utilizing multiple sequence alignments predicted a revised topology for cytochrome bd, adding two transmembrane helices to subunit I to the seven that were previously indicated by the analysis of the sequence of the oxidase from E. coli. This revised topology has the effect of relocating the N-terminus and C-terminus to the periplasmic and cytoplasmic sides of the membrane, respectively. The new topology repositions I-H19, the putative ligand for heme b595, close to the periplasmic edge of the membrane, which suggests that the heme b595/heme d active site of the oxidase is located near the outer (periplasmic) surface of the membrane. The most highly conserved region of the sequence of subunit I contains the sequence GRQPW and is located in a predicted periplasmic loop connecting the eighth and ninth transmembrane helices. The potential importance of this region of the protein was previously unsuspected, and it may participate in the binding of either quinol or heme d. There are two very highly conserved glutamates in subunit I, E99 and E107, within the third transmembrane helix (E. coli cytochrome bd-I numbering). It is speculated that these glutamates may be part of a proton channel leading from the cytoplasmic side of the membrane to the heme d oxygen-reactive site, now placed near the periplasmic surface. The revised topology and newly revealed conserved residues provide a clear basis for further experimental tests of these hypotheses. Phylogenetic analysis of the new sequences of cytochrome bd reveals considerable deviation from the 16sRNA tree, suggesting that a large amount of horizontal gene transfer has occurred in the evolution of cytochrome bd.
... The last step of UQ biosynthesis pathway in strain KLS-1 was probably regulated by CydABX (Fig. 5D & Table S4), a complex consisting of cytochrome bd ubiquinol oxidase subunit I, cytochrome bd ubiquinol oxidase subunit II and cytochrome bd-I ubiquinol oxidase subunit X [78]. While subunits I and II of the terminal oxidase produce a proton motive force by the vectorial transfer of protons across the membrane, subunit X is required for correct functioning of cytochrome bd-I oxidase [48]. Recent studies showed that both UbiJ and UbiK were also required for efficient UQ biosynthesis under aerobic conditions [1,12]. ...
The wide distribution of p-hydroxybenzoic acid (PHBA) in the environments has attracted great concerns due to its potential risks to organisms. Bioremediation is considered a green way to remove PHBA from environment. Here, a new PHBA-degrading bacterium Herbaspirillum aquaticum KLS-1was isolated and its PHBA degradation mechanisms were fully evaluated. Results showed that strain KLS-1 could utilize PHBA as the sole carbon source and completely degrade 500 mg/L PHBA within 18 h. The optimal conditions for bacterial growth and PHBA degradation were pH values of 6.0–8.0, temperatures of 30 °C-35 °C, shaking speed of 180 rpm, Mg2+ concentration of 2.0 mM and Fe2+ concentration of 1.0 mM. Draft genome sequencing and functional gene annotations identified three operons (i.e., pobRA, pcaRHGBD and pcaRIJ) and several free genes possibly participating in PHBA degradation. The key genes pobA, ubiA, fadA, ligK and ubiG involved in the regulation of protocatechuate and ubiquinone (UQ) metabolisms were successfully amplified in strain KLS-1 at mRNA level. Our data suggested that PHBA could be degraded by strain KLS-1 via the protocatechuate ortho-/meta-cleavage pathway and UQ biosynthesis pathway. This study has provided a new PHBA-degrading bacterium for potential bioremediation of PHBA pollution.
... Another difference between the enzymes is found in the nature of the distal ligands of the remaining 7 haem groups. In contrast to HAO, where all haems except for the active site show bis-histidinyl coordination, haem 7 of the ¸HAOs is ligated by a methionine, a feature commonly found in the widespread electron carrier cytochrome c195 , the eponym for all proteins containing c-type haems, or b-type cytochromes196 , but was at the time unprecedented for octahaem cytochromes. A substrate/product channel can be identified, spanning from an entry site, close to the active site, to an exit site inside the central pore. ...
Nitrogen, an essential component of most biomolecules, used to be a scarce nutrient in many ecosystems since the beginning of life on Earth. The increasing use of artificial fertilizers has led to significant nitrogen pollution in many parts of the world. The resulting inorganic nitrogen species are interconverted by different microorganisms. The reactions involved are summarized in the biological nitrogen cycle. One of these reactions is the dissimilatory nitrate reduction to ammonium (DNRA), a pathway used by various bacteria and archaea for energy conservation. One of the key enzymes of DNRA is the pentahaem cytochrome c nitrite reductase (NrfA), which catalyses the reduction of nitrite to ammonium in a concerted six-electron reduction. NrfA is a particularity in the diverse family of cytochrome c proteins, as the catalytic haem is bound to an unusual CXXCK motif that requires an additional maturation system. Besides nitrite reduction to ammonium, many other reactions in the nitrogen cycle are catalysed by multihaem cytochromes (MCC). Most of these MCCs are evolutionary related, with NrfA as the common ancestor. The latest member of these related proteins is the octahaem hydroxylamine oxidoreductase (HAO). Several intermediate proteins from this transition have been identified and characterised to date. One such intermediate is represented by a family of proteins identified in the class of -proteobacteria. Because of their sequence homology with HAO, these enzymes were annotated as HAOs, although recent studies showed that they actually catalyse the reduction of nitrite to ammonium. The -proteobacterium Geobacter metallireducens, well known for its capability to utilise various metal ions as terminal electron acceptors, contains two genes encoding canonical NrfA proteins, as well as a protein assigned to be a member of the HAO-family. This octahaem cytochrome, called GmNOR, has previously been crystallised and the structure was solved by X-ray crystallography. Structural similarity shows the relation of GmNOR, to the HAOs but the investigation of its catalytic properties revealed low nitrite reductase activity. In this work, GmNOR was characterised as an enzyme that reduces nitric oxide to ammonium with high specificity and at high rates by UV-vis spectroscopy-based enzymatic activity assays. Although this reaction is a side reactivity of nitrite-reducing enzymes such as NrfA or HOAs, no enzyme with such a clear selectivity for the reduction of nitric oxide has been reported yet. Furthermore, NrfA from Geobacter metallireducens was heterologously expressed in E. coli, by a novel expression approach for the maturation of unusual haem-binding motifs. The enzyme’s structure was determined by X-ray crystallography to a resolution of 1.9 Å. The structure showed that GmNrfA is another member of the recently described family of Ca2+-independent NrfAs. Additionally, the active site revealed the presence of an unprecedented loop. This loop harbours an aspartate, an active site residue unseen in any previously characterised NrfA protein. The catalytic relevance of this aspartate was investigated by kinetic studies with the wild type and two variants of the enzyme.
... E. coli strain GO105/pTK1 was kindly provided by R. B. Gennis (University of Illinois at Urbana Champaign, USA). This strain overproduces cytochrome bd I and lacks cytochrome bo 3 , which makes it possible to obtain preparations of cytochrome bd I without admixtures of bo 3 oxidase [61]. ...
Cytochrome bd-I is a terminal oxidase of the Escherichia coli respiratory chain. This integral membrane protein contains three redox-active prosthetic groups (hemes b558, b595, and d) and couples the electron transfer from quinol to molecular oxygen to the generation of proton motive force, as one of its important physiological functions. The study was aimed at examining the effect of the membrane environment on the ligand-binding properties of cytochrome bd-I by absorption spectroscopy. The membrane environment was found to modulate the ligand-binding characteristics of the hemoprotein in both oxidized and reduced states. Absorption changes upon the addition of exogenous ligands, such as cyanide or carbon monoxide (CO), to the detergent-solubilized enzyme were much more significant and heterogeneous than those observed with the membrane-bound enzyme. In the native membranes, both cyanide and CO interacted mainly with heme d. An additional ligand-binding site (heme b558) appeared in the isolated enzyme, as was evidenced by more pronounced changes in the absorption in the Soret band. This additional reactivity could also be detected after treatment of E. coli membranes with a detergent. The observed effect did not result from the enzyme denaturation, since reconstitution of the isolated enzyme into azolectin liposomes restored the ligand-binding pattern close to that observed for the intact membranes.
... cytochrome bd Ubiquinol Oxidase which has three heme prosthetic groups. One of these, heme bggg is a 6 co-ordinate, low spin component, with histidine and methionine ligation (177). However, comparison of the spin signals from flavocytochrome bggg with those from other heme-binding proteins tends to exclude this possibility (169). ...
Phagocytic cells play a key role in the immune system, engulfing and sequestering bacteria in a phagocytic vacuole. There they are subjected to a barrage of degradative enzymes and superoxide, O2-. Superoxide is generated by an electron transport chain called flavocytochrome b558, which spans the wall of the vacuole. The reactive products from superoxide, and the alteration in pH that these reactions cause, act together for successful bacterial killing. With its associated cytosolic factors, flavocytochrome b558 forms a complex called the NADPH Oxidase. If a component of the NADPH oxidase is missing or defective, so that superoxide is not produced, then the individual is severely immune-compromised, leading to the condition of Chronic Granulomatous Disease (CGD). Flavocytochrome is composed of two subunit types, p22phox and gp91phox. Together, these form an electron transport chain which uses cytosolic NADPH, via FAD and heme cofactors, to reduce molecular oxygen in the vacuolar space to superoxide. The subunits are closely associated and copurify through stringent conditions. There are now known to be two non-identical heme groups in the complex, and the ratio of heme to FAD is 2:1. The NADPH and FAD binding sites are on gp91phox, and the accepted view has been that heme was associated with p22phox. The work described in this thesis investigates the structure of flavocytochrome b558. First, the subunit stoichiometry was determined by a variety of complementary approaches. This data showed that p22phox and gp91phox were present in neutrophil membrane in a 1:1 ratio. Second, topology studies were carried out to analyze the glycosylation sites of gp91phox using site directed mutagenesis and in vitro protein synthesis. This information was used to construct a model of the amino-terminal, membrane associated part of gp91phox. The model is consistent with having both heme associated with gp91phox, along with the FAD and NADPH binding sites, implicating the p22phox subunit as having a structural and/or regulatory role in the oxidase.
... The two axial ligands of heme b 558 are Met 393.A and His 186.A (Fig. 2C). The location and coordination of this heme are equivalent to the G. th enzyme (12,13). Propionate A of heme b 558 is coordinated by the conserved residues Lys 252.A and Lys 183.A . ...
Hemes switch spots in a terminal oxidase
Reduction of molecular oxygen to water is the driving force for respiration in aerobic organisms and is catalyzed by several distinct integral membrane complexes. These include an exclusively prokaryotic enzyme, cytochrome bd–type quinol oxidase, which is a potential antimicrobial target. Safarian et al. determined a high-resolution cryo–electron microscopy structure of this enzyme from the enteric bacterium Escherichia coli . Comparison to a homolog reveals a complete relocation of the site of oxygen binding and reduction caused by a change in the arrangement of heme cofactors and channels in the protein scaffold. This switch illustrates the diversity of structure and function in this family of enzymes and might reflect different biochemical roles of these homologs.
Science , this issue p. 100
... Site-directed mutagenesis studies have been used alongside spectroscopic methods to reveal that highly conserved Histidine-186 and Methionine-393 of CydA are axial ligands of b 558 (Fang, Lin, & Gennis, 1989;Kaysser, Ghaim, Georgiou, & Gennis, 1995;Spinner et al., 1995). The positive charge of the conserved Arginine-391 residue has a role in stabilising the reduced form of haem b 558 and is required for oxidase activity (Zhang, Hellwig, Osborne, & Gennis, 2004), and Histidine-19 of CydA provides the essential axial ligand for b 595 (Sun et al., 1996). ...
The CydDC complex of Escherichia coli is a heterodimeric ATP-binding cassette type transporter (ABC transporter) that exports the thiol-containing redox-active molecules cysteine and glutathione. These reductants are thought to aid redox homeostasis of the periplasm, permitting correct disulphide folding of periplasmic and secreted proteins. Loss of CydDC results in the periplasm becoming more oxidising and abolishes the assembly of functional bd-type respiratory oxidases that couple the oxidation of ubiquinol to the reduction of oxygen to water. In addition, CydDC-mediated redox control is important for haem ligation during cytochrome c assembly. Given the diverse roles for CydDC in redox homeostasis, respiratory metabolism and the maturation of virulence factors, this ABC transporter is an intriguing system for researchers interested in both the physiology of redox perturbations and the role of low-molecular-weight thiols during infection.
... All together, these data show that i) cytochrome bd is not inactivated by up to 100 μM ONOO − and ii) the enzyme in turnover with O 2 is able to rapidly metabolize ONOO − with an apparent turnover rate increasing at higher concentrations of the reducing substrates. These conclusions are fully consistent with the results obtained in control experiments carried out with cells of the E. coli GO105/pTK1 strain, expressing plasmid-encoded cytochrome bd as the only respiratory terminal oxidase [70]. As shown in Fig. 6, similar to the isolated enzyme (Fig. 1A), addition of ONOO − to the cells respiring on endogenous substrates led to a short-term evolution of O 2 , due to contaminant H 2 O 2 in the ONOO − solution. ...
Cytochrome bd is a prokaryotic respiratory quinol oxidase phylogenetically unrelated to heme-copper oxidases, that was found to promote virulence in some bacterial pathogens. Cytochrome bd from Escherichia coli was previously reported to contribute not only to proton motive force generation, but also to bacterial resistance to nitric oxide (NO) and hydrogen peroxide (H2O2). Here, we investigated the interaction of the purified enzyme with peroxynitrite (ONOO−), another harmful reactive species produced by the host to kill invading microorganisms. We found that addition of ONOO− to cytochrome bd in turnover with ascorbate and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) causes the irreversible inhibition of a small (≤ 15%) protein fraction, due to the NO generated from ONOO− and not to ONOO− itself. Consistently, addition of ONOO− to cells of the E. coli strain GO105/pTK1, expressing cytochrome bd as the only terminal oxidase, caused only a minor (≤ 5%) irreversible inhibition of O2 consumption, without measurable release of NO. Furthermore, by directly monitoring the kinetics of ONOO− decomposition by stopped-flow absorption spectroscopy, it was found that the purified E. coli cytochrome bd in turnover with O2 is able to metabolize ONOO− with an apparent turnover rate as high as ~ 10 mol ONOO− (mol enzyme)− 1 s− 1 at 25 °C. To the best of our knowledge, this is the first time that the kinetics of ONOO− decomposition by a terminal oxidase has been investigated. These results strongly suggest a protective role of cytochrome bd against ONOO− damage.
... The low-spin hexacoordinate haem b 558 seems to be directly involved in quinol oxidation, and the two quinol protons are released into the periplasm (Mogi et al., 2006). His 186 and Met 393 of subunit I are identified as its axial ligands (Kaysser, Ghaim, Georgiou, & Gennis, 1995). The high-spin haem d is the core of the enzyme where oxygen is bound (Poole et al., 1983), activated and reduced into H 2 O. ...
Escherichia coli is a facultatively anaerobic bacterium. With glucose if no external electron acceptors are available, ATP is produced by substrate level phosphorylation. The intracellular redox balance is maintained by mixed-acid fermentation, that is, the production and excretion of several organic acids. When oxygen is available, E. coli switches to aerobic respiration to achieve redox balance and optimal energy conservation by proton translocation linked to electron transfer. The switch between fermentative and aerobic respiratory growth is driven by extensive changes in gene expression and protein synthesis, resulting in global changes in metabolic fluxes and metabolite concentrations. This oxygen response is determined by the interaction of global and local genetic regulatory mechanisms, as well as by enzymatic regulation. The response is affected by basic physical constraints such as diffusion, thermodynamics and the requirement for a balance of carbon, electrons and energy (predominantly the proton motive force and the ATP pool). A comprehensive systems level understanding of the oxygen response of E. coli requires the integrated interpretation of experimental data that are pertinent to the multiple levels of organization that mediate the response. In the pan-European venture, Systems Biology of Microorganisms (SysMO) and specifically within the project Systems Understanding of Microbial Oxygen Metabolism (SUMO), regulator activities, gene expression, metabolite levels and metabolic flux datasets were obtained using a standardized and reproducible chemostat-based experimental system. These different types and qualities of data were integrated using mathematical models. The approach described here has revealed a much more detailed picture of the aerobic-anaerobic response, especially for the environmentally critical microaerobic range that is located between unlimited oxygen availability and anaerobiosis.
... These oxidases are also proposed to function as oxygen scavengers under H 2 O 2 and NO stress241242243. Crystal structures have not been resolved for cytochrome bd oxidases but spectroscopic and mutational analyses suggest that the three heme groups are located in CydA and together with the quinol oxidation site are situated close to the P-phase244245246247248249250251Fig. 6 . ...
Membrane integrated respiratory metallo-enzymes occur in all three domains of life and perform the bioenergetic reactions sustaining the living state of the cell. To this end these enzymes catalyze redox chemical reactions that require tightly controlled electron transfer, proton binding and proton translocation events. The great quantity of structural and kinetic studies performed in the last two decades has provided the molecular detail needed for a basic understanding as to how respiratory enzymes convert redox free energy into a proton-motive force. In this review these recent developments are discussed within the framework of Peter Mitchell's Chemiosmotic Hypothesis that is by now a Theory.
... DTT, Q 1 , dithionite and N-lauroyl-sarcosine were from Sigma-Aldrich. Recombinant cyt bd was expressed in the E. coli strain GO105/ pTK1 devoid of cytochrome bo 3 and cyt bd oxidases according to Ref [67]. Enzyme purification was carried out following the protocol described in Refs [68,69]. ...
The cytochrome bd ubiquinol oxidase from Escherichia coli couples the exergonic two-electron oxidation of ubiquinol and four-electron reduction of O(2) to 2H(2)O to proton motive force generation by transmembrane charge separation. The oxidase contains two b-type hemes (b(558) and b(595)) and one heme d, where O(2) is captured and converted to water through sequential formation of a few intermediates. The spectral features of the isolated cytochrome bd at steady-state have been examined by stopped-flow multiwavelength absorption spectroscopy. Under turnover conditions, sustained by O(2) and dithiothreitol (DTT)-reduced ubiquinone, the ferryl and oxy-ferrous species are the mostly populated catalytic intermediates, with a residual minor fraction of the enzyme containing ferric heme d and possibly one electron on heme b(558). These findings are unprecedented and differ from those obtained with mammalian cytochrome c oxidase, in which the oxygen intermediates were not found to be populated at detectable levels under similar conditions [M.G. Mason, P. Nicholls, C.E. Cooper, The steady-state mechanism of cytochrome c oxidase: redox interactions between metal centres, Biochem. J. 422 (2009) 237-246]. The data on cytochrome bd are consistent with the observation that the purified enzyme has the heme d mainly in stable oxy-ferrous and ferryl states. The results are here discussed in the light of previously proposed models of the catalytic cycle of cytochrome bd.
... E. coli strain GO105 (cyd AB∷kan, cyo, recA) devoid of cytochrome bo 3 and cytochrome bd quinol oxidases [48] was used as the host strain for expressing both the wild type and E445A mutant cytochrome bd from a plasmid. In both cases, plasmid pTK1 containing the whole operon encoding cytochrome bd and the ampicillin resistance gene was introduced into the strain [47]. ...
Cytochrome bd is a terminal quinol:O2 oxidoreductase of respiratory chains of many bacteria. It contains three hemes, b558, b595, and d. The role of heme b595 remains obscure. A CO photolysis/recombination study of the membranes of Escherichia coli containing either wild type cytochrome bd or inactive E445A mutant was performed using nanosecond absorption spectroscopy. We compared photoinduced changes of heme d-CO complex in one-electron-reduced, two-electron-reduced, and fully reduced states of cytochromes bd. The line shape of spectra of photodissociation of one-electron-reduced and two-electron-reduced enzymes is strikingly different from that of the fully reduced enzyme. The difference demonstrates that in the fully reduced enzyme photolysis of CO from heme d perturbs ferrous heme b595 causing loss of an absorption band centered at 435 nm, thus supporting interactions between heme b595 and heme d in the di-heme oxygen-reducing site, in agreement with previous works. Photolyzed CO recombines with the fully reduced enzyme monoexponentially with τ ∼ 12 μs, whereas recombination of CO with one-electron-reduced cytochrome bd shows three kinetic phases, with τ ∼ 14 ns, 14 μs, and 280 μs. The spectra of the absorption changes associated with these components are different in line shape. The 14 ns phase, absent in the fully reduced enzyme, reflects geminate recombination of CO with part of heme d. The 14-μs component reflects bimolecular recombination of CO with heme d and electron backflow from heme d to hemes b in ∼ 4% of the enzyme population. The final, 280-μs component, reflects return of the electron from hemes b to heme d and bimolecular recombination of CO in that population. The fact that even in the two-electron-reduced enzyme, a nanosecond geminate recombination is observed, suggests that namely the redox state of heme b595, and not that of heme b558, controls the pathway(s) by which CO migrates between heme d and the medium.
... In the air-oxidized condition, heme d is actually in a reduced state and coordinates a molecular oxygen (10), as an Fe 2ϩ -O 2 diamagnetic EPR-inactive state. Heme b 558 is claimed as a sixcoordinated ferric low-spin heme component of the oxidase which has been shown to be contained within subunit I (11,12) and has been implicated in the oxidation of the substrate, ubiquinol-8, in the cytoplasmic membrane. Heme b 595 is a ferric high-spin center (13,14). ...
The heme axial ligands of bd-type ubiquinol oxidase of Escherichia coli were studied by EPR and optical spectroscopies using nitric oxide (NO) as a monitoring probe. We found that NO bound to ferrous heme d of the air-oxidized and fully reduced enzymes with very high affinity and to ferrous heme b595 of the fully reduced enzyme with low affinity. EPR spectrum of the 14NO complex of the reduced enzyme exhibited an axially symmetric signal with g-values at g = 2.041 and g = 1.993 and a clear triplet of triplet (or a triplet of doublet for the 15NO complex) superhyperfine structure originating from a nitrogenous proximal ligand trans to NO was observed. This EPR species was assigned to the ferrous heme d-NO complex. This suggests that the proximal axial ligand of heme d is a histidine residue in an anomalous condition or other nitrogenous amino acid residue. Furthermore, the EPR line shape of the ferrous heme d-NO was slightly influenced by the oxidation state of the heme b595. This indicates that heme d exists in close proximity to heme b595 forming a binuclear center. Another axially symmetric EPR signal with g-values at g(parallel) = 2.108 and g(perpendicular) = 2.020 appeared after prolonged incubation of the reduced enzyme with NO and was attributed to the ferrous heme b595-NO complex.
... Therefore, the protein is expected to provide four axial ligands; two to the lowspin haem b 558 and one each to the high-spin haems b 595 and d. Three out of the four expected ligands have been identified with a reasonable degree of certainty by a combination of site-directed mutagenesis and a variety of spectroscopic methods (Kaysser et al., 1995; Fang et al., 1989; Jiang et al., 1993; Spinner et al., 1995). These studies have identified His-186 and Met-393 of CydA as the ligands to haem b 558 . ...
The structural genes for the cyanide-insensitive terminal oxidase (CIO) of Pseudomonas aeruginosa were sequenced. The locus comprised two open reading frames, cioA and cioB, coding for gene products of 488 and 335 amino acid residues with predicted molecular masses of 54241 and 37016 Da respectively. These genes were encoded by a 2.7 kb transcript and probably comprise an operon. Upstream of a major transcriptional start site is a -10 promoter region and, approximately at nucleotides -50 and +13, there are sequences homologous to the binding site of the transcriptional regulator Anr. The deduced amino acid sequences of CioA and CioB are homologous to the cytochrome bd quinol oxidases of Escherichia coli and Azotobacter vinelandii. However, no cytochrome d-like signals were found in wild-type P. aeruginosa strains. An atypical cytochrome d-like signal was seen under low-aeration growth conditions but only in strains in which the cioAB genes were present on a high-copy-number plasmid. The appearance of these cytochrome d-like signals was not paralleled by a concomitant increase in CIO activity. These data support the hypothesis that the CIO of P. aeruginosa does not contain haem d. This raises the possibility that there is a family of bacterial quinol oxidases related to the cytochrome bd of E. coli that can differ in their haem composition from the E. coli paradigm.
... Both subunits of the bd-type oxidase are integral membrane proteins with seven and eight putative transmembrane helices, respectively, as revealed by membrane topology studies (239). SUI (CydA) contains the low-spin heme B (cytochrome b 558 ), which is thought to be the quinol oxidation site (87,175). Heme D and a high-spin heme B (cytochrome b 595 ) are shared between the two subunits and are believed to form a bimetallic center where oxygen is reduced to H 2 O (75, 144). ...
Biogenesis of respiratory cytochromes is defined as consisting of the posttranslational processes that are necessary to assemble apoprotein, heme, and sometimes additional cofactors into mature enzyme complexes with electron transfer functions. Different biochemical reactions take place during maturation: (i) targeting of the apoprotein to or through the cytoplasmic membrane to its subcellular destination; (ii) proteolytic processing of precursor forms; (iii) assembly of subunits in the membrane and oligomerization; (iv) translocation and/or modification of heme and covalent or noncovalent binding to the protein moiety; (v) transport, processing, and incorporation of other cofactors; and (vi) folding and stabilization of the protein. These steps are discussed for the maturation of different oxidoreductase complexes, and they are arranged in a linear pathway to best account for experimental findings from studies concerning cytochrome biogenesis. The example of the best-studied case, i.e., maturation of cytochrome c, appears to consist of a pathway that requires at least nine specific genes and more general cellular functions such as protein secretion or the control of the redox state in the periplasm. Covalent attachment of heme appears to be enzyme catalyzed and takes place in the periplasm after translocation of the precursor through the membrane. The genetic characterization and the putative biochemical functions of cytochrome c-specific maturation proteins suggest that they may be organized in a membrane-bound maturase complex. Formation of the multisubunit cytochrome bc, complex and several terminal oxidases of the bo3, bd, aa3, and cbb3 types is discussed in detail, and models for linear maturation pathways are proposed wherever possible.
... Purification of the cytochrome bd oxidase was performed as described previously [9], but with minor modifications which are described by Kaysser et al. [10]. Purification of cytochrome bo 3 was performed as described previously [11], whereas the His-tagged version of this enzyme was purified on a Qiagen Ni2+-nitrilotriacetic acid (NTA) agarose column by low pressure nickel affinity chromatography [12]. ...
Matrix-assisted laser desorption ionization (MALDI) mass spectrometry has been used to obtain accurate molecular weight information for each subunit of several hydrophobic integral membrane proteins: cytochrome bo3 (4 subunits) and cytochrome bd (2 subunits) from E. coli, and the bc1 complex (3 subunits) and the cytochrome c oxidase (3 subunits) from Rhodobacter sphaeroides. The results demonstrate that the MALDI method is a convenient, quick, sensitive and reliable means for obtaining the molecular masses of the subunits of purified multisubunit membrane proteins.
... Analysis of the deduced sequences of B. subtilis cydA and cydB indicated that they are similar to the products of the E. coli cydA and cydB genes, respectively (41 and 36% identity, respectively), which encode the two subunits of the cytochrome bd-type quinol oxidase of E. coli. Studies using site-directed mutagenesis and various spectroscopic methods indicate that in E. coli CydA, His-19, His-186, and Met-393 provide three of the four axial ligands to the iron of the three hemes in the cytochrome bd complex (9,27,45,47). These amino acid residues are preserved in B. subtilis CydA (His-18, His-183, and Met-334). ...
Under aerobic conditions Bacillus subtilis utilizes a branched electron transport chain comprising various cytochromes and terminal oxidases. At present there is evidence for three types of terminal oxidases in B. subtilis: a caa3-, an aa3-, and a bd-type oxidase. We report here the cloning of the structural genes (cydA and cydB) encoding the cytochrome bd complex. Downstream of the structural genes, cydC and cydD are located. These genes encode proteins showing similarity to bacterial ATP-binding cassette (ABC)-type transporters. Analysis of isolated cell membranes showed that inactivation of cydA or deletion of cydABCD resulted in the loss of spectral features associated with cytochrome bd. Gene disruption experiments and complementation analysis showed that the cydC and cydD gene products are required for the expression of a functional cytochrome bd complex. Disruption of the cyd genes had no apparent effect on the growth of cells in broth or defined media. The expression of the cydABCD operon was investigated by Northern blot analysis and by transcriptional and translational cyd-lacZ fusions. Northern blot analysis confirmed that cydABCD is transcribed as a polycistronic message. The operon was found to be expressed maximally under conditions of low oxygen tension.
... Second, it is now evident that in the purified cytochrome bd complex (but not in membranes), even the "native" sixcoordinate, low-spin ferric heme b 558 interacts significantly with CNat high concentrations of the latter. The interaction of low-spin heme b 558 with CNis perhaps not surprising since the heme iron in cytochrome b 558 has His and Met as axial ligands, rather than the more usual case of two histidines as axial ligands (61,64). Methionine is a relatively weak axial ligand for the ferric heme iron and can be displaced by strong exogenous ligands such as CN -. ...
The interactions of the fully reduced and fully oxidized cytochrome bd from E. coli with ligands CO, NO, and CN- have been studied by a combination of absorption and magnetic circular dichroism (MCD) spectroscopy. In the reduced cytochrome bd, MCD resolves individual bands due to the high-spin heme b595 and the low-spin heme b558 components of the enzyme, allowing one to separately monitor their interactions along with ligand binding to the heme d component. The data show that at low concentrations, the ligands bind almost exclusively to heme d. At high concentrations, the ligands begin to interact with the low-spin heme b558. At the same time, no evidence for significant binding of the ligands to the high-spin heme b595 is revealed in either the reduced or the fully oxidized cytochrome bd complex. The data support the model [Borisov, V. B., Gennis, R. B., and Konstantinov, A. A. (1995) Biochemistry (Moscow) 60, 231-239] according to which the two high-spin hemes d and b595 share a high-affinity ligand binding site with a capacity for only a single molecule of the ligand; i.e., there is a strong negative cooperativity with respect to ligand binding to these two hemes with cytochrome d having an intrinsic ligand affinity much higher than that of heme b595.
Biogenesis of respiratory cytochromes is defined as consisting of the posttranslational processes that are necessary to assemble apoprotein, heme, and sometimes additional cofactors into mature enzyme complexes with electron transfer functions. Different biochemical reactions take place during maturation: (i) targeting of the apoprotein to or through the cytoplasmic membrane to its subcellular destination; (ii) proteolytic processing of precursor forms; (iii) assembly of subunits in the membrane and oligomerization; (iv) translocation and/or modification of heme and covalent or noncovalent binding to the protein moiety; (v) transport, processing, and incorporation of other cofactors; and (vi) folding and stabilization of the protein. These steps are discussed for the maturation of different oxidoreductase complexes, and they are arranged in a linear pathway to best account for experimental findings from studies concerning cytochrome biogenesis. The example of the best-studied case, i.e., maturation of cytochrome c, appears to consist of a pathway that requires at least nine specific genes and more general cellular functions such as protein secretion or the control of the redox state in the periplasm. Covalent attachment of heme appears to be enzyme catalyzed and takes place in the periplasm after translocation of the precursor through the membrane. The genetic characterization and the putative biochemical functions of cytochrome c-specific maturation proteins suggest that they may be organized in a membrane-bound maturase complex. Formation of the multisubunit cytochrome bc, complex and several terminal oxidases of the bo3, bd, aa3, and cbb3 types is discussed in detail, and models for linear maturation pathways are proposed wherever possible.
Like most bacteria, Escherichia coli has a flexible and branched respiratory chain that enables the prokaryote to live under a variety of environmental conditions, from highly aerobic to completely anaerobic. In general, the bacterial respiratory chain is composed of dehydrogenases, a quinone pool, and reductases. Substrate-specific dehydrogenases transfer reducing equivalents from various donor substrates (NADH, succinate, glycerophosphate, formate, hydrogen, pyruvate, and lactate) to a quinone pool (menaquinone, ubiquinone, and dimethylmenoquinone). Then electrons from reduced quinones (quinols) are transferred by terminal reductases to different electron acceptors. Under aerobic growth conditions, the terminal electron acceptor is molecular oxygen. A transfer of electrons from quinol to O₂ is served by two major oxidoreductases (oxidases), cytochrome bo₃ encoded by cyoABCDE and cytochrome bd encoded by cydABX. Terminal oxidases of aerobic respiratory chains of bacteria, which use O₂ as the final electron acceptor, can oxidize one of two alternative electron donors, either cytochrome c or quinol. This review compares the effects of different inhibitors on the respiratory activities of cytochrome bo₃ and cytochrome bd in E. coli. It also presents a discussion on the genetics and the prosthetic groups of cytochrome bo₃ and cytochrome bd. The E. coli membrane contains three types of quinones that all have an octaprenyl side chain (C₄₀). It has been proposed that the bo₃ oxidase can have two ubiquinone-binding sites with different affinities. "WHAT'S NEW" IN THE REVISED ARTICLE: The revised article comprises additional information about subunit composition of cytochrome bd and its role in bacterial resistance to nitrosative and oxidative stresses. Also, we present the novel data on the electrogenic function of appBCX-encoded cytochrome bd-II, a second bd-type oxidase that had been thought not to contribute to generation of a proton motive force in E. coli, although its spectral properties closely resemble those of cydABX-encoded cytochrome bd.
The heme axial ligands of bd-type ubiquinol oxidase of Escherichia coli were studied by EPR and optical spectroscopies using nitric oxide (NO) as a monitoring probe. We found that NO bound to ferrous heme d of the air-oxidized and fully reduced enzymes with very high affinity and to ferrous heme b(595) of the fully reduced enzyme with low affinity. EPR spectrum of the (NO)-N-14 complex of the reduced enzyme exhibited an axially symmetric signal with g-values at g(perpendicular to) = 2.041 and g(parallel to) = 1.993 and a clear triplet of triplet (or a triplet of doublet for the (NO)-N-15 complex) superhyperfine structure originating from a nitrogenous proximal ligand trans to NO was observed. This EPR species was assigned to the ferrous heme d-NO complex. This suggests that the proximal axial ligand of heme d is a histidine residue in an anomalous condition or other nitrogenous amino acid residue. Furthermore, the EPR line shape of the ferrous heme d-NO was slightly influenced by the oxidation state of the heme b(595). This indicates that heme d exists in close proximity to heme b(595) forming a binuclear center. Another axially symmetric EPR signal with g-values at g(parallel to) = 2.108 and g(perpendicular to) = 2.020 appeared after prolonged incubation of the reduced enzyme with NO and was attributed to the ferrous heme b(595)-NO complex.
Oxygen as Acceptor, Page 1 of 2
Abstract
Like most bacteria, Escherichia coli has a flexible and branched respiratory chain that enables the prokaryote to live under a variety of environmental conditions, from highly aerobic to completely anaerobic. In general, the bacterial respiratory chain is composed of dehydrogenases, a quinone pool, and reductases. Substrate specific dehydrogenases transfer reducing equivalents from various donor substrates (NADH, succinate, glycerophoshate, formate, hydrogen, pyruvate, and lactate) to a quinone pool (menaquinone, ubiquinone, and demethylmenoquinone). Then electrons from reduced quinones (quinols) are transferred by terminal reductases to different electron acceptors. Under aerobic growth conditions, the terminal electron acceptor is molecular oxygen. A transfer of electrons from quinol to O2 is served by two major oxidoreductases (oxidases), cytochrome bo3 and cytochrome bd. Terminal oxidases of aerobic respiratory chains of bacteria, which use O2 as the final electron acceptor, can oxidize one of two alternative electron donors, either cytochrome c or quinol. This review compares the effects of different inhibitors on the respiratory activities of cytochrome bo3 and cytochrome bd in E. coli. It also presents a discussion on the genetics and the prosthetic groups of cytochrome bo3 and cytochrome bd. The E. coli membrane contains three types of quinones which all have an octaprenyl side chain (C40). It has been proposed that the bo 3 oxidase can have two ubiquinone-binding sites with different affinities. The spectral properties of cytochrome bd-II closely resemble those of cydAB-encoded cytochrome bd.
Cyanide-insensitive terminal quinol oxidase (CIO) is a subfamily of cytochrome bd present in bacterial respiratory chain. We purified CIO from the Gluconobacter oxydans membranes and characterized its properties. The air-oxidized CIO showed some or weak peaks of reduced hemes b and of oxygenated and ferric heme d, differing from cytochrome bd. CO- and NO-binding difference spectra suggested that heme d serves as the ligand binding site of CIO. Notably the purified CIO showed an extraordinary high ubiquinol-1 oxidase activity with the pH optimum of pH 5 to 6. The apparent Vmax value of CIO was 17-fold higher than that of G. oxydans cytochrome bo3. In addition, compared with Escherichia coli cytochrome bd, the quinol oxidase activity of CIO was much more resistant to cyanide, but sensitive to azide. The Km value for O2 of CIO was 7- to 10-fold larger than that of G. oxydans cytochrome bo3 or E. coli cytochrome bd. Our results suggest that CIO has unique features attributable to the structure and properties of the O2-binding site, and thus forms a new sub-group distinct from cytochrome bd. Furthermore, CIO of acetic acid bacteria may play some specific role for rapid oxidation of substrates under acidic growth conditions.
Azotobacter vinelandii is an obligately aerobic bacterium in which aerotolerant dinitrogen fixation requires cytochrome bd. This oxidase comprises two polypeptide subunits and three hemes, but no copper, and has been studied extensively. However,
there remain apparently conflicting reports on the reactivity of the high spin hemeb
595 with ligands. Using purified cytochromebd, we show that absorption changes induced by CO photodissociation from the fully reduced cytochrome bd at low temperatures demonstrate binding of the ligand with hemeb
595. However, the magnitude of these changes corresponds to the reaction with CO of only about 5% of the heme. CO binding with
a minor fraction of heme b
595 is also revealed at room temperature by time-resolved studies of CO recombination. The data resolve the apparent discrepancies
between conclusions drawn from room and low temperature spectroscopic studies of the CO reaction with cytochrome bd. The results are consistent with the proposal that hemes b
595and d form a diheme oxygen-reducing center with a binding capacity for a single exogenous ligand molecule that partitions between
the hemes d and b
595 in accordance with their intrinsic affinities for the ligand. In this model, the affinity of heme b
595 for CO is about 20-fold lower than that of heme d.
The quinol-linked cytochrome bd oxidases are terminal oxidases in respiration. These oxidases harbor a low spin heme b558 that donates electrons to a binuclear heme b595/heme d center. The reaction with O2 and subsequent catalytic steps of the Escherichia coli cytochrome bd-I oxidase were investigated by means of ultra-fast freeze-quench trapping followed by EPR and UV-visible spectroscopy. After
the initial binding of O2, the O–O bond is heterolytically cleaved to yield a kinetically competent heme d oxoferryl porphyrin π-cation radical intermediate (compound I) magnetically interacting with heme b595. Compound I accumulates to 0.75–0.85 per enzyme in agreement with its much higher rate of formation (∼20,000 s−1) compared with its rate of decay (∼1,900 s−1). Compound I is next converted to a short lived heme d oxoferryl intermediate (compound II) in a phase kinetically matched to the oxidation of heme b558 before completion of the reaction. The results indicate that cytochrome bd oxidases like the heme-copper oxidases break the O–O bond in a single four-electron transfer without a peroxide intermediate.
However, in cytochrome bd oxidases, the fourth electron is donated by the porphyrin moiety rather than by a nearby amino acid. The production of reactive
oxygen species by the cytochrome bd oxidase was below the detection level of 1 per 1000 turnovers. We propose that the two classes of terminal oxidases have
mechanistically converged to enzymes in which the O–O bond is broken in a single four-electron transfer reaction to safeguard
the cell from the formation of reactive oxygen species.
Purple sulfur bacteria, which are known to be the most ancient among anoxygenic phototrophs, play an important role in the global sulfur cycle. Allochromatium vinosum oxidizes reduced sulfur compounds such as hydrogen sulfide, elemental sulfur and thiosulfide. At low oxygen concentrations, A. vinosum can grow chemotrophically using oxygen as the terminal electron acceptor. Being also a nitrogen fixer, A. vinosum is faced with the paradox of co-existence of aerobic metabolism and nitrogen fixation. Due to growth difficulties, only a few studies have dealt with the aerobic metabolism of the organism and, until now, there has been no information about the genes involved in the respiratory metabolism of purple sulfur bacteria. In this article we show the first terminal oxidase gene for A. vinosum. The presence of a Bd type of quinol oxidase is necessary to protect nitrogenases against the inhibitory effects of oxygen. In this case, a nitrogen fixation related gene is part of the cyd operon and this gene is co-transcribed with cydAB genes. Bd oxidase of A. vinosum may be the earliest form of oxidase where the function of the enzyme is to scavenge the contaminant oxygen during nitrogen fixation. This may be an important clue about the early evolution of oxygenic photosynthesis, perhaps as a protective mechanism for nitrogen fixation.
Cellular energy generation uses membrane-localized electron transfer chains for ATP synthesis. Formed ATP in turn is consumed for the biosynthesis of cellular building blocks. In contrast, heme cofactor biosynthesis was found driving ATP generation via electron transport after initial ATP consumption. The FMN enzyme protoporphyrinogen IX oxidase (HemG) of Escherichia coli abstracts six electrons from its substrate and transfers them via ubiquinone, cytochrome bo(3) (Cyo) and cytochrome bd (Cyd) oxidase to oxygen. Under anaerobic conditions electrons are transferred via menaquinone, fumarate (Frd) and nitrate reductase (Nar). Cyo, Cyd and Nar contribute to the proton motive force that drives ATP formation. Four electron transport chains from HemG via diverse quinones to Cyo, Cyd, Nar, and Frd were reconstituted in vitro from purified components. Characterization of E. coli mutants deficient in nar, frd, cyo, cyd provided in vivo evidence for a detailed model of heme biosynthesis coupled energy generation.
In this thesis, the role of the Escherichia coli YidC protein in membrane biogenesis is investigated with a focus on subunits of respiratory complexes. A directed proteomics approach was applied to investigate the effect of YidC depletion on components of the respiratory chains expressed under anaerobic conditions. It was found that YidC depletion under these conditions caused a cessation in growth and that a number of respiratory complexes were negatively affected. This was further investigated, where metabolic labeling with 15N/14N was employed to study the membrane proteome-wide changes upon YidC depletion under both aerobic and anaerobic respiratory conditions. This allowed the identification of putative YidC substrates and the in vitro insertion requirements of subunit K of complex I were characterized. It was found that NuoK absolutely requires the Sec translocase and YidC for insertion. Essential membrane-embedded negative charges contained in NuoK were shown to confer YidC-dependence. Using the mechanosensitive channel of large conductance, MscL, it was shown further that in vitro translation and membrane insertion systems can be exploited to produce fully active oligomeric protein complexes in a cell-free system. Since YidC has been shown to interact with the SecDFYajC complex the effect of SecDFYajC depletion on the membrane proteome of E. coli was also investigated. The findings are discussed in the context of our current knowledge of the Oxa1/Alb3/YidC family of proteins with particular focus on the diverse functioning of YidC in membrane biogenesis in E. coli.
Microbial metal reduction contributes to biogeochemical cycling, and reductive precipitation provides the basis for bioremediation strategies designed to immobilize radionuclide contaminants present in the subsurface. Facultatively anaerobic ×-proteobacteria of the genus Shewanella are present in many aquatic and terrestrial environments and are capable of respiration on a wide range of compounds as terminal electron acceptor including transition metals, uranium and transuranics. S. putrefaciens is readily cultivated in the laboratory and a genetic system was recently developed to study U(VI) reduction in this organism. U(VI) reduction-deficient S. putrefaciens point mutant Urr14 (hereafter referred to as CCMB1) was found to retain the ability to respire several alternate electron acceptors. In the present study, CCMB1 was tested on a suite of electron acceptors and found to retain growth on electron acceptors with high reduction potential (E¡¬0) [O2, Fe(III)-citrate, Mn(IV), Mn(III)-pyrophosphate, NO3-] but was impaired for anaerobic growth on electron acceptors with low E¡¬0 [NO2-, U(VI), dimethyl sulfoxide, trimethylamine N-oxide, fumarate, ×-FeOOH, SO32-, S2O32-]. Genetic complementation and sequencing analysis revealed that CCMB1 contained a point mutation (H108Y) in a CcmB homolog, an ABC transporter permease subunit required for c-type cytochrome maturation in E. coli. The periplasmic space of CCMB1 contained low levels of cytochrome c and elevated levels of free thiol equivalents (-SH), an indication that redox homeostasis was disrupted. Anaerobic growth ability, but not cytochrome c maturation activity, was restored to CCMB1 by adding exogenous disulfide bond-containing compounds (e.g., cystine) to the growth medium. To test the possibility that CcmB transports heme from the cytoplasm to the periplasm in S. putrefaciens, H108 was replaced with alanine, leucine, methionine and lysine residues via site-directed mutagenesis. Anaerobic growth, cytochrome c biosynthesis or redox homeostasis was disrupted in each of the site-directed mutants except H108M. The results of this study demonstrate, for the first time, that S. putrefaciens requires CcmB to produce c-type cytochromes under U(VI)-reducing conditions and maintain redox homeostasis during growth on electron acceptors with low E¡¬0. The present study is the first to examine CcmB activity during growth on electron acceptors with widely-ranging E¡¬0, and the results suggest that cytochrome c or free heme maintains periplasmic redox poise during growth on electron acceptors with E¡¬0 < 0.36V such as in the subsurface engineered for rapid U(VI) reduction or anoxic environments dominated by sulfate-reducing bacteria. A mechanism for CcmB heme translocation across the S. putrefaciens cytoplasmic membrane via heme coordination by H108 is proposed. Ph.D. Committee Chair: Thomas J. DiChristina; Committee Member: Frank E. Loeffler; Committee Member: John R. Kirby; Committee Member: Martial Taillefert; Committee Member: Roger Wartell
The genome of the green sulfur bacterium Chlorobaculum (Cba.) tepidum, a strictly anaerobic photolithoautotroph, is predicted to encode more than ten genes whose products are potentially involved in protection from reactive oxygen species and an oxidative stress response. The encoded proteins include cytochrome bd quinol oxidase, NADH oxidase, rubredoxin oxygen oxidoreductase, several thiol peroxidases, alkyl hydroperoxide reductase, superoxide dismutase, methionine sulfoxide reductase, and rubrerythrin. To test the physiological functions of some of these proteins, ten genes were insertionally inactivated. Wild-type Cba. tepidum cells were very sensitive to oxygen in the light but were remarkably resistant to oxygen in the dark. When wild-type and mutant cells were subjected to air for various times under dark or light condition, significant decreases in viability were detected in most of the mutants relative to wild type. Treatments with hydrogen peroxide (H(2)O(2)), tert-butyl hydroperoxide (t-BOOH) and methyl viologen resulted in more severe effects in most of the mutants than in the wild type. The results demonstrated that these putative antioxidant proteins combine to form an effective defense against oxygen and reactive oxygen species. Reverse-transcriptase polymerase chain reaction studies showed that the genes with functions in oxidative stress protection were constitutively transcribed under anoxic growth conditions.
Cytochrome bd is a cyanide-resistant terminal quinol oxidase under micro-aerophilic growth conditions and generates a proton motive force via scalar protolytic reactions. Protons used for dioxygen reduction are taken up from the cytoplasm and delivered to haem d through a proton channel. Electrons are transferred from quinols to haem d through haem b558 and haem b595. All three haems are bound to subunit I but only the axial ligand of haem d remains to be determined. Haems b595 and d form a haem-haem binuclear centre and substitutions of either His19 in helix I (haem b595 ligand) and Glu99 in helix III eliminated or severely reduced both haems. To probe the location of the haem d ligand, we introduced mutations around His19 and Glu99 and examined the cyanide-resistance of the oxidase activity and spectroscopic properties. In contrast to mutations around His19, I98F and L101T reduced the IC50 for cyanide to 0.18 and 0.41 mM, respectively, from 1.4 mM of the wild-type. Blue shifts in the alpha peak of I98F suggest that Ile98 is in the vicinity of the haem d-binding site. Our data are consistent with the proposal that Glu99 serves as a haem d ligand of cytochrome bd.
Cytochromes bo and bd are terminal ubiquinol oxidases in the aerobic respiratory chain of Escherichia coli and generate proton motive force across the membrane. To probe roles of haem species in the oxidation of quinols, intramolecular
electron transfer and the dioxygen reduction, we replaced b-haems with haem O by using the haem O synthase-overproducing system, which can accumulate haem O in cytoplasmic membranes.
Characterizations of spectroscopic properties of cytochromes bo and bd isolated from BL21 (DE3)/pLysS and BL21 (DE3)/pLysS/pTTQ18-cyoE after 4 h of the aerobic induction of haem O synthase (CyoE)
showed the specific incorporation of haem O into the low-spin haem-binding site in both oxidases. We found that the resultant
haem oo- and obd-type oxidase severely reduced the ubiquinol-1 oxidase activity due to the perturbations of the quinol oxidation site. Our
observations suggest that haem B is required at the low-spin haem site for the oxidation of quinols by cytochromes bo and bd.
In the aerobic respiratory chain of the cyanobacterium Synechocystis sp. PCC 6803, cytochrome c oxidase serves as a major terminal oxidase while cyanide-resistant cytochrome bd serves as an alternative oxidase and evades the over-reduction of the plastoquinone pool under stress conditions. Here we expressed Synechocystis cytochrome bd in Escherichia coli and characterized enzymatic and spectroscopic properties. Cyanobacterial cytochrome bd showed the higher activity with ubiquinols than with decyl-plastoquinol and K(m) values for quinols were 2-fold smaller than those of E. coli cytochrome bd (CydAB). The dioxygen reduction site was resistant to cyanide as in E. coli oxidase while the quinol oxidation site was more sensitive to antimycin A and quinolone inhibitors. Spectroscopic analysis showed the presence of the haem b(595)-d binuclear centre but the sequence analysis indicates that cyanobacterial cytochrome bd is structurally related to cyanide-insensitive oxidase (CioAB), which does not show typical spectral changes upon reduction and ligand binding. Our data indicate that cyanobacterial cytochrome bd has unique enzymatic and structural properties and we hope that our findings will help our understanding the role and properties of CydAB and CioAB quinol oxidases in other bacterial species.
Cytochrome bd oxidase is a bacterial terminal oxidase that contains three cofactors: a low-spin heme (b558), a high-spin heme (b595), and a chlorin d. The center of dioxygen reduction has been proposed to be a binuclear b595/d site, whereas b558 is mainly involved in transferring electrons from ubiquinol to the oxidase. Information on the nature of the axial ligands of the three heme centers has come from site-directed mutagenesis and spectroscopy, which have implicated a His/Met coordination for b558 (Spinner, F., Cheesman, M. R., Thomson, A. J., Kaysser, T., Gennis, R. B., Peng, Q., & Peterson, J. (1995) Biochem. J. 308, 641-644; Kaysser, T. M., Ghaim, J. B., Georgiou, C., & Gennis, R. B. (1995) Biochemistry 34, 13491-13501), but the ligands to b595 and d are not known with certainty. In this work, the three heme chromophores of the fully reduced cytochrome bd oxidase are studied individually by selective enhancement of their resonance Raman (rR) spectra at particular excitation wavelengths. The rR spectrum obtained with 413.1-nm excitation is dominated by the bands of the 5cHS b595(2+) cofactor. Excitation close to 560 nm yields a rR spectrum dominated by the 6cLS b558(2+) heme. Wavelengths between these values enhance contributions from both b595(2+) and b558(2+) chromophores. The rR bands of the ferrous chlorin become the major features with red laser excitation (595-650 nm). The rR data indicate that d2+ is a 5cHS system whose axial ligand is either a weakly coordinating protein donor or a water molecule. In the low-frequency region of the 441.6-nm spectrum, we assign a rR band at 225 cm-1 to the (b595)Fe(II)-N(His) stretching vibration, based on its 1.2-cm(-1) upshift in the 54Fe-labeled enzyme. This observation provides the first physical evidence that the proximal ligand of b595 is a histidine. Site-directed mutagenesis had suggested that His 19 is associated with either b595 or d (Fang, H., Lin, R. -J., & Gennis, R. B. (1989) J. Biol. Chem. 264, 8026-8032). On the basis of the present study, we propose that the proximal ligand of b595 is His 19. We have also studied the reaction of cyanide with the fully reduced cytochrome bd oxidase. In approximately 700-fold excess cyanide (approximately 35 mM), the 629-nm UV/vis band of d2+ is blue-shifted to 625 nm and diminished in intensity. However, the rR spectra at each of three different gamma(0) (413.1, 514.5, and 647.1 nm) are identical with or without cyanide, thus indicating that both b595 and d remain as 5cHS species in the presence of CN-. This observation leads to the proposal that a native ligand of ferrous chlorin d is replaced by CN- to form the 5cHS d2+ cyano adduct. These findings corroborate our companion study of the "as-isolated" enzyme in which we proposed a 5cHS d3+ cyano adduct (Sun, J., Osborne, J. P., Kahlow, M. A., Kaysser, T. M., Hill, J. J., Gennis, R. B., & Loehr, T. M. (1995) Biochemistry 34, 12144-12151). To further characterize the unusual and unexpected nature of these proposed high-spin cyanide adducts, we have obtained EPR spectral evidence that binding of cyanide to fully oxidized cytochrome bd oxidase perturbs a spin-state equilibrium in the chlorin d3+ to yield entirely the high-spin form of the cofactor.
Decyl-aurachin D is a near-stoichiometric inhibitor of cytochrome bd from Azotobacter vinelandii. Interaction of decyl-aurachin D with the oxidase induces a redshift of the alpha-band and Soret band of a b-type cytochrome, probably b-558, suggesting close proximity of the inhibitor binding site to this haem and hence to the proposed quinol binding domain. The compound does not affect the oxygen binding site directly as judged from unchanged CO recombination kinetics to haem d in dithionite-reduced enzyme. Although in the presence of ubiquinol-1 a decyl-aurachin D containing sample generates levels of haem reduction and catalytic intermediates similar to the control, the approach to this steady state is severely inhibited. In addition to the spectral effect on b-558, decyl-aurachin D raises the midpoint potential of haem b-558, but also lowers that of haem b-595. Consistent with the shift in midpoint potentials, electron backflow from haem d to the b-type haems can be observed in decyl-aurachin D inhibited samples following photolysis of the mixed-valence CO-ligated form of the enzyme. The data show that decyl-aurachin D acts on the donor side of haem b-558 without substantially affecting internal electron transfer rates or the oxygen reduction site.
Cytochrome bd' has been implicated in having an important role in microaerobic nitrogen fixation in the enteric bacterium Klebsiella pneumoniae, where it is expressed under all conditions that permit diazotrophy. In this paper the sequence of the genes encoding this terminal oxidase (cydAB) of Klebsiella pneumoniae and the characterization of a cyd mutant are reported. The deduced amino acid sequences support the proposal that His 19, His 186 and Met 393 provide three of the four axial ligands to the Fe of the three haems in the oxidase complex. The nitrogen-fixing ability of the mutant was severely impaired in the presence of low concentrations of oxygen compared with the wild-type bacterium. Only the wild-type organism was capable of microaerobic nitrogenase activity supported by fermentation products. It is proposed that formate dehydrogenase-O may be involved in supplying electrons to a respiratory chain terminated by the bd-type oxidase, which would remove inhibitory oxygen and supply ATP for nitrogenase activity.
We identified a protein, Aer, as a signal transducer that senses intracellular energy levels rather than the external environment and that transduces signals for aerotaxis (taxis to oxygen) and other energy-dependent behavioral responses in Escherichia coli. Domains in Aer are similar to the signaling domain in chemotaxis receptors and the putative oxygen-sensing domain of some transcriptional activators. A putative FAD-binding site in the N-terminal domain of Aer shares a consensus sequence with the NifL, Bat, and Wc-1 signal-transducing proteins that regulate gene expression in response to redox changes, oxygen, and blue light, respectively. A double mutant deficient in aer and tsr, which codes for the serine chemoreceptor, was negative for aerotaxis, redox taxis, and glycerol taxis, each of which requires the proton motive force and/or electron transport system for signaling. We propose that Aer and Tsr sense the proton motive force or cellular redox state and thereby integrate diverse signals that guide E. coli to environments where maximal energy is available for growth.
Gram-positive thermophilic Bacillus species contain cytochrome caa3-type cytochrome c oxidase as their main terminal oxidase in the respiratory chain. We previously identified and purified an alternative oxidase, cytochrome bd-type quinol oxidase, from a mutant of Bacillus stearothermophilus defective in the caa3-type oxidase activity (J. Sakamoto et al., FEMS Microbiol. Lett. 143 (1996) 151-158). Compared with proteobacterial counterparts, B. stearothermophilus cytochrome bd showed lower molecular weights of the two subunits, shorter wavelength of alpha-band absorption maximum due to heme D, and lower quinol oxidase activity. Preincubation with menaquinone-2 enhanced the enzyme activity up to 40 times, suggesting that, besides the catalytic site, there is another quinone-binding site which largely affects the enzyme activity. In order to clarify the molecular basis of the differences of cytochromes bd between B. stearothermophilus and proteobacteria, the genes encoding for the B. stearothermophilus bd was cloned based on its partial peptide sequences. The gene for subunit I (cbdA) encodes 448 amino acid residues with a molecular weight of 50195 Da, which is 14 and 17% shorter than those of Escherichia coli and Azotobacter vinelandii, respectively, and CbdA lacks the C-terminal half of the long hydrophilic loop between the putative transmembrane segments V and VI (Q loop), which has been suggested to include the substrate quinone-binding site for the E. coli enzyme. The gene for subunit II (cbdB) encodes 342 residues with a molecular weight of 38992 Da. Homology search indicated that the B. stearothermophilus cbdAB has the highest sequence similarity to ythAB in B. subtilis genome rather than to cydAB, the first set of cytochrome bd genes identified in the genome. Sequence comparison of cytochromes bd and their homologs from various organisms demonstrates that the proteins can be classified into two subfamilies, a proteobacterial type including E. coli bd and a more widely distributed type including the B. stearothermophilus enzyme, suggesting that the latter type is evolutionarily older.
Disulfide bond formation is catalyzed in vivo by DsbA and DsbB. Here we reconstitute this oxidative folding system using purified components. We have found the sources of oxidative power for protein folding and show how disulfide bond formation is linked to cellular metabolism. We find that disulfide bond formation and the electron transport chain are directly coupled. DsbB uses quinones as electron acceptors, allowing various choices for electron transport to support disulfide bond formation. Electrons flow via cytochrome bo oxidase to oxygen under aerobic conditions or via cytochrome bd oxidase under partially anaerobic conditions. Under truly anaerobic conditions, menaquinone shuttles electrons to alternate final electron acceptors such as fumarate. This flexibility reflects the vital nature of the disulfide catalytic system.
Interaction of the two high-spin hemes in the oxygen reduction site of the bd-type quinol oxidase from Escherichia coli has been studied by femtosecond multicolor transient absorption spectroscopy. The previously unidentified Soret band of ferrous heme b(595) was determined to be centered around 440 nm by selective excitation of the fully reduced unliganded or CO-bound cytochrome bd in the alpha-band of heme b(595). The redox state of the b-type hemes strongly affects both the line shape and the kinetics of the absorption changes induced by photodissociation of CO from heme d. In the reduced enzyme, CO photodissociation from heme d perturbs the spectrum of ferrous cytochrome b(595) within a few ps, pointing to a direct interaction between hemes b(595) and d. Whereas in the reduced enzyme no heme d-CO geminate recombination is observed, in the mixed-valence CO-liganded complex with heme b(595) initially oxidized, a significant part of photodissociated CO does not leave the protein and recombines with heme d within a few hundred ps. This caging effect may indicate that ferrous heme b(595) provides a transient binding site for carbon monoxide within one of the routes by which the dissociated ligand leaves the protein. Taken together, the data indicate physical proximity of the hemes d and b(595) and corroborate the possibility of a functional cooperation between the two hemes in the dioxygen-reducing center of cytochrome bd.
The crystal structure of the His-175-->Gly (H175G) mutant of cytochrome-c peroxidase (EC 1.11.1.5), missing its only heme ligand, reveals that the histidine is replaced by solvent to give a bisaquo heme protein. This protein retains some residual activity, which can be stimulated or inhibited by addition of exogenous ligands. Structural analysis confirms the binding of imidazole to the heme at the position of the wild-type histidine ligand. This imidazole complex reacts readily with hydrogen peroxide to produce a radical species with novel properties. However, reactivation in this complex is incomplete (approximately 5%), which, in view of the very similar structures of the wild-type and the H175G/imidazole forms, implies a critical role for tethering of the axial ligand in catalysis. This study demonstrates the feasibility of constructing heme enzymes with no covalent link to the protein and with unnatural ligand replacements. Such enzymes may prove useful in studies of electron transfer mechanisms and in the engineering of novel heme-based catalysts.
Azotobacter vinelandii is a free-living nitrogen-fixing bacterium that has one of the highest respiratory rates of all aerobic organisms. Based on various physiological studies, a d-type cytochrome has been postulated to be the terminal oxidase of a vigorously respiring but apparently uncoupled branch of the electron transport system in the membranes of this organism. We cloned and characterized the structural genes of the two subunits of this oxidase. The deduced amino acid sequences of both subunits of the A. vinelandii oxidase have extensive regions of homology with those of the two subunits of the Escherichia coli cytochrome d complex. Most notably, the histidine residues proposed to be the axial ligands for the b hemes of the E. coli oxidase and an 11-amino-acid stretch proposed to be part of the ubiquinone binding site are all conserved in subunit I of the A. vinelandii oxidase. The A. vinelandii cytochrome d was expressed in a spectrally and functionally active form in the membranes of E. coli, under the control of the lac or tac promoter. The spectral features of the A. vinelandii cytochrome d expressed in E. coli are very similar to those of the E. coli cytochrome d. The expressed oxidase was active as a quinol oxidase and could reconstitute an NADH to oxygen electron transport chain.
The Escherichia coli acid phosphatase gene appA is expressed in response to oxygen deprivation and is positively controlled by the product of appR (katF) which encodes a putative new sigma transcription-initiation factor. However, transcription of appA from its nearest promoter (P1) did not account for total pH 2.5 acid phosphatase expression and was not subject to regulation. The cloned region upstream of appA was extended and analyzed by insertions of transposon TnphoA and by fusions with lacZ. It contains two new genes, appC and appB, which both encode extracytoplasmic proteins. appC and appB are expressed from a promoter (P2) lying just upstream of appC. Both genes are regulated by oxygen, as is appA, and by appR gene product exactly as previously shown for appA. Analysis of the nucleotide sequence and of the origins of transcription have confirmed that the P2-appC-appB- (ORFX)-P1-appA region is organized on the chromosome as an operon transcribed clockwise from P2 and that P1 is a minor promoter for appA alone. Genes appC and appB encode proteins of Mr 58,133 and 42,377, respectively, which have the characteristics of integral membrane proteins. The deduced amino acid sequences of appC and appB show 60% and 57% homology, respectively, with subunits I and II of the E. coli cytochrome d oxidase (encoded by genes cydA and cydB). The notion that the AppC and AppB proteins constitute a new cytochrome oxidase or a new oxygen-detoxifying system is supported by the observation of enhanced sensitivity to oxygen of mutants lacking all three genes, cyo (cytochrome o oxidase), cyd (cytochrome d oxidase) and appB, compared to that of cyo cyd double mutants.
The aerobic respiratory chain of Escherichia coli contains two terminal oxidases: the cytochrome d complex and the cytochrome o complex. Each of these enzymes catalyzes the oxidation of ubiquinol-8 within the cytoplasmic membrane and the reduction of molecular oxygen to water. Both oxidases are coupling sites in the respiratory chain; electron transfer from ubiquinol to oxygen results in the generation of a proton electrochemical potential difference across the membrane. The cytochrome d complex is a heterodimer (subunits I and II) that has three heme prosthetic groups. Previous studies characterized two monoclonal antibodies that bind to subunit I and specifically block the ability of the enzyme to oxidize ubiquinol. In this paper, the epitopes of both of these monoclonal antibodies have been mapped to within a single 11-amino acid stretch of subunit I. The epitope is located in a large hydrophilic loop between the fifth and sixth putative membrane-spanning segments. Binding experiments with these monoclonal antibodies show this polypeptide loop to be periplasmic. Such localization suggests that the loop may be close to His186, which has been identified as one of the axial ligands of cytochrome b558. Together, these data begin to define a functional domain in which ubiquinol is oxidized near the periplasmic surface of the membrane.
The genome of Azotobacter vinelandii contains DNA sequences homologous to the structural genes for the Escherichia coli cytochrome bd terminal oxidase complex. Two recombinant clones bearing cydA- and cydB-like sequence were isolated from an A. vinelandii gene library and subcloned into the plasmid vector pACYC184. Physical mapping demonstrated that the cydA- and cydB-like regions in A. vinelandii are contiguous. The cydAB and flanking DNA was mutagenized by the insertion of Tn5-B20. Mutations in the cydB-hybridizing region resulted in the loss of spectral features associated with cytochromes b595 and d. A new locus, cydB, encoding cytochromes b595 and d in A. vinelandii is proposed. A second region adjacent to cydB was also involved in expression of the cytochrome bd complex in A. vinelandii, since mutations in this region resulted in an increase in the levels of both cytochrome b595 and cytochrome d. The regions involved in expression of the cytochrome bd complex and cydB are transcribed in the same direction. Mutants deficient in cytochromes b595 and d were unable to grow on N-deficient medium when incubated in air but could fix nitrogen when the environmental O2 concentration was reduced to 1.5% (vol/vol). It is proposed that the branch of the respiratory chain terminated by the cytochrome bd complex supports the high respiration rates required for the respiratory protection of nitrogenase.
The cytochrome d complex from Escherichia coli has been reconstituted in proteoliposomes. Previous studies have shown that the enzyme rapidly oxidizes ubiquinol-8 within the bilayer as well as the soluble homologue, ubiquinol-1, and that quinol oxidase activity is accompanied by the formation of a transmembrane potential across the vesicle bilayer. In this work, the proton pumping activity of the cytochrome in the reconstituted vesicles is examined. Ubiquinol-1 oxidase activity is shown to be accompanied by the net alkalinization of the interior space of the reconstituted vesicles and by the release of protons in the external volume. H+/O ratios varying from 0.6 to 1.2 were measured in different preparations, by the oxygen pulse technique. Antibodies which bind specifically to subunit I (cytochrome b558) of the 2-subunit oxidase were used to estimate the topology of the reconstituted oxidase in the vesicles. It was concluded that 70-85% of the molecules were oriented with subunit I facing the outside and that this population of molecules is responsible for the observed proton release. Correction for the fraction of the oxidase which pumps protons into the vesicle interior yields an estimate of H+/O = 1.7 +/- 0.2. It is proposed that the enzyme does not function as an actual proton pump, but that the enzyme oxidizes ubiquinol and reduces oxygen (to water) on opposite faces of the membrane. Hence, scalar chemistry would yield H+/O = 2 and an electrogenic reaction by virtue of the transmembrane electron transfer between the proposed active sites.
Oxygenated and peroxy states of the cytochrome d complex of Escherichia coli have been proposed as intermediates in the reaction mechanism of this ubiquinol oxidase. In this report, several stable states of the purified enzyme were examined spectroscopically at room temperature. As purified, the cytochromecytochrome d complex exists in an oxygenated state characterized by an absorbance band at 650 nm. Removal of oxygen results in loss of absorbance at this wavelength, which is restored upon the return of oxygen. The presence of one oxygen molecule in the oxygenated state was quantified by measuring oxygen released when excess hydrogen peroxide was added to the oxygenated state by passage of argon generates a “partially reduced” state with an absorbance peak at 628 nm, apparently due to reduced cytochrome d. Addition of equimolar hydrogen peroxide to the fully oxidized state produces the peroxy state. This peroxy state is also formed upon addition of excess hydrogen peroxide to the oxygenated state via a stable intermediate termed “peroxy intermediate.”
It is likely that 1) the oxygenated state consists of one molecule of oxygen bound to reduced heme d, and 2) there are at least two stable states that have bound peroxide at room temperature, the peroxy state and a newly discovered peroxy intermediate.
The cytochrome d terminal oxidase complex is one of two terminal oxidases which are components of the aerobic respiratory chain of Escherichia coli. This membrane-bound enzyme catalyzes the two-electron oxidation of ubiquinol and the four-electron reduction of oxygen to water. Enzyme turnover generates proton and voltage gradients across the bilayer. The oxidase is a heterodimer containing 2 mol of protoheme IX and 1 or 2 mol of heme d per mol of complex. To explain the functional properties of the enzyme, a simple model has been proposed in which it is speculated that the heme prosthetic groups define two separate active sites on opposite sides of the membrane at which the oxidation of quinol and the reduction of water, respectively, are catalyzed. This paper represents an initial effort to define the axial ligands of each of the three or four hemes within the amino acid sequence of the oxidase subunits. Each of the 10 histidine residues has been altered by site-directed mutagenesis with the expectation that histidine residues are likely candidates for heme ligands. Eight of the 10 histidine residues are not essential for enzyme activity, and 2 appear to function as heme axial ligands. Histidine 186 in subunit I is required for the cytochrome b558 component of the enzyme. This residue is likely to be located near the periplasmic surface of the membrane. Histidine 19, near the amino terminus of subunit I also appears to be a heme ligand.
It is concluded that two of the four or five expected heme axial ligands have been tentatively identified, although further work is required to confirm these conclusions. A minimum of two additional axial ligands must be residues other than histidine.
The ESR signals of all the major components of the aerobic respiratory chain of Escherichia coli were measured and assigned at liquid helium temperature. Cytochrome b-556 gives a weak high-spin signal at g = 6.0. The terminal oxidase cytochrome b-562 . o complex gives signals at g = 6.0, 3.0 and 2.26, and the terminal oxidase cytochrome b-558 . d complex gives signals at g = 6.0, 2.5 and 2.3. A signal derived from cupric ions in the purified cytochrome b-562 . o complex was observed near g = 2.0. It was shown by the effects of KCN or NaN3 on cytochromes under the air-oxidized conditions that cytochrome o has a high-spin heme and cytochrome d has a low-spin heme. The E'm values for cytochromes b-558 and d, respectively, determined by potentiometric titration of the ESR signals were 140 and 240 mV in the membrane preparation, and 30 and 240 mV in the purified preparation. The oxidized cytochrome d gave intense low-spin signals at g = 2.5 and 2.3, while cytochrome d under the air-oxidized conditions gave corresponding signals of only very low intensity. These results suggested that most of the cytochrome d under the air-oxidized conditions contains a diamagnetic iron atom with a bound dioxygen.
The ESR signals of the cytochromes in the Escherichia coli terminal oxidase cytochrome d complex were studied at cryogenic temperature. The intensities and g values of the rhombic high-spin signals changed when the electronic state of cytochrome d was changed from the oxidized state to the reduced or oxygen-binding or CO-binding state. These rhombic signals were therefore assigned to cytochrome b-595, which is located near cytochrome d in the oxidase complex. This assignment was supported by the finding that the Em value of the rhombic signals differed from that of cytochrome d (Hata, A. et al. (1985) Biochim. Biophys. Acta 810, 62-72). Photolysis and ligand-exchange experiments with the reduced CO complex of the oxidase were performed in the presence of oxygen at -140 degrees C. The ESR spectra of three intermediate forms trapped by controlled low temperatures were detected. These forms were designated as the oxygen-binding intermediate I (ESR-silent), oxygen-binding intermediate II (giving ESR signals at g = 6.3, 5.5 and 2.15), and oxygen-binding intermediate III (giving signals at g = 6.3, 5.5 and 6.0). From these results, electron flow in the cytochrome d complex is proposed to proceed in the order, cytochrome b-558----cytochrome b-595----cytochrome d----O2. A model of the mechanism of four-electron chemistry for oxidation of ubiquinol-8 and formation of H2O by the cytochrome d complex is presented.
The cytochrome d complex is a component of the aerobic respiratory system of Escherichia coli. The enzyme functions as a terminal oxidase, oxidizing ubiquinol-8 within the cytoplasmic membrane and reducing oxygen to water. The enzyme is of particular interest because it is a coupling site in the electron transfer chain. The electron transfer reaction catalyzed by this enzyme is coupled to the translocations of protons across the membrane (H+/e-approximately equal to 1). The oxidase contains two subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, with molecular weights of 58,000 and 43,000. In this paper, the question of the quaternary structure is addressed. Quantitative N-terminal analysis of the isolated enzyme and relative mass quantitation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicate the subunits are present in equimolar amounts. Sedimentation velocity and sedimentation equilibrium studies were used to characterize the hydrodynamic properties of the purified enzyme solubilized in Triton X-100, under conditions where the enzyme is active. It is concluded that the active enzyme in Triton X-100 is a heterodimer, containing one copy of each subunit. This is likely the structure of the enzyme in the E. coli membrane.
The cytochrome d terminal oxidase complex was recently purified from Escherichia coli membranes (Miller, M. J., and Gennis , R. B. (1983) J. Biol. Chem. 258, 9159-1965). The complex contains two polypeptides, subunits I and II, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and three spectroscopically defined cytochromes, b558 , a1, and d. A mutant that failed to oxidize N,N,N',N'-tetramethyl-p-phenylenediamine was obtained which was lacking this terminal oxidase complex and was shown to map at a locus called cyd on the E. coli genome. In this paper, localized mutagenesis was used to generate a series of mutants in the cytochrome d terminal oxidase. These mutants were isolated by a newly developed selection procedure based on their sensitivity to azide. Two classes of mutants which map to the cyd locus were obtained, cydA and cydB . The cydA phenotype included the lack of all three spectroscopically detectable cytochromes as well as the absence of both polypeptides, determined by immunological criteria. Strains manifesting the cydB phenotype lacked cytochromes a1 and d, but had a normal amount of cytochrome b558 . Immunological analysis showed that subunit I (57,000 daltons) was present in the membranes, but that subunit II (43,000 daltons) was missing. These data justify the conclusion that subunit I of this two-subunit complex can be identified as the cytochrome b558 component of the cytochrome d terminal oxidase complex.
Partial purification of a cytochrome bd complex from Azotobacter vinelandii grown under high aeration was achieved by isolating respiratory particles enriched in this hemoprotein via differential centrifugation and detergent extraction. The cytochrome bd complex was subsequently solubilized from the inner membrane with dodecyl maltoside and purified to near homogeneity via DEAE-Sepharose chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the complex consisted of two subunits, with sizes in good agreement with those predicted from the cloned cyd locus (59.7 and 42 kDa). Spectral analysis of the purified complex indicated that the heme components present were cytochromes b560, b595, and d; CO difference spectral studies identified cytochrome d as a CO-reactive component. The complex had a Km for ubiquinol-1 approximately seven times larger than that for the analogous bd complex from Escherichia coli, and O2 consumption curves revealed a Km value for O2 three times greater than that which we determined for the E. coli bd complex.
A strategy has been developed to express and purify a recombinant, nonfunctional axial-ligand mutant of iso-1-cytochrome c (Met-80-->Ala) in Saccharomyces cerevisiae in quantities necessary for extensive biophysical characterization. It involves coexpressing in the same plasmid (YEp213) the nonfunctional gene with a functional gene copy for complementation in a selective medium. The functional gene encodes a product with an engineered metal-chelating dihistidine site (His-39 and Leu-58-->His) that enables efficient separation of the two isoforms by immobilized metal-affinity chromatography. The purified Met-80-->Ala protein possesses a binding site for dioxygen and other exogenous ligands. Absorption spectra of several derivatives of this mutant show striking similarities to those of corresponding derivatives of horseradish peroxidase, myoglobin, and cytochrome P450. The use of a dual-gene vector for cytochrome c expression together with metal-affinity separation opens the way for the engineering of variants with dramatically altered structural and catalytic properties.
The aerobic respiratory chain of Escherichia coli can function with either of two different membrane-bound NADH dehydrogenases (NDH-1 and NDH-2) and with either of two ubiquinol oxidases (bd-type and bo-type). The amounts of each of these enzymes present in the E. coli membrane depend on growth conditions in general and particularly on the dissolved oxygen concentration. Previous in vitro studies have established that NDH-1 and NDH-2 differ in the extent to which they are coupled to the generation of an energy-conserving proton motive force. The same is true for the two ubiquinol oxidases. Hence, the bioenergetic efficiency of the aerobic respiratory chain must depend on the electron flux through each of the specific enzyme components which are being utilized. In this work, the specific rates of oxygen consumption for cells growing under glucose-limited conditions are reported for a series of isogenic strains in which one or more respiratory components are genetically eliminated. The results are compatible with the proton translocation values of the various components reported from in vitro measurements. The data show that (i) the bd-type oxidase is less efficient than is the bo-type oxidase, but the former is still a coupling site in the respiratory chain; and (ii) under the conditions employed, the wild-type strain uses both the NDH-1 and NDH-2 NADH dehydrogenases to a significant degree, but most of the electron flux is directed through the bo-type oxidase.
The cytochrome bd complex is a ubiquinol oxidase, which is part of the aerobic respiratory chain of Escherichia coli. This enzyme is structurally unrelated to the heme-Cu oxidases such as cytochrome c oxidase. While the cytochrome bd complex contains no copper, it does have three heme prosthetic groups: heme b558, heme b595, and heme d (a chlorin). Heme b558 appears to be involved in the oxidation of quinol, and heme d is known to be the site where oxygen binds and is reduced to water. The role of heme b595, which is high spin, is not known. In this paper, CO is used to probe the oxygen-binding site by use of Fourier transform infrared spectroscopy to monitor the stretching frequency of CO bound to the enzyme. Photodissociation at low temperature (e.g., 20 K) of the CO-heme d adduct results in CO associated with the protein within the heme binding pocket. This photodissociated CO can subsequently relax to form a kinetically trapped CO-heme b595 adduct. The data clearly show that heme d and heme b595 must reside within a common binding pocket in the enzyme. The catalytic active site where oxygen is reduced to water is, thus, properly considered to be a heme d-heme b595 binuclear center. This is analogous to the heme alpha 3-Cu(B) binuclear center in the heme-Cu oxidases. Heme b595 may play roles analogous to those proposed for the Cu(B) component of cytochrome c oxidase.
The proximal bond between the iron atom of the heme group and the N epsilon of histidine F8 in myoglobin (Mb) and hemoglobin (Hb) is presumed to be an important determinant of heme binding, protein structure, and oxygen binding. Here a system is described in which the proximal ligand is provided intermolecularly by the histidine side chain mimic imidazole. The proximal ligand of sperm whale Mb is replaced with glycine (H93G) using site-directed mutagenesis. The addition of imidazole to Escherichia coli expressing this gene reconstitutes myoglobin function. H93G Mb purified in the presence of imidazole is spectroscopically similar to wild-type Mb in combination with a wide variety of distal ligands. The crystal structure of H93G Mb, determined in the presence of imidazole, reveals that an imidazole molecule is bonded to the heme iron on the proximal side, substituting in trans for the side-chain function of the proximal histidine of wild-type Mb. Although H93G Mb is similar in spectroscopic and gross structural detail to wild-type Mb, subtle differences exist in the orientation of imidazole with respect to the heme group. trans-Complementation of proximal ligand function will allow the proximal bond in hemoproteins to be chemically substituted beyond the limits of the genetic code.
Heme d has been isolated from the terminal oxidase complex of Escherichia coli strain and purified by high-pressure liquid chromatography. The infrared spectrum indicated that carbonyls in the chlorin skeleton of this isolated heme existed as carboxylic acids. Earlier work on the iron-free chlorin had demonstrated the presence of a spirolactone substituent. This may have arisen from a cyclization reaction from a dicarboxylic acid, diol precursor. Although the free heme in extracts can exist as a diol, this does not prove that the diol as such is the precise form in the enzyme complex. Visible and fluorescence spectra are reported for a variety of derivatives and complexes of heme d to establish a spectral library that may be used to prove the presence of this structure in other enzymes or cells. Association constants have been measured for complexes of heme d with cyanide, imidazole, and pyridine and are contrasted to available data for protoheme.
The heme d prosthetic group of the Escherichia coli terminal oxidase has been proposed to be a hydroxychlorin γ-spirolactone. This "lactochlorin" structure has now been verified by total synthesis. Vicinal dihydroxylation of pyrrole ring C of protoporphyrin was accomplished by OsO4 oxidation of 2,4-bis(2-chloroethyl)deuteroporphyrin followed by dehydrochlorination. Hydroxychlorins substituted with a geminal propionic ester group have a propensity to lactonize under general-base catalysis. Mild bases such as NaOAc cyclize the geminal groups without inversion of configuration while prolonged contact with silica gel invariably gives the trans diastereomer. 1H NMR spectra have provided important information on the conformation of the cis and trans spirolactones. This work strongly supports the argument that the lactone structure of the isolated chromophore is an artifact. The true ligand structure of heme d in the enzyme is most likely 5,6-dihydroxyprotochlorin IX, the cis and trans isomers of which have also been synthesized.
The aerobic respiratory chain of Escherichia coli couples the oxidation of organic substrates, such as succinate, to the generation of a proton motive force across the cytoplasmic membrane. The mechanism by which this is accomplished is quite different than found in the mitochondrial respiratory system, as predicted by D. Keilin1 nearly 50 years ago. Aerobically grown E. coli has no cytochrome c, and no equivalent to the mitochondrial Complex III (bc1 complex) or Complex IV (cytochrome c oxidase). Instead, two enzymes in the E. coli cytoplasmic membrane, the cytochrome o and cytochrome d complexes, oxidize ubiquinol and directly reduce molecular oxygen to water, concomitantly generating an electrochemical proton gradient across the membrane.
Two procedures for analyzing overlapping optical spectra of mixtures of pyridine hemochromes are described, and extinction coefficients of pyridine hemochromes are provided for use with these methods. In the first procedure, absorbance is measured at a number of wavelengths equal to the number of components to be analyzed. This is the minimum amount of spectral data from which the concentration of each species can be calculated. In the second procedure, absorbance is measured at a number of wavelengths greater than the number of components to be analyzed. This redundancy of information makes it impossible to fit spectra which contain contributions from additional components, unless the spectra of the additional components are equal to linear combinations of the spectra of the species being analyzed. These two procedures are generally applicable to analyses of absolute or difference spectra of mixtures of components obeying Beer's law. The sensitivity to error in the absorbance measurements is only slightly greater than that for measuring a pure component at a single wavelength.
A number of gene replacements at different loci were constructed using covalently closed circular (ccc) plasmid DNA in he recB21 recC22 sbcB15 asbcC201 mutant of Escherichia coli (JC7623). Selected constructs representing deletions and insertion mutations formed from double-crossover events involving the ccc plamid molecules and the genome were confirmed by Southern blots, and the frequency of double-crossover events was evaluated. It is reported that such mutants may be constructed without linearizing plasmid DNA, as described previously.
Restriction endonucleases Dpn I and Dpn II are produced by two distinct strains of Diplococcus pneumoniae. The two enzymes show complementary specificity with respect to methylation of sites in DNA. From the identity of its cleavage site with that of Mbo I, it appears that Dpn II cleaves at the unmodified sequence 5′-G-A-T-C-3′. Dpn I cleaves at the same sequence when the adenine residue is methylated. Both enzymes produce only double-strand breaks in susceptible DNA.Their susceptibility to Dpn 1 and not Dpn II shows that essentially all the G-A-T-C sequences are methylated in DNA from the pneumococcal strain that produces Dpn II as well as in DNA from Hemophilus influenzae and Escherichia coli. In the dam-3 mutant of E. coli none of these sequences appear to be methylated. Residual adenine methylation in the dam-3 mutant DNA most likely occurs at different sites. Different but characteristic degrees of methylation at G-A-T-C sites are found in the DNA of bacterial viruses grown in E. coli. DNAs from mammalian cells and viruses are not methylated at this sequence. Mitochondrial DNA from Paramecium aurelia is not methylated, but a small proportion of G-A-T-C sequences in the macronuclear DNA of this eukaryote appear to be methylated. Possible roles of sequence-specific methylation in the accommodation of plasmids, in the replication of DNA, in the regulation of gene function and in the restriction of viral infection are discussed.
The cytochrome d complex is a two-subunit, membrane-bound terminal oxidase in the aerobic respiratory chain of Escherichia coli. The enzyme catalyzes the two-electron oxidation of ubiquinol and the four-electron reduction of oxygen to water. Previous work demonstrated that the site for ubiquinol oxidation was selectively inactivated by limited proteolysis by trypsin, which cleaves at a locus within subunit I. This work is extended to show that a similar phenomenon is observed with limited chymotrypsin proteolysis of the complex. The cleavage patterns are similar whether one uses the purified oxidase in nondenaturing detergent or reconstituted in proteoliposomes or uses spheroplasts of E. coli as the substrate for the proteolysis. Hence, the protease-sensitive locus is periplasmic in the cell. Fragments resulting from proteolysis were characterized by N-terminal sequencing and by immunoblotting with the use of a monoclonal antibody of known epitope within subunit I. The data indicate that inactivation of the ubiquinol oxidase activity results from cleavage at specific residues with a hydrophilic region previously defined as the Q loop. This domain has been already implicated in ubiquinol oxidation by the use of inhibitory monoclonal antibodies. Electrochemical and HPLC analysis of the protease-cleaved oxidase suggests no global changes in either the quaternary or tertiary structure of the enzyme. It is likely that the Q loop is directly involved in forming a portion of the ubiquinol binding site near the periplasmic surface of the membrane.
The cytochrome d complex of Escherichia coli is a heterodimer located in the bacterial cytoplasmic membrane, where it functions as a terminal oxidase of the aerobic respiratory chain. The topology of each of the two subunits of the cytochrome d complex was analysed by the genetic method involving alkaline phosphatase gene fusions. These fusions were generated by both an in vivo method using the transposon TnphoA and an in vitro method of construction. A total of 48 unique fusions were isolated and the whole-cell alkaline phosphatase-specific activities were determined. Data from these fusions, in combination with information from other studies, provide the basis for two-dimensional models for each of the two subunits, defining the way in which the subunits fold in the inner membrane of E. coli.
Proton translocation coupled to oxidation of ubiquinol by O2 was studied in spheroplasts of two mutant strains of Escherichia coli, one of which expresses cytochrome d, but not cytochrome bo, and the other expressing only the latter. O2 pulse experiments revealed that cytochrome d catalyzes separation of the protons and electrons of ubiquinol oxidation but is not a proton pump. In contrast, cytochrome bo functions as a proton pump in addition to separating the charges of quinol oxidation. E. coli membranes and isolated cytochrome bo lack the CuA center typical of cytochrome c oxidase, and the isolated enzyme contains only 1Cu/2Fe. Optical spectra indicate that high-spin heme o contributes less than 10% to the reduced minus oxidized 560-nm band of the enzyme. Pyridine hemochrome spectra suggest that the hemes of cytochrome bo are not protohemes. Proteoliposomes with cytochrome bo exhibited good respiratory control, but H+/e- during quinol oxidation was only 0.3-0.7. This was attributed to an "inside out" orientation of a significant fraction of the enzyme. Possible metabolic benefits of expressing both cytochromes bo and d in E. coli are discussed.
The cytochrome d terminal oxidase complex is a heterodimer located in the cytoplasmic membrane of Escherichia coli. Subunit II of the cytochrome d terminal oxidase complex was expressed independently of subunit I of the complex. It was found that the polypeptide is produced and is associated with the cytoplasmic membrane in the absence of subunit I, and is not associated with any of the three cytochrome components of the complex. Oxidase activity and heme binding are restored when the subunit I is expressed in the same cells using a second compatible plasmid. It has been previously demonstrated that subunit I, expressed in the absence of subunit II, contains cytochrome b-558, one of the three heme prosthetic groups found in the oxidase. Association of the two other heme moieties, cytochromes b-595 and d, apparently requires the association of the two subunits, and must be a late step in the assembly of the membrane-bound protein. It was also shown that under heme-deficient conditions, the two polypeptide subunits are expressed and are associated with the cytoplasmic membrane.
We have examined the thermodynamic and EPR properties of one of the ubiquinol oxidase systems (the cytochrome d complex) of Escherichia coli, and have assigned the EPR-detectable signals to the optically identified cytochromes. The axial high spin g = 6.0 signal has been assigned to cytochrome d based on the physicochemical properties of this signal and those of the optically defined cytochrome d. A rhombic low spin species at gx,y,z = 1.85, 2.3, 2.5 exhibited similar properties but was present at only one-fifth the concentration of the axial high spin species. Both species have an Em7 of 260 mV and follow a -60 mV/pH unit dependence from pH 6 to 10. The rhombic high spin signal with gy,z = 5.5 and 6.3 has been assigned to cytochrome b-595. This component has an Em7 of 136 mV and follows a -30 mV/pH unit dependence from pH 6 to 10. Lastly, the low spin gz = 3.3 signal which titrates with an Em7 of 195 mV and follows a -40 mV/pH unit dependence from pH 6 to 10 has been assigned to cytochrome b-558. Spin quantitation of the high-spin signals indicates that cytochrome d and b-595 are present in approximately equal amounts. These observations are discussed in terms of the stoichiometry of the prosthetic groups and its implications on the mechanism of electron transport.
We describe an in vitro selection procedure for oligodeoxynucleotide-directed mutagenesis, which produces mutants at frequencies of greater than 90%, facilitating the identification of mutants directly by nucleotide sequencing. The method is based on the selective methylation of the mutant strand by the incorporation of 5-methyl-dCTP. Restriction endonuclease digestion of the resulting hemimethylated DNA with MspI results in the nicking of only the nonmethylated-parental strand. The parental strand is removed by treatment with exonuclease III. The mutants are recovered by transformation of a mcrAB strain of Escherichia coli with the nascent strand.
Coulometric and spectroscopic analyses were performed on the three cytochrome components (cytochrome d, cytochrome b558, and the cytochrome previously described as cytochrome a1) of the purified cytochrome d complex, a terminal oxidase of the Escherichia coli aerobic respiratory chain. On the basis of heme extraction, spectroscopic, and coulometric data, the "cytochrome a1" component was identified as a b-type cytochrome: cytochrome b595. The pyridine hemochromogen technique revealed the presence of two molecules of protoheme IX per cytochrome d complex. This quantity of protoheme IX fully accounted for the sum of the cytochrome b558 and cytochrome b595 components as determined coulometrically. The renaming of cytochrome a1 as cytochrome b595 was further indicated by the lack of any heme a in the complex and by its resolved reduced-minus-oxidized spectrum. The latter was found to be similar to that of cytochrome c peroxidase, which contains protoheme IX. Coulometric titrations and carbon monoxide binding titrations revealed that there are two molecules of cytochrome d per complex. A convenient measurement of the amount of cytochrome b558 was found to be the beta-band at 531 nm since cytochrome b558 was observed to be the only component of the cytochrome d complex with a peak at this wavelength. By use of this method and the extinction coefficient for the purified cytochrome b558, it was estimated that there is one molecule of cytochrome b595 and one of cytochrome b558 per cytochrome complex.
This article describes the present status of information on bacterial aerobic respiratory chains, with special reference to recent biochemical and molecular biological studies of aerobic cytochromes. Citations of works on mitochondrial electron transfer systems are made only when required for comprehensive discussion. Anaerobic electron transfer systems in bacteria are scarcely discussed because of limitation of space. For this subject readers should refer to comprehensive reviews.
A recombination proficient strain ofEscherichia coli which is recB− recC− sbcB− has been subjected to mutagenesis by nitrosoguanidine. Among the recombination deficient mutants isolated one was sbcB+, three were recA∼ and 11 were mutants in at least four newrec genes: recF, recJ, recK and recL. recF143 and recL152 are cotransducible with ilv but they lie on opposite sides of the ilv operons as determined by Fstudies. recF, recL and recK are not involved in the RecBC pathway of recombination since a recB+recC+sbcB− strain carrying a mutation in one of these genes is recombination proficient. Hence the hypothesis that a RecF pathway of recombination can operate as a partially independent substitute for the RecBC pathway of recombination is supported. recF−recB+ and recF+recB− single mutants are sensitive to u.v. irradiation while the recF−recB− double mutant is more sensitive than either single mutant. The sensitivity of the recB−recC−sbcB−recF− strain approaches the sensitivity of a recA− single mutant. This is interpreted to mean that there are partially independent RecF and RecBC pathways for the repair of u.v. damage. recJ and mutations were not mapped precisely; hence the mutant properties they confer can not be stated conclusively.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
Two plasmids containing the two structural genes for the inner-membrane-bound cytochrome d complex (Cyd) have been isolated from the Clarke and Carbon Escherichia coli DNA bank. A 5.4-kb DNA fragment from one plasmid was subcloned in both orientations into pBR322. The promoter(s) and both genes must have been present within this fragment since the two orientations yielded similar levels of Cyd. Recombination and transduction studies indicated that the cyd gene locus had been isolated. These results demonstrate that cyd contains all the structural information for the complex. Overproduction of Cyd has yielded a visual screening procedure for plasmids bearing cyd that is unique to colored proteins like cytochromes. Colonies of E. coli bearing the cloned cyd gene are yellow-green. The cyd gene can, therefore, be used as a vehicle for detection of inserted DNA fragments.
Escherichia coli strains K12 and B, and a new strain designated D, each encode a characteristic restriction and modification enzyme. These enzymes (EcoK, EcoB and presumably EcoD) comprise three subunits of which one, that encoded by the so-called specificity gene (hsdS), is responsible for recognition of the DNA sequence specific to that system. The other two subunits, encoded by hsdR and hsdM, are interchangeable between systems, and the available molecular evidence suggests that the hsdR and hsdM genes are highly conserved. The DNA sequence of a segment of the hsd region that includes the hsdS gene has been determined for each of the three strains. The hsdS gene varies in length from 1335 to 1425 base-pairs and the only regions showing obvious homology, one of about 100 base-pairs and a second of about 250 base-pairs, are highly conserved. The remainder of each hsd S gene shares little, or no, homology with either of the other related specificity genes. Thus, the specificity subunits, though components of a family of closely related enzymes with very similar functions, have remarkably dissimilar primary structure.
The cytochrome d terminal oxidase complex is one of the two terminal oxidases in the aerobic respiratory chain of Escherichia coli. In this paper, effects of pH and detergent on the electrochemical and kinetic properties of the enzyme are investigated. There are two significant conclusions. (1) The oxidation-reduction midpoint potential of the cytochrome b-558 component is sensitive to the detergent used to solubilize the complex. In particular, it is shown that octylglucoside and cholate cause a large decrease in the midpoint potential of cytochrome b-558, while they also result in the reversible inactivation of the oxidase. (2) The midpoint potentials of the cytochrome b-558, a1 and d components are sensitive to pH. More acidic solutions result in stabilizing the reduced forms of the redox-active groups, i.e., raising their midpoint potentials. This may be significant in view of the fact that it has been demonstrated that this enzyme catalyzes an electrogenic reaction and appears to function as a proton pump.
The role of the proximal ligand in determining the structure and ligand binding properties of sperm whale myoglobin has been investigated using the mutant H93G(L), where the proximal histidine has been replaced with glycine, creating a cavity which can be occupied by a variety of exogenous ligands, L, to the iron [Barrick, D. (1994) Biochemistry 33, 6546-6554; DePillis, G.D., Decatur, S.M., Barrick, D., & Boxer, S.G. (1994) J. Am. Chem. Soc. 116, 6981-6982]. In this report, we present the assignments of selected protons of the heme and heme pocket residues in the metcyano complexes of H93G with Im and a series of methyl-substituted Ims [H93G(Im)CN, H93G(N-MeIm)CN, H93G(2-MeIm)CN, H93G-(4-MeIm)CN]. Each complex has a unique 1H NMR spectrum, providing a fingerprint for documenting the ligand exchange phenomenon. Moreover, the identification of NOEs between the protons of proximal ligands and protons of proximal pocket amino acid residues confirms that the new ligand occupies the proximal cavity in solution. The pattern of hyperfine-shifted heme methyl resonances in H93G(Im)CN is very different from that of wild-type Mb, consistent with the differences compared to wild-type in is very different from that of wild-type Mb, consistent with the differences compared to wild-type in orientation of the proximal imidazole observed in the X-ray crystal structure of H93G(Im) [Barrick, D. (1994) Biochemistry 33, 6546-6554]. Addition of deuterated Im to H93G(Im)CN permits direct observation of exchange of proximal ligands with ligands from solution; exchange of Im for deuterated Im in the metcyano complex occurs with half-life of around 10 min.(ABSTRACT TRUNCATED AT 250 WORDS)
When grown under O2-limited conditions, Escherichia coli expresses a cytochrome bd quinol oxidase that has an unusually high affinity for O2. We have studied the reaction of cytochrome bd with CO and O2 by rapid-reaction spectrophotometry. The reduced enzyme forms a photosensitive ferrocytochrome d-CO complex, and following photolysis, CO recombines with the reduced enzyme with a bimolecular rate of 8 x 10(7) M-1 s-1. Reaction of CO-bound enzyme with O2 gives a CO off-rate of 1.6 s-1. The O2 reaction is followed by a flow-flash procedure in which CO-ligated enzyme is mixed with O2, and the reaction commenced by photolysis of cytochrome d-CO. In the presence of O2, two processes are resolved on a time-scale of 300 microseconds. The absorbance at 645 nm first increases at a rate that is dependent on O2 concentration with a value of 2 x 10(9) M-1 s-1. The second phase results in decreased absorbance at 645 nm and increased absorbance at 680 nm. The rate of the second process is independent from O2 concentration above 50 microM O2 and reaches a first-order limit of 1 x 10(4) s-1. A model for the reaction of the cytochrome bd quinol oxidase with O2 is proposed in which an initial ferrocytochrome d-oxy adduct forms, and then decays to a ferryl-oxo species. The oxidation of the low-spin cytochrome b component of the oxidase, monitored at 560 nm, occurs at the same time as the ferryl species forms. We suggest that the suitability of the cytochrome bd quinol oxidase to function at low O2 concentration is conferred by its rapid rate of binding O2.
Antimycin A and UHBDT inhibit the activity of the purified cytochrome bd complex from Azotobacter vinelandii. Inhibition of activity is non-competitive and antimycin A binding induces a shift to the red in the spectrum of a b-type haem. No inhibitory effects were seen with myxothiazol. Steady-state experiments indicate that the site of inhibition for antimycin A lies on the low-potential side of haem b558. In the presence of antimycin A at concentrations sufficient to inhibit respiration, some direct electron transfer from ubiquinol-1 to haem b595 and haem d still occurs. The results are consistent with a branched electron transfer pathway from ubiquinol to the oxygen reduction site.
The bo-type ubiquinol oxidase of Escherichia coli is a member of the superfamily of heme-copper oxidases which also includes the aa3-type cytochrome c oxidases. The oxygen-binding binuclear center of cytochrome bo is located in subunit I and consists of a heme (heme o; heme a3 in the aa3-type oxidases) and a copper (Cu(B)). Previous spectroscopic studies have shown that heme o is bound to the protein via a single histidine residue. Site-directed mutagenesis of conserved histidine residues in subunit I has identified two residues (H284 and H419) which are candidates for the ligand of heme o, while spectroscopic studies of mutants at H284 definitively demonstrated that this residue cannot be the axial ligand. Consequently, the single remaining conserved histidine in subunit I (H419) was assigned as the ligand for the heme of the binuclear center. In this paper, this assignment is tested by characterization of additional mutants in which the putative heme o axial ligand, H419, is replaced by other amino acids. All mutations at H419 result in the loss of enzyme activity. Analyses via UV-visible and Fourier transform infrared spectroscopies reveal that substantial perturbation has occurred at the binuclear center as a result of the amino acid substitutions. In contrast with the wild-type enzyme, the mutant enzymes bind very little carbon monoxide. Three other amino acid residues which are potential ligands for heme o are shown tob e nonessential for enzyme activity. Mutations in these residues do not perturb the UV-visible or FTIR spectroscopic characteristics of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
- F Spinner
- M R Cheesman
- A J Thomson
- T Kaysser
- R B Gennis
- Q Peng
- J Peterson
Spinner, F., Cheesman, M. R., Thomson, A. J., Kaysser, T., Gennis,
R. B., Peng, Q., & Peterson, J. (1995) Biochem. J. 308, 641-644.
- R Timkovich
- M S Cork
- R B Gennis
- P Y Johnson
Timkovich, R., Cork, M. S., Gennis, R. B., & Johnson, P. Y. (1985)
J. Am. Chem. SOC. 107, 6069-6075.