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pH-Dependent Structures of the Manganese Binding Sites in Oxalate Decarboxylase as Revealed by High-Field Electron Paramagnetic Resonance
Abstract
A high-field electron paramagnetic resonance (HFEPR) study of oxalate decarboxylase (OxdC) is reported. OxdC breaks down oxalate to carbon dioxide and formate and possesses two distinct manganese(II) binding sites, referred to as site-1 and -2. The Mn(II) zero-field interaction was used to probe the electronic state of the metal ion and to examine chemical/mechanistic roles of each of the Mn(II) centers. High magnetic-fields were exploited not only to resolve the two sites, but also to measure accurately the Mn(II) zero-field parameters of each of the sites. The spectra exhibited surprisingly complex behavior as a function of pH. Six different species were identified based on their zero-field interactions, two corresponding to site-1 and four states to site-2. The assignments were verified using a mutant that only affected site-1. The speciation data determined from the HFEPR spectra for site -2 was consistent with a simple triprotic equilibrium model, while the pH dependence of site-1 could be described by a single pK(a). This pH dependence was independent of the presence of the His-tag and of whether the preparations contained 1.2 or 1.6 Mn per subunit. Possible structures of the six species are proposed based on spectroscopic data from model complexes and existing protein crystallographic structures obtained at pH 8 are discussed. Although site-1 has been identified as the active site and no role has been assigned to site-2, the pronounced changes in the electronic structure of the latter and its pH behavior, which also matches the pH-dependent activity of this enzyme, suggests that even if the conversion of oxalate to formate is carried out at site-1, site-2 likely plays a catalytically relevant role.
... In part, this reflects the fact that only one of the two bound manganese ions (located in the N-terminal domain) likely mediates catalysis, 20-23 which has greatly complicated any spectroscopic characterization of the enzyme using EPR methods ( Figure S1 of the Supporting Information). [24][25][26][27] Although prior EPR studies did find weak evidence of the presence of Mn(III), 27 any clear mechanistic interpretation of that data is complicated by the presence of Mn(III) in the C-terminal site of wild-type (wt) OxDC at high pH. 20,24 We now report parallel mode EPR measurements that unambiguously demonstrate Mn(III) formation in wt OxDC under V max conditions (100 mM oxalate at pH 4.2), which have been made possible by the development of new methods for obtaining high concentrations (0.5 mM) of the pure enzyme. ...
... [24][25][26][27] Although prior EPR studies did find weak evidence of the presence of Mn(III), 27 any clear mechanistic interpretation of that data is complicated by the presence of Mn(III) in the C-terminal site of wild-type (wt) OxDC at high pH. 20,24 We now report parallel mode EPR measurements that unambiguously demonstrate Mn(III) formation in wt OxDC under V max conditions (100 mM oxalate at pH 4.2), which have been made possible by the development of new methods for obtaining high concentrations (0.5 mM) of the pure enzyme. ...
... At pH 8.5, OxDC does not catalyze substrate decarboxylation because only the monoprotonated form of oxalate is the true substrate. 8 In agreement with previous EPR observations, 24 the sample exhibited a characteristic signal for the hyperfine-split transition of Mn(II) at g = 2.0 in perpendicular mode EPR together with a sextet feature near g eff = 4.3 that can be attributed to the half-field transition ( Figure 1A). The parallel mode EPR spectrum of wt OxDC at pH 8.5 exhibited a distinctive sextet signal, corresponding to the Δm s = ±2 transition of Mn(II), but no Mn(III) signal was present under these conditions ( Figure 1B), confirming the findings of other EPR studies. ...
Oxalate decarboxylase (OxDC) catalyzes the disproportionation of oxalic acid monoanion into CO2 and formate. The enzyme has long been hypothesized to utilize dioxygen to form mononuclear Mn(III) or Mn(IV) in the catalytic site during turnover. Recombinant OxDC, however, contains only tightly-bound Mn(II), and direct spectroscopic detection of the metal in higher oxidation states under optimal catalytic conditions (pH 4.2) has not yet been reported. Using parallel mode EPR spectroscopy, we now show that substantial amounts of Mn(III) are indeed formed in OxDC, but only in the presence of oxalate and dioxygen under acidic conditions. These observations provide the first direct support for proposals in which Mn(III) removes an electron from the substrate to yield a radical intermediate in which the barrier to C-C bond cleavage is significantly decreased. Thus, OxDC joins a small list of enzymes capable of stabilizing and controlling the reactivity of the powerful oxidizing species Mn(III).
... EPR has been used successfully to probe radical intermediates during turnover and observing different coordination environments of the two Mn ions [6,[10][11][12][13][14][15]. The resting state oxidation number of the Mn ions is predominantly þ 2 [7,15,16]. ...
... EPR has been used successfully to probe radical intermediates during turnover and observing different coordination environments of the two Mn ions [6,[10][11][12][13][14][15]. The resting state oxidation number of the Mn ions is predominantly þ 2 [7,15,16]. High-field multi-frequency EPR provided evidence for pH dependent conformational changes as seen by changes in the Mn coordination environment [15]. Tabares [11]. ...
... The resting state oxidation number of the Mn ions is predominantly þ 2 [7,15,16]. High-field multi-frequency EPR provided evidence for pH dependent conformational changes as seen by changes in the Mn coordination environment [15]. Tabares [11]. ...
Oxalate decarboxylase, a bicupin enzyme coordinating two essential manganese ions per subunit, catalyzes the decomposition of oxalate into carbon dioxide and formate in the presence of oxygen. Current efforts to elucidate its catalytic mechanism are focused on EPR studies of the Mn. We report on a new immobilization strategy linking the enzyme's N-terminal His6-tag to a Zn-loaded immobilized metal affinity resin. Activity is lowered somewhat due to the expected crowding effect. High-field EPR spectra of free and immobilized enzyme show that the resin affects the coordination environment of the active site Mn ions only minimally. The immobilized preparation was used to study the effect of varying pH on the same sample. Repeated freeze-thaw cycles lead to break down of the resin beads and some enzyme loss from the sample. However, the EPR signal increases due to higher packing efficiency on the sample column.
... There are, however, enzymes containing mononuclear Mn(II) that have been studied fruitfully by HFEPR. These include reduced MnSOD [97][98][99][100], and oxalate oxidase (OxOx)/oxalate decarboxylase (OxDC) [101][102][103], which exhibited |D| B 0.4 cm -1 , as helpfully summarized by Tabares et al. [101]. Changes in zfs as a function of biochemically important events, such as pH changes and substrate binding, could be monitored by HFEPR [101]. ...
... There are, however, enzymes containing mononuclear Mn(II) that have been studied fruitfully by HFEPR. These include reduced MnSOD [97][98][99][100], and oxalate oxidase (OxOx)/oxalate decarboxylase (OxDC) [101][102][103], which exhibited |D| B 0.4 cm -1 , as helpfully summarized by Tabares et al. [101]. Changes in zfs as a function of biochemically important events, such as pH changes and substrate binding, could be monitored by HFEPR [101]. ...
... These include reduced MnSOD [97][98][99][100], and oxalate oxidase (OxOx)/oxalate decarboxylase (OxDC) [101][102][103], which exhibited |D| B 0.4 cm -1 , as helpfully summarized by Tabares et al. [101]. Changes in zfs as a function of biochemically important events, such as pH changes and substrate binding, could be monitored by HFEPR [101]. ...
This minireview describes high-frequency and high-field electron paramagnetic resonance (HFEPR) spectroscopy in the context of its application to bioinorganic chemistry, specifically to metalloproteins and model compounds. HFEPR is defined as frequencies above ~100 GHz (i.e., above W-band) and a resonant field reaching 25 T and above. The ability of HFEPR to provide high-resolution determination of g values of S = 1/2 is shown; however, the main aim of the minireview is to demonstrate how HFEPR can extract spin Hamiltonian parameters [zero-field splitting (zfs) and g values] for species with S > 1/2 with an accuracy and precision unrivalled by other physical methods. Background theory on the nature of zfs in S = 1, 3/2, 2, and 5/2 systems is presented, along with selected examples of HFEPR spectroscopy of each that are relevant to bioinorganic chemistry. The minireview also provides some suggestions of specific systems in bioinorganic chemistry where HFEPR could be rewardingly applied, in the hope of inspiring workers in this area.
... Efforts to observe the proposed interaction of dioxygen with the metal in OxDC have also been complicated by the fact that the two Mn(II) centers in the enzyme exhibit similar EPR properties. 7,8 In an effort to assess the existence of the putative dioxygen binding site in OxDC, we examined the interaction of nitric oxide (NO) with the enzyme using a new continuous assay based on membrane inlet mass spectrometry (MIMS). 9 Thus, wild type Bacillus subtilis OxDC bearing a C-terminal His 6 tag was expressed and purified using standard procedures. ...
... These observations seem to indicate that NO does not exert its inhibitory effect on decarboxylase activity by displacing dioxygen from the metal center or by forming a heptacoordinate Mn(II) species, at least for the site that is visible in the X-band EPR spectrum. High-frequency EPR analysis of OxDC by other workers has also suggested that the Mn(II) site exhibiting the smaller fine structure value Disactually located in the N-terminal domain of the enzyme, 8 this assignment being based on spectroscopic evidence for the presence of a pentacoordinate Mn(II) center in OxDC at high solution pH. 5 If this interpretation is correct then our findings would seem to exclude NO binding at the solvent accessible catalytically active N-terminal site. 14 These observations are also consistent with the remarkably small number of well characterized mononuclear {Mn-NO} 6 complexes that have been reported. ...
... 18 On the other hand, it remains possible that any Mn(II)/NO interaction is masked by the complexity of the X-band EPR spectrum for OxDC. 7,8 If NO is exerting its inhibitory effect without binding to either of the two Mn centers, other explanations need to be considered for the molecular interaction of NO with OxDC leading to inhibition. It seems unlikely that covalent modification of tyrosine residues (such as Tyr-200 in the putative catalytic site of OxDC) as observed for Mn-dependent superoxide dismutase, 19 is responsible for OxDC inhibition because such changes would be expected to be irreversible. ...
Membrane inlet mass spectrometry (MIMS) has been employed to assay the catalytic activity of oxalate decarboxylase (OxDC), allowing us to demonstrate that nitric oxide (NO) reversibly inhibits the enzyme under dioxygen-depleted conditions. X-band EPR measurements do not provide any direct evidence for the interaction of NO with either of the Mn(II) centers in OxDC raising the possibility that there is a separate dioxygen-binding pocket in the enzyme.
... Numerous biophysical, biochemical, and structural studies have provided insights into how metal ions promote catalysis, highlighting their involvement in bond polarization and stabilization of transition states and intermediates (Andreini et al., 2008). Moreover, in the case of certain redox enzymes, for example manganese superoxide dismutase (Abreu and Cabelli, 2010) and oxalate decarboxylase (Tabares et al., 2009), changes in the oxidation state of the metal ions have been associated with variations in ligand coordination geometry during the reaction (Andreini et al., 2008). However, there is still much to be learned about the exact roles that different metal ions play in driving enzyme catalysis, specifically how the exquisite specificity with which they are selected is matched to the chemistry in which they are involved. ...
... methane monooxygenase (Rosenzweig et al., 1993) and hemoglobin (Perutz, 1970). Although five-coordinate Mn(II) centers and changes in coordination states have also been observed in the structures of both manganese superoxide dismutase and oxalate decarboxylase (Tabares et al., 2005(Tabares et al., , 2009) their roles, unlike IGPD, are associated with changes in metal redox state during the reaction. To our knowledge, the work on IGPD presented here is the first description of how an enzyme can exploit the unique ability of transition metals to switch coordination states at specific steps in catalysis. ...
Imidazoleglycerol-phosphate dehydratase (IGPD) catalyzes the Mn(II)-dependent dehydration of imidazoleglycerol phosphate (IGP) to 3-(1H-imidazol-4-yl)-2-oxopropyl dihydrogen phosphate during biosynthesis of histidine. As part of a program of herbicide design, we have determined a series of high-resolution crystal structures of an inactive mutant of IGPD2 from Arabidopsis thaliana in complex with IGP. The structures represent snapshots of the enzyme trapped at different stages of the catalytic cycle and show how substrate binding triggers a switch in the coordination state of an active site Mn(II) between six- and five-coordinate species. This switch is critical to prime the active site for catalysis, by facilitating the formation of a high-energy imidazolate intermediate. This work not only provides evidence for the molecular processes that dominate catalysis in IGPD, but also describes how the manipulation of metal coordination can be linked to discrete steps in catalysis, demonstrating one way that metalloenzymes exploit the unique properties of metal ions to diversify their chemistry.
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... Moreover, oxalate decarboxylase activity may convert into oxalate oxidase activity by forming H 2 O 2 due to a mutation in the amino acids of the lid region . Earlier it was proposed that the activity of B. subtilis oxalate decarboxylase to convert oxalate into formate and CO 2 is conserved in its N-terminal domain (Just et al. 2004;Burrell et al. 2007;Svedruzic et al. 2007) but later evidence showed that both N-and C-terminal domains may catalyze the decarboxylation reaction (Tabares et al. 2009). The structural and spectroscopic studies revealed that site 1 acts as the catalytic site, in the presence of two manganese-binding sites in B. subtilis. ...
Nephrolithiasis is a terrible pathological condition marked by the presence and formation of kidney stones. It affects around 3–20% of the community in the world. Several environmental, physiological, and nutritional conditions influence this disease. Not only the food sources but also the body’s own metabolism add up oxalate content in the human body. The increased intake of oxalate leads to hyperoxaluria, which often results in the formation of calcium oxalate stones, commonly known as kidney stones. The incidences of kidney stone are very common, and the current therapeutic measure of its cure is not much effective. Therefore, new therapeutic approaches are needed. In the last few years, the use of gut microbiome with oxalate-degrading activity has emerged as an excellent therapeutic approach to treat kidney stones. As the genes responsible for oxalate-degrading enzymes are not found in humans use of bacterial enzymes with the ability to degrade oxalate in intestinal digestion has a significant therapeutic impact. This chapter summarizes the roles of microbial enzymes produced by gut microflora involved in the solubilization of the dietary oxalates, and their potential applications in kidney stone diseases.
... With increasing ZFS the X-band spectrum becomes broader and more complicated and the low-field conditions make straightforward interpretation difficult. To simplify and better understand the spectra, one can record the spectra at higher microwave frequencies such as 35 GHz (Q-band) [41,42], 95 GHz (Wband) or even higher, up to THz which is commonly known as high-field EPR (HFEPR) [37,41,[43][44][45][46][47][48][49][50][51][52]. ...
We report the synthesis of [Mn(tacud)2](OTf)2 (1) (tacud = 1,4,8-triazacycloundecane), [Mn(tacd)2](OTf)2 (2) (tacd = 1,4,7-triazacyclodecane), and [Mn(tacn)2](OTf)2 (3) (tacn = 1,4,7-triazacyclononane). Electrochemical measurements on the MnIII/II redox couple show that complex 1 has the largest anodic potential of the set (E1/2 = 1.16 V vs NHE, ΔEp = 106 mV) compared to 2 (E1/2 = 0.95 V, ΔEp = 108 mV) and 3 (E1/2 = 0.93 V, ΔEp = 96 mV). This is due to the fact that 1 has the fewest 5-membered chelate rings and thus is least stabilized. Magnetic studies of 1–3 revealed that all complexes remain high spin throughout the temperature range investigated (2–300 K). X-band EPR investigations in methanol glass indicated that the manganese(II) centers for 2 and 3 resided in a more distorted octahedral geometric configuration compared to 1. To ease spectral interpretation and extract ZFS parameters, we performed high-frequency high-field EPR (HFEPR) at frequencies above 200 GHz and a field of 7.5 T. Simulation of the spectral data yielded g = 2.0013 and D = −0.031 cm⁻¹ for 1, g = 2.0008, D = −0.0824 cm⁻¹, |E/D| = 0.12 for 2, and g = 2.00028, D = −0.0884 cm⁻¹ for 3. These results are consistent with 3 possessing the most distorted geometry. Calculations (PBE0/6-31G(d)) were performed on 1–3. Results show that 1 has the largest HOMO-LUMO gap energy (6.37 eV) compared to 2 (6.12 eV) and 3 (6.26 eV). Complex 1 also has the lowest HOMO energies indicating higher stability.
... The Mn is not released from the enzyme at low pH. The pH dependence of the high-field spectra show very little if any change unlike what has been observed in OxDC [43]. The biggest change is seen in the X-band spectra, where the sharp features between 100 and 200 mT diminish at low pH. ...
oxalate oxidase (CsOxOx) is the first bicupin enzyme identified that catalyzes manganese-dependent oxidation of oxalate. In previous work, we have shown that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated. CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC) and the 241-244DASN region of the N-terminal Mn binding domain of CsOxOx is analogous to the lid region of OxDC that has been shown to determine reaction specificity. We have prepared a series of CsOxOx mutants to probe this region and to identify the carboxylate residue implicated in catalysis. The pH profile of the D241A CsOxOx mutant suggests that the protonation state of aspartic acid 241 is mechanistically significant and that catalysis takes place at the N-terminal Mn binding site. The observation that the D241S CsOxOx mutation eliminates Mn binding to both the N- and C- terminal Mn binding sites suggests that both sites must be intact for Mn incorporation into either site. The introduction of a proton donor into the N-terminal Mn binding site (CsOxOx A242E mutant) does not affect reaction specificity. Mutation of conserved arginine residues further support that catalysis takes place at the N-terminal Mn binding site and that both sites must be intact for Mn incorporation into either site.
... Fig. 1A shows the broadband NMR spectra, obtained by 95-GHz ENDOR spectroscopy, of phosphorus and proton nuclei near the Mn(II) ions in intact cells of D. radiodurans from cultures at exponential (OD 600 ϭ 0.4) or stationary (OD 600 ϭ 2.0) growth phase. As a consequence of the high magnetic fields used and judicious selection of temperatures (22), they are simpler and more resolved than the previous 35-GHz yeast spectra (14). At the 31 P resonance frequency (58 MHz), the 95-GHz D. radiodurans Davies ENDOR spectra had an intense pair of resonances split by 4.3 MHz, similar to the pair reported for yeast (14) (Fig. 1, A and B). ...
High magnetic field high frequency electron paramagnetic resonance techniques were used to measure in situ Mn(II) speciation in Deinococcus radiodurans, a radiation-resistant bacteria capable of accumulating high concentrations of Mn(II). It was possible to identify and quantify
the evolution of Mn(II) species in intact cells at various stages of growth. Aside from water, 95-GHz high field electron
nuclear double resonance showed that the Mn(II) ions are bound to histidines and phosphate groups, mostly from fructose-1,6-bisphosphate
but also inorganic phosphates and nucleotides. During stationary growth phase, 285-GHz continuous wave EPR measurements showed
that histidine is the most common ligand to Mn(II) and that significant amounts of cellular Mn(II) in D. radiodurans are bound to peptides and proteins. As much as 40% of the total Mn(II) was in manganese superoxide dismutase, and it is this
protein and not smaller manganese complexes, as has been suggested recently, that is probably the primary defense against
superoxide.
... A series of His 6 -tagged OxDC mutants was therefore prepared with which to probe the functional importance of the interactions involving the Ser-161 and Thr-165 side chains (Table 1). We note that recent EPR studies have shown that the presence of the His 6 -tag at the C-terminus of the enzyme does not perturb the electronic properties of the Mn(II) centers [21]. ...
EPR spin trapping experiments on bacterial oxalate decarboxylase from Bacillus subtilis under turn-over conditions are described. The use of doubly (13)C-labeled oxalate leads to a characteristic splitting of the observed radical adducts using the spin trap N-tert-butyl-α-phenylnitrone linking them directly to the substrate. The radical was identified as the carbon dioxide radical anion which is a key intermediate in the hypothetical reaction mechanism of both decarboxylase and oxidase activities. X-ray crystallography had identified a flexible loop, SENS161-4, which acts as a lid to the putative active site. Site directed mutagenesis of the hinge amino acids, S161 and T165 was explored and showed increased radical trapping yields compared to the wild type. In particular, T165V shows approximately ten times higher radical yields while at the same time its decarboxylase activity was reduced by about a factor of ten. This mutant lacks a critical H-bond between T165 and R92 resulting in compromised control over its radical chemistry allowing the radical intermediate to leak into the surrounding solution.
... It contains two distinct Mn-binding sites both of which have to be populated for enzymatic activity [61]. High-field EPR has revealed a remarkable number of distinct Mn(II) species dependent on pH and buffer con- ditions [62,63] . The obvious advantage of using the H@iBuT 8 standard in this case over the commonly used Mn(II) standard lies in the fact that it shows only little overlap with the spectrum of interest . ...
We introduce atomic hydrogen trapped in an octaisobutylsilsesquioxane nanocage (H@iBuT₈) as a new molecular high-precision magnetic field standard for high-field EPR spectroscopy of organic radicals and other systems with signals around g=2. Its solid-state EPR spectrum consists of two 0.2 mT wide lines separated by about 51 mT and centered at g≈2. The isotropic g factor is 2.00294(3) and essentially temperature independent. The isotropic ¹H hyperfine coupling constant is 1416.8(2) MHz below 70 K and decreases slightly with increasing temperature to 1413.7(1) MHz at room temperature. The spectrum of the standard does not overlap with those of most organic radicals, and it can be easily prepared and is stable at room temperature.
... In fact, a mutation of the amino acids in the lid region converts the ODC activity into H 2 O 2 -producing OXO activity (Burrell et al. 2007). Recent data suggest that both N-and C-terminal domains may catalyze the decarboxylation reaction (Tabares et al. 2009). ...
Oxalate decarboxylase (ODC) is a manganese-containing, multimeric enzyme of the cupin protein superfamily. ODC is one of the three enzymes identified to decompose oxalic acid and oxalate, and within ODC catalysis, oxalate is split into formate and CO(2). This primarily intracellular enzyme is found in fungi and bacteria, and currently the best characterized enzyme is the Bacillus subtilis OxdC. Although the physiological role of ODC is yet unidentified, the feasibility of this enzyme in diverse biotechnological applications has been recognized for a long time. ODC could be exploited, e.g., in diagnostics, therapeutics, process industry, and agriculture. So far, the sources of ODC enzyme have been limited including only a few fungal and bacterial species. Thus, there is potential for identification and cloning of new ODC variants with diverse biochemical properties allowing e.g. more enzyme fitness to process applications. This review gives an insight to current knowledge on the biochemical characteristics of ODC, and the relevance of oxalate-converting enzymes in biotechnological applications. Particular emphasis is given to fungal enzymes and the inter-connection of ODC to fungal metabolism of oxalic acid.
Although not as important as iron, manganese is one of the key transition metal (d block) elements in biochemistry. The role of Mn in photosynthesis is perhaps its “starring role,” but has been extensively covered elsewhere and will not be discussed in this chapter. In contrast to iron, for which ⁵⁷Fe Mӧssbauer effect (nuclear gamma resonance) spectroscopy is available, ⁵⁵Mn (100% abundant) lacks this ability. Instead, the defining technique for Mn is electron paramagnetic resonance (EPR) spectroscopy, as Mn(II), Mn(IV), and even Mn(III), as well as certain di-Mn systems are generally EPR active. This chapter describes how EPR and its advanced derivatives, electron spin echo envelope modulation (ESEEM) and electron–nuclear/electron–electron double resonance (ENDOR/ELDOR) spectroscopies, provide valuable biochemical information on many Mn-containing systems. These include endogenous Mn in mono- and di-Mn (protein) enzymes as well as brief mention of applications to Mn in non-protein biological systems.
The hexameric low-pH stress response enzyme oxalate decarboxylase catalyzes the decarboxylation of the oxalate mono-anion in the soil bacterium Bacillus subtilis. A single protein subunit contains two Mn-binding cupin domains, and catalysis depends on Mn(III) at the N-terminal site. The present study suggests a mechanistic function for the C-terminal Mn as an electron hole donor for the N-terminal Mn. The resulting spatial separation of the radical intermediates directs the chemistry toward decarboxylation of the substrate. A π-stacked tryptophan pair (W96/W274) links two neighboring protein subunits together, thus reducing the Mn-to-Mn distance from 25.9 Å (intra-subunit) to 21.5 Å (inter-subunit). Here, we used theoretical analysis of electron hole-hopping paths through redox-active sites in the enzyme combined with site-directed mutagenesis and X-ray crystallography to demonstrate that this tryptophan pair supports effective electron hole hopping between the C-terminal Mn of one subunit and the N-terminal Mn of the other subunit through two short hops of ∼8.5 Å. Replacement of W96, W274, or both with phenylalanine led to a large reduction in catalytic efficiency, while replacement with tyrosine led to recovery of most of this activity. W96F and W96Y mutants share the wild-type tertiary structure. Two additional hole-hopping networks were identified leading from the Mn ions to the protein surface, potentially protecting the enzyme from high Mn oxidation states during turnover. Our findings strongly suggest that multi-step hole hopping transport between the two Mn ions is required for enzymatic function, adding to the growing examples of proteins that employ aromatic residues as hopping stations.
Dioxygen activation at manganese centers is well known in nature, but synthetic manganese systems capable of utilizing O2 as an oxidant are relatively uncommon. These present investigations probe the dioxygen activation pathways of two mononuclear MnII complexes supported by pentacoordinate amide-containing ligands, [MnII(dpaq)](OTf) and the sterically modified [MnII(dpaq2Me)](OTf). Dioxygen titration experiments demonstrate that [MnII(dpaq)](OTf) reacts with O2 to form [MnIII(OH)(dpaq)](OTf) according to a 4 : 1 Mn : O2 stoichiometry. This stoichiometry is consistent with a pathway involving comproportionation between a MnIV–oxo species and residual MnII complex to form a (μ-oxo)dimanganese(III,III) species that is hydrolyzed by water to give the MnIII–hydroxo product. In contrast, the sterically modified [MnII(dpaq2Me)](OTf) complex was found to react with O2 according to a 2 : 1 Mn : O2 stoichiometry. This stoichiometry is indicative of a pathway in which a MnIV–oxo intermediate abstracts a hydrogen atom from solvent instead of undergoing comproportionation with the MnII starting complex. Isotopic labeling experiments, in which the oxygenation of the MnII complexes was carried out in deuterated solvent, supported this change in pathway. The oxygenation of [MnII(dpaq)](OTf) did not result in any deuterium incorporation in the MnIII–hydroxo product, while the oxygenation of [MnII(dpaq2Me)](OTf) in d3-MeCN showed [MnIII(OD)(dpaq2Me)]⁺ formation. Taken together, these observations highlight the use of steric effects as a means to select which intermediates form along dioxygen activation pathways.
The new tetradentate L⁷BQ ligand (L⁷BQ = 1,4-di(quinoline-8-yl)-1,4-diazepane) has been synthesized and shown to support MnII and MnIII-peroxo complexes. X-ray crystallography of the [MnII(L⁷BQ)(OTf)2] complex shows a monomeric MnII center with the L⁷BQ ligand providing four donor nitrogen atoms in the equatorial field, with two triflate ions bound in the axial positions. When this species is treated with H2O2 and Et3N at −40 °C, a MnIII-peroxo adduct, [MnIII(O2)(L⁷BQ)]⁺ is formed. The formation of this new intermediate is supported by a variety of spectroscopic techniques, including electronic absorption, Mn K-edge X-ray absorption and electron paramagnetic resonance methods. Evaluation of extended X-ray absorption fine structure data for [MnIII(O2)(L⁷BQ)]⁺ resolved Mn–O bond distances of 1.85 Å, which are on the short end of those previously reported for crystallographically characterized MnIII-peroxo adducts. An analysis of the X-ray pre-edge region of [MnIII(O2)(L⁷BQ)]⁺ revealed a large pre-edge area of 20.8 units. Time-dependent density functional theory computations indicate that the pre-edge intensity is due to Mn 4p–3d mixing caused by geometric distortions from centrosymmetry induced by both the peroxo and L⁷BQ ligands. The reactivity of [MnIII(O2)(L⁷BQ)]⁺ towards aldehydes was assessed through reaction with cyclohexanecarboxaldehyde and 2-phenylpropionaldehyde. From these experiments, it was determined that [MnIII(O2)(L⁷BQ)]⁺ only reacts with aldehydes in the presence of acid. Specifically, the addition of cyclohexanecarboxylic acid to [MnIII(O2)(L⁷BQ)]⁺ converts the MnIII-peroxo adduct to a new intermediate that could be responsible for the observed aldehyde deformylation activity. These observations underscore the challenges in identifying the reactive metal species in aldehyde deformylation reactions.
This perspective reviews our recent efforts towards the self-assembly of polynuclear clusters with ditopic and tritopic multidentate ligands HL¹ (2-phenyl-4,5-bis{6-(3,5-dimethylpyrazol-1-yl)pyrid-2-yl}-1H-imidazole) and H2L² (2,6-bis-[5-(2-pyridinyl)-1H-pyrazole-3-yl]pyridine), both of which are planar and rigid molecules. HL¹ was found to be an excellent support for tetranuclear [Fe4] complexes, [FeII4(L¹)4](BF4)4 ([FeII4]) and [FeIII2FeII2(L¹)4](BF4)6 ([FeIII2FeII2]). The homovalent system was found to exhibit multistep spin crossover (SCO), while the mixed-valence [FeIII2FeII2] complex shows wavelength-dependent tuneable light-induced excited spin state trapping (LIESST). For H2L², a variety of polynuclear complexes were obtained through complexation with different transition metal ions, allowing the isolation of rings, grids, and helix structures. The rigidity of the ligand, difference in its coordination sites, and affinity for different metal ions dictates its coordination behaviour. In this paper, we summarise these ligand pre-programmed self-assembled clusters and their diverse physical properties.
This article introduces the foundations and application ranges of very-high-frequency EPR experiments. The benefits of VHF-EPR for the resolution of small g-tensor anisotropies and the determination of large zero-field splittings are introduced. In view of these fields of application, different design principles of VHF-EPR spectrometers are described and compared.
This contribution describes EPR experiments on Mn(III) in oxalate decarboxylase of Bacillus subtilis, an interesting enzyme that catalyzes the redox-neutral dissociation of oxalate into formate and carbon dioxide. Chemical redox cycling provides strong evidence that both Mn centers can be oxidized although the N-terminal Mn(II) appears to have the lower reduction potential and is most likely the carrier of the +3 oxidation state under moderate oxidative conditions in agreement with the general view that it represents the active site. Significantly, Mn(III) was observed in untreated OxDC in succinate and acetate buffers while it could not be directly observed in citrate buffer. Quantitative analysis showed that up to 16% of the EPR visible Mn is in the +3 oxidation state at low pH in the presence of succinate buffer. The fine structure and hyperfine structure parameters of Mn(III) are affected by small carboxylate ligands that can enter the active site and have been recorded for formate, acetate, and succinate. The results from a previous report by Zhu et al. (2016) Biochemistry 55, 429-434 could therefore be reinterpreted as evidence for formate-bound Mn(III) after the enzyme is allowed to turn over oxalate. The pH dependence of the Mn(III) EPR signal compares very well with that of enzymatic activity providing strong evidence that the catalytic reaction of oxalate decarboxylase is driven by Mn(III) which is generated in the presence of dioxygen.
High-spin GdIII and MnII complexes have emerged as alternatives to the standard nitroxide radical spin-labels for measuring nanometric distances using pulsed electron-electron double resonance (PELDOR or DEER) at high-fields/frequencies. For certain complexes, particularly those with relatively small Zero-Field Splitting (ZFS) and short distances between the two metal centers, the pseudo-secular term of the dipolar coupling Hamiltonian was found to be non-negligible. However, in general the contribution from this term during conventional data analysis has been masked by the flexibility of the molecule of interest and/or the long tethers connecting them to the spin-labels. We report here the efficient synthesis of a model system consisting of two [Mn(DOTA)]2-, directly connected to the ends of a central rod like oligo(phenylene-ethynylene) (OPE) spacer. The rigidity of the OPE was confirmed by Q-band PELDOR measurements on a bis-nitroxide analogue. The MnII-MnII distance distribution profile determined by W-band PELDOR was in fair agreement with one simulated using a simple rotamer analysis. The small degree of flexibility arising from the linking MnDOTA arm appeared to outweigh the contribution from the pseudo-secular term at this inter-spin distance. This study illustrates the potential of MnDOTA-based spin-labels for measuring fairly short nanometric distances, and also presents an interesting candidate for in-depth studies of pulse dipolar spectroscopy methods on MnII-MnII systems.
Pulse electron-electron double resonance (PELDOR) is a versatile technique for probing the structures and functions of complex biological systems. Despite the recent interest in high-spin metal-ions for high field/frequency applications, PELDOR measurements of Mn(II) remain relatively underexplored. Here we present Mn(II)-Mn(II) PELDOR distance measurements at 94 GHz on polyproline II (PPII) helices doubly spin-labeled with Mn(II)DOTA, which are distinguished by their small zero-field interaction. The measured Mn-Mn distances and distribution profiles were in good agreement with the expected values from molecular models. Additional features in the frequency-domain spectra became apparent at certain combinations of detect and pump frequencies. Spin-Hamiltonian calculations showed that they likely arose from contributions from the pseudo-secular component of the dipolar interaction that were found to be non-negligible for Mn(II)DOTA. However, the influence of the pseudo-secular component on the distance distribution profiles apparently was limited. The results show the potential of Mn(II)DOTA spin labels for high-field PELDOR distance measurements in proteins and other biological systems.
The chapter reports on recent studies of metal complexes that have the phenolate group as a ligand. The synthesis and characterization of many low molecular weight complexes is reviewed and commented on. Special attention is devoted to biomimetic models, for a deeper understanding of metalloprotein structures and functions. The chapter is divided into two parts, related to oxidant and antioxidant activities and to the hydrolysis of phospatediesters, respectively.
Activity of phenols as antioxidants and V IV O ²⁺ , Mn II , Mn III , Mn IV ,Co II , Co III , Ni II , Cu I , Cu II and Zn II complexes with phenolato‐containing Schiff bases as biomimetic for biological systems, is the focus of the first part. A section is dedicated to selected Cu II and Pt II complexes having pharmaceutical active molecules as ligands, from the OXICAM family of non‐steroidal anti‐inflammatory drugs. The second part reports on metal–phenolato complexes as models for phosphodiesterase enzymes. Dinuclear complexes based on Zn II , Fe III /Zn II , Zn II /Mg II metal centers are reviewed and commented on
D. radiodurans accumulates large quantities of Mn(II), which is believed to form low molecular weight complexes with phosphate and metabolites that protect D. radiodurans from radiation damage. The concentration of Mn(II) species in D. radiodurans during exponential and stationary phase were determined using high-field EPR and biochemical techniques. In the exponential growth phase cells a large fraction of the manganese was in the form of Mn(II)-orthophosphate complexes. By contrast, the intracellular concentrations of these compounds in stationary phase cells was less than 16 μM, while Mn superoxide dismutase was 320 μM and another, yet unidentified, Mn(II) protein 250 μM. Stationary cells were found to be equally resistant to irradiation as the exponential cells in spite of having significant lower Mn(II)-orthophosphate concentrations. Gamma irradiation induced no changes in the Mn(II) speciation. During stationary growth phase D. radiodurans favours the production of the two Mn-proteins over low molecular weight complexes suggesting that the latter were not essential for radio-resistance at this stage of growth
The manganese(ii) speciation in intact cells of D. radiodurans, E. coli, S. cerevisiae and Arabidopsis thaliana seeds was measured using high-field electron paramagnetic resonance techniques. The majority of the Mn(ii) ions in these organisms were six-coordinate, bound predominately by water, phosphates and nitrogen-based molecules. The relative distribution of the different phosphates in bacteria and S. cerevisiae was the same and dominated by monophosphate monoesters. Mn(ii) was also found bound to the phosphate backbone of nucleic acids in these organisms. Phosphate ligation in Arabidopsis seeds was dominated by phytate. The extent of nitrogen ligation in the four organisms was also determined. On average, the Mn(ii) in D. radiodurans had the most nitrogen ligands followed by E. coli. This was attributed to higher concentrations of Mn(ii) bound to proteins in these species. Although constitutively expressed in all four organisms, MnSOD was only detected in D. radiodurans. As previously reported, D. radiodurans also accumulates a second abundant Mn containing protein species. The high concentration of proteinaceous Mn(ii) is a unique feature of D. radiodurans.
Oxalate decarboxylase (OxDC) catalyzes the Mn-dependent conversion of the oxalate monoanion into CO2 and formate. EPR-based strategies for investigating the catalytic mechanism of decarboxylation are complicated by the difficulty of assigning the signals associated with the two Mn(II) centers located in the N- and C-terminal cupin domains of the enzyme. We now report a mutational strategy that has established the assignment of EPR fine structure parameters to each of these Mn(II) centers at pH 8.5. These experimental findings are also used to assess the performance of a multistep strategy for calculating the zero-field splitting (zfs) parameters of protein-bound Mn(II) ions. Despite the known sensitivity of calculated D and E values to the computational approach, we demonstrate that good estimates of these parameters can be obtained using cluster models taken from carefully optimized DFT/MM structures. Overall, our results provide new insights into the strengths and limitations of theoretical methods for understanding electronic properties of protein-bound Mn(II) ions, thereby setting the stage for future EPR studies on the electronic properties of the Mn(II) centers in OxDC and site-specific variants.
Three polypyridine ligand-supported multinuclear iron complexes, [Fe5], [Fe7] and [Fe17], were synthesized and their physical properties were investigated. The complexes had triple-stranded helical structures with pseudo threefold symmetry, and were stabilized by varying degrees of intramolecular π-π stacking. The pentanuclear iron complex consisted of two Fe(II) and three Fe(III) ions, supported by three ligands, while the heptanuclear complex comprised four Fe(II) centres, three Fe(III) ions, and six ligands, and the heptadecanuclear complex contained seventeen Fe(III) ions and nine ligands. Electrochemical studies revealed that the pentanuclear and heptanuclear iron complexes showed pseudo-reversible three- and five-step redox behaviours, respectively. Magnetic measurements conducted on the pentanuclear and heptanuclear complexes revealed that antiferromagnetic interactions were operative between neighbouring iron ions through the oxo- and pyrazole-bridges.
The chemical speciation of 2-amino-N-hydroxypropanamide (β-alaninohydroxamic acid, HL) and vanadium(V) in aqueous solution has been investigated through calculations of the thermodynamic properties and the (51)V NMR chemical shifts of the species formed at equilibrium. The results have been compared directly with the experimental (51)V NMR data. The (51)V NMR chemical shifts have been calculated by using a DFT approach accounting for relativistic corrections and solvent effects. All tautomers of the 1:1 and 1:2 VO2(+)/β-ala complexes with different degrees of protonation have been calculated and thermodynamic and structural properties are presented for the most stable species. The system is better modeled as tautomeric equilibria and species lying down in the range of 10 kcal.mol(-1) cannot be neglected at the BP/TZ2P/COSMO approach. In fact, the metal complex speciation in aqueous solution should not be investigated based solely on the thermodynamic analysis, but together with spectroscopic calculations such as NMR.
A common feature of a large majority of the manganese metalloenzymes, as well as many synthetic biomimetic complexes, is the bonding between the manganese ion and imidazoles. This interaction was studied by examining the nature and structure of manganese(II) imidazole complexes in frozen aqueous solutions using 285 GHz high magnet-field continuous-wave electron paramagnetic resonance (cw-HFEPR) and 95 GHz pulsed electron-nuclear double resonance (ENDOR) and pulsed electron-double resonance detected nuclear magnetic resonance (PELDOR-NMR). The (55)Mn hyperfine coupling and isotropic g values of Mn(II) in frozen imidazole solutions continuously decreased with increasing imidazole concentration. ENDOR and PELDOR-NMR measurements demonstrated that the structural basis for this behavior arose from the imidazole concentration-dependent distribution of three six-coordinate and two four-coordinate species: [Mn(H2O)6](2+), [Mn(imidazole)(H2O)5](2+), [Mn(imidazole)2(H2O)4](2+), [Mn(imidazole)3(H2O)](2+), and [Mn(imidazole)4](2+). The hyperfine and g values of manganese proteins were also fully consistent with this imidazole effect. Density functional theory methods were used to calculate the structures, spin and charge densities, and hyperfine couplings of a number of different manganese imidazole complexes. The use of density functional theory with large exact-exchange admixture calculations gave isotropic (55)Mn hyperfine couplings that were semiquantitative and of predictive value. The results show that the covalency of the Mn-N bonds play an important role in determining not only magnetic spin parameters but also the structure of the metal binding site. The relationship between the isotropic (55)Mn hyperfine value and the number of imidazole ligands provides a quick and easy test for determining whether a protein binds an Mn(II) ion using histidine residues and, if so, how many are involved. Application of this method shows that as much as 40% of the Mn(II) ions in Deinococcus radiodurans are ligated to two histidines (Tabares, L. C.; Un, S. J. Biol. Chem2013, in press).
Oxalate decarboxylase, an oxalate degradation enzyme used for medical diagnosis and decreasing the oxalate level in the food or paper industry, was covalently immobilized to Eupergit C. Different immobilization parameters, including ratio of enzyme to support, ammonia sulfate concentration, pH, and incubation time, were optimized. Under the condition of enzyme/support ratio at 1:20, pH 9, with 1.5 mol/L (NH(4))(2)SO(4), room temperature, and shaking at 30 rpm for 24 hr, activity recovery of immobilized Oxdc reached 90% with an apparent specific activity of 0.44 U/mg support. The enzymatic properties of immobilized Oxdc were investigated and compared with those of the soluble enzyme. Both shared a similar profile of optimum conditions; the optimum pH and temperature for soluble and immobilized Oxdc were 3.5 and 50°C, respectively. The immobilized enzyme was more stable at lower pH and higher temperatures. The kinetic parameters for soluble and immobilized enzyme were also determined.
One of the most puzzling questions of manganese and iron superoxide dismutases (SODs) is what is the basis for their metal-specificity. This review summarizes our findings on the Mn(II) electronic structure of SODs and related synthetic models using high-field high-frequency electron paramagnetic resonance (HFEPR), a technique that is able to achieve a very detailed and quantitative information about the electronic structure of the Mn(II) ions. We have used HFEPR to compare eight different SODs, including iron, manganese and cambialistic proteins. This comparative approach has shown that in spite of their high structural homology each of these groups have specific spectroscopic and biochemical characteristics. This has allowed us to develop a model about how protein and metal interactions influence protein pK, inhibitor binding and the electronic structure of the manganese center. To better appreciate the thermodynamic prerequisites required for metal discriminatory SOD activity and their relationship to HFEPR spectroscopy, we review the work on synthetic model systems that functionally mimic Mn-and FeSOD. Using a single ligand framework, it was possible to obtain metal-discriminatory "activity" as well as variations in the HFEPR spectra that parallel those found in the proteins. Our results give new insights into protein-metal interactions from the perspective of the Mn(II) and new steps towards solving the puzzle of metal-specificity in SODs.
This review summarizes the recent discovery of the cupin superfamily (from the Latin term "cupa," a small barrel) of functionally diverse proteins that initially were limited to several higher plant proteins such as seed storage proteins, germin (an oxalate oxidase), germin-like proteins, and auxin-binding protein. Knowledge of the three-dimensional structure of two vicilins, seed proteins with a characteristic beta-barrel core, led to the identification of a small number of conserved residues and thence to the discovery of several microbial proteins which share these key amino acids. In particular, there is a highly conserved pattern of two histidine-containing motifs with a varied intermotif spacing. This cupin signature is found as a central component of many microbial proteins including certain types of phosphomannose isomerase, polyketide synthase, epimerase, and dioxygenase. In addition, the signature has been identified within the N-terminal effector domain in a subgroup of bacterial AraC transcription factors. As well as these single-domain cupins, this survey has identified other classes of two-domain bicupins including bacterial gentisate 1, 2-dioxygenases and 1-hydroxy-2-naphthoate dioxygenases, fungal oxalate decarboxylases, and legume sucrose-binding proteins. Cupin evolution is discussed from the perspective of the structure-function relationships, using data from the genomes of several prokaryotes, especially Bacillus subtilis. Many of these functions involve aspects of sugar metabolism and cell wall synthesis and are concerned with responses to abiotic stress such as heat, desiccation, or starvation. Particular emphasis is also given to the oxalate-degrading enzymes from microbes, their biological significance, and their value in a range of medical and other applications.
The Bacillus subtilis oxalate decarboxylase (EC 4.1.1.2), YvrK, converts oxalate to formate and CO2. YvrK and the related hypothetical proteins YoaN and YxaG from B. subtilis have been successfully overexpressed in Escherichia coli. Recombinant YvrK and YoaN were found to be soluble enzymes with oxalate decarboxylase activity only when expressed in the
presence of manganese salts. No enzyme activity has yet been detected for YxaG, which was expressed as a soluble protein without
the requirement for manganese salts. YvrK and YoaN were found to catalyze minor side reactions: oxalate oxidation to produce
H2O2; and oxalate-dependent, H2O2-independent dye oxidations. The oxalate decarboxylase activity of purified YvrK was O2-dependent. YvrK was found to contain between 0.86 and 1.14 atoms of manganese/subunit. EPR spectroscopy showed that the metal
ion was predominantly but not exclusively in the Mn(II) oxidation state. The hyperfine coupling constant (A = 9.5 millitesla) of the maing = 2 signal was consistent with oxygen and nitrogen ligands with hexacoordinate geometry. The structure of YvrK was modeled
on the basis of homology with oxalate oxidase, canavalin, and phaseolin, and its hexameric oligomerization was predicted by
analogy with proglycinin and homogentisate 1,2-dioxygenase. Although YvrK possesses two potential active sites, only one could
be fully occupied by manganese. The possibility that the C-terminal domain active site has no manganese bound and is buried
in an intersubunit interface within the hexameric enzyme is discussed. A mechanism for oxalate decarboxylation is proposed,
in which both Mn(II) and O2are cofactors that act together as a two-electron sink during catalysis.
Oxalate decarboxylase (EC 4.1.1.2) catalyzes the conversion of oxalate to formate and carbon dioxide and utilizes dioxygen as a cofactor. By contrast, the evolutionarily related oxalate oxidase (EC 1.2.3.4) converts oxalate and dioxygen to carbon dioxide and hydrogen peroxide. Divergent free radical catalytic mechanisms have been proposed for these enzymes that involve the requirement of an active site proton donor in the decarboxylase but not the oxidase reaction. The oxidase possesses only one domain and manganese binding site per subunit, while the decarboxylase has two domains and two manganese sites per subunit. A structure of the decarboxylase together with a limited mutagenesis study has recently been interpreted as evidence that the C-terminal domain manganese binding site (site 2) is the catalytic site and that Glu-333 is the crucial proton donor (Anand, R., Dorrestein, P. C., Kinsland, C., Begley, T. P., and Ealick, S. E. (2002) Biochemistry 41, 7659-7669). The N-terminal binding site (site 1) of this structure is solvent-exposed (open) and lacks a suitable proton donor for the decarboxylase reaction. We report a new structure of the decarboxylase that shows a loop containing a 3(10) helix near site 1 in an alternative conformation. This loop adopts a "closed" conformation forming a lid covering the entrance to site 1. This conformational change brings Glu-162 close to the manganese ion, making it a new candidate for the crucial proton donor. Site-directed mutagenesis of equivalent residues in each domain provides evidence that Glu-162 performs this vital role and that the N-terminal domain is either the sole or the dominant catalytically active domain.
Deinococcus radiodurans is extremely resistant to ionizing radiation. How this bacterium can grow under chronic gamma radiation [50 grays (Gy) per hour] or recover from acute doses greater than 10 kGy is unknown. We show that D. radiodurans accumulates very high intracellular manganese and low iron levels compared with radiation-sensitive bacteria and that resistance exhibits a concentration-dependent response to manganous chloride [Mn(II)]. Among the most radiation-resistant bacterial groups reported, Deinococcus, Enterococcus, Lactobacillus, and cyanobacteria accumulate Mn(II). In contrast, Shewanella oneidensis and Pseudomonas putida have high iron but low intracellular manganese concentrations and are very sensitive. We propose that Mn(II) accumulation facilitates recovery from radiation injury.
A new variant of lipoxygenases, one containing manganese instead of iron, is characterized by electron paramagnetic resonance (EPR) at two frequencies. In the manganous state (S(e) = 5/2), maganese lipoxygenase (MnLO) yields very broad X-band (9.2 GHz) EPR signals, extending over about 800 mT. In contrast, at W-band (94 GHz), the signal is much simplified, consisting of nested transitions centered near the free electron g-value. Computer simulation has been employed to derive estimates of the zero-field splittings for MnLO, with data from these two EPR frequencies. The general features of both X- and W-band spectra are fit, first, by simulations with S(e) = 5/2, but no nuclear hyperfine splitting. The simulations are then refined by inclusion of the hyperfine splitting. On the basis of the simulations, the ranges of zero-field splitting parameters are D = +0.07 to +0.10 cm(-1), and E/D = 0.13 to 0.23. Comparison of the value of D for MnLO with that of other manganese-containing proteins suggests that MnLO has three N-ligands to the metal center and O-ligands in the remainder of 6 coordination positions. The coordination environment of MnLO is similar to that in iron lipoxygenases.
Two areas of research have recently converged to highlight important roles for Mn(2+) in pathogenesis: the recognition that both bacterial Nramp homologs and members of LraI family of proteins are Mn(2+) transporters. Their mutation is associated with decreased virulence of various bacterial species. Thus, Mn(2+) appears to be essential for bacterial virulence. This review describes what is currently known about Mn(2+) transport in prokaryotes and how prokaryotic Mn(2+) transport is regulated. Some of the phenotypes that arise when microorganisms lack Mn(2+) are then discussed, with an emphasis on those phenotypes involving pathogenesis. The concluding section describes possible enzymatic roles for Mn(2+) that might help explain why Mn(2+) is necessary for virulence.
Oxalate decarboxylase (EC 4.1.1.2) catalyses the conversion of oxalate into carbon dioxide and formate. It requires manganese and, uniquely, dioxygen for catalysis. It forms a homohexamer and each subunit contains two similar, but distinct, manganese sites termed sites 1 and 2. There is kinetic evidence that only site 1 is catalytically active and that site 2 is purely structural. However, the kinetics of enzymes with mutations in site 2 are often ambiguous and all mutant kinetics have been interpreted without structural information. Nine new site-directed mutants have been generated and four mutant crystal structures have now been solved. Most mutants targeted (i) the flexibility (T165P), (ii) favoured conformation (S161A, S164A, D297A or H299A) or (iii) presence (Delta162-163 or Delta162-164) of a lid associated with site 1. The kinetics of these mutants were consistent with only site 1 being catalytically active. This was particularly striking with D297A and H299A because they disrupted hydrogen bonds between the lid and a neighbouring subunit only when in the open conformation and were distant from site 2. These observations also provided the first evidence that the flexibility and stability of lid conformations are important in catalysis. The deletion of the lid to mimic the plant oxalate oxidase led to a loss of decarboxylase activity, but only a slight elevation in the oxalate oxidase side reaction, implying other changes are required to afford a reaction specificity switch. The four mutant crystal structures (R92A, E162A, Delta162-163 and S161A) strongly support the hypothesis that site 2 is purely structural.
Fe2+ has traditionally been considered the most important divalent cation involved in host-pathogen interactions. However, recent research indicates a previously unappreciated role for transition metal divalent cations other than Fe2+ during infection. Recent studies have identified an absolute requirement for Mn2+ in bacterial pathogens that are Fe2+-independent, indicating an important role for Mn2+ in pathogenesis. Potential roles for Mn2+ in pathogenesis include effects on the detoxification of reactive oxygen intermediates (ROIs), as a cofactor for enzymes involved in intermediary metabolism and signal transduction, and as a stimulus for virulence gene regulation. This review focuses on how these possible roles for Mn2+ may affect bacterial pathogenesis and the outcome of an infection.
Crystals of Zn2+/Mn2+ yeast enolase with the inhibitor PhAH (phosphonoacetohydroxamate) were grown under conditions with a slight preference for binding of Zn2+ at the higher affinity site, site I. The structure of the Zn2+/Mn2+-PhAH complex was solved at a resolution of 1.54 A, and the two catalytic metal binding sites, I and II, show only subtle displacement compared to that of the corresponding complex with the native Mg2+ ions. Low-temperature echo-detected high-field (W-band, 95 GHz) EPR (electron paramagnetic resonance) and 1H ENDOR (electron-nuclear double resonance) were carried out on a single crystal, and rotation patterns were acquired in two perpendicular planes. Analysis of the rotation patterns resolved a total of six Mn2+ sites, four symmetry-related sites of one type and two out of the four of the other type. The observation of two chemically inequivalent Mn2+ sites shows that Mn2+ ions populate both sites I and II and the zero-field splitting (ZFS) tensors of the Mn2+ in the two sites were determined. The Mn2+ site with the larger D value was assigned to site I based on the 1H ENDOR spectra, which identified the relevant water ligands. This assignment is consistent with the seemingly larger deviation of site I from octahedral symmetry, compared to that of site II. The ENDOR results gave the coordinates of the protons of two water ligands, and adding them to the crystal structure revealed their involvement in a network of H bonds stabilizing the binding of the metal ions and PhAH. Although specific hyperfine interactions with the inhibitor were not determined, the spectroscopic properties of the Mn2+ in the two sites were consistent with the crystal structure. Density functional theory (DFT) calculations carried out on a cluster representing the catalytic site, with Mn2+ in site I and Zn2+ in site II, and vice versa, gave overestimated D values on the order of the experimental ones, although the larger D value was found for Mn2+ in site II rather than in site I. This discrepancy was attributed to the high sensitivity of the ZFS parameters to the Mn-O bond lengths and orientations, such that small, but significant, differences between the optimized and crystal structures alter the ZFS considerably, well above the difference between the two sites.
The origin of the zero‐field splittings of the orbitally nondegenerate ground states of the transition metal ions (3d3, 3d5, and 3d8 solutes) has been studied on the assumption that these splittings are due to the combined action of an electric field gradient and the spin—spin interaction. A relation between the splitting parameters D and E of the conventional spin Hamiltonian and the field gradients q∣∣ and ηq∣∣, respectively, has been deduced on the above basis using hydrogenic wavefunctions and d→d, d→g, and d→s excitations. A reasonably good agreement with the splitting has been obtained for Mn2+ in corundum but an apparent disagreement has been found for Fe3+ in Al2O3, considering the known values of the field gradients in these cases. For Cr3+ (d3 solute) and Ni2+ (d8 solute) in corundum, the major sources of splitting seem to be the spin—orbit interaction and the distortion of the ground state wavefunctions due to covalent π bonding and not the spin—spin interaction. The cause of the apparent disagreement for Fe3+ in Al2O3 is discussed. A definite understanding of the cause of the zero‐field splittings in the transition metal ions depends on more accurate knowledge of the field gradients acting on these ions in different crystalline environments.
Although a considerable body of data exists on the parametrization of the ground-state splittings of S-state ions in crystals, relatively little progress has been made in obtaining a quantitative understanding of the mechanisms which determine these parameters. In the course of summarizing our present understanding, we emphasize the need for making planned experiments explicitly aimed at testing theoretical models, such as those proposed in this article. The variable frequency E.P.R. technique is described in some detail, as it has proved to be particularly useful in this respect.
W-band EPR (lambda almost-equal-to 3 mm, nu almost-equal-to 95 GHz) investigations of frozen solutions of various semiquinone anion radicals in vitro are presented. Among the quinones studied are ubiquinone-10 and the primary electron acceptors of photosystem I and II of plant photosynthesis, vitamin K1, and plastoquinone-9, respectively. In addition, W-band spectra of the primary quinone Q(A).- and of the radical pair P865.+Q(A).- in Zn-substituted reaction centers of Rhodobacter sphaeroides R-26 are shown. The electron Zeeman. interaction at resonant B0 fields in the W-band is increased by a factor of 10 as compared with conventional X-band, leading to almost completely resolved g-tensor components in all quinone radicals. By means of computer simulations of the EPR spectra of the immobilized radical ions, anisotropic g tensor components, anisotropic line width contributions, and, in some cases, hyperfine tensor information could be obtained. The data are partially interpreted by semiempirical MO methods including solvent effects. In all quinone radical anions studied, the measured g-tensor components are assigned to the molecular axes as follows: g(xx) > g(yy) > g(zz), where x is along the C-O bond direction and z is perpendicular to the quinone plane.
Complexes of the general formulation [M(bispicam)2]X2 [where bispicam is bis(2-pyridylmethyl)amine, C12H13N3, and M is Mn(II), Zn(II), or Cd(II)] have been synthesized and characterized by X-ray crystallography and by EPR and C-13 NMR spectroscopy. All of the complexes are facial isomers, but three distinct structural types have been discovered. The manganese complex [Mn(bispicam)2](ClO4)2 (1) crystallizes in the space group C2/c of the monoclinic system with four molecules in a cell of dimensions a = 20.594 (4) angstrom, b = 11.01 1 (2) angstrom, c = 13.715 (3) angstrom, and beta = 114.845 (15)-degrees. The manganese atom in 1 sits on a crystallographic 2-fold axis. The cadmium complex [Cd-(bispicaM)2](ClO4)2 (2) is isomorphous with 1, with cell dimensions a = 20.670 (4) angstrom, b = 10.911 (3) angstrom, c = 13.953 (2) angstrom, and beta = 114.493 (13)-degrees. The zinc complex [Zn(bispicam)2](ClO4)2 (3) also crystallizes in the space group C2/c of the monoclinic system but with eight molecules in a cell of dimensions a = 23.715 (6) angstrom, b = 9.006 (2) angstrom, c = 26.052 (I 1) angstrom, and beta = 91.52 (3)-degrees. There are two crystallographically independent cations in the crystals of 3, in one of which the zinc atom sits on a crystallographic 2-fold axis as in 1 and 2 and in the other of which zinc sits on a crystallographic inversion center. The chloride salt of the zinc complex, [Zn(bispicam)2]Cl2.6H2O (4), crystallizes in the space group P2(1)/c of the monoclinic system with two molecules in a cell of dimensions a = 9.175 (2) angstrom, b = 10.088 (2) angstrom, c = 16.472 (3) angstrom, and beta = 94.540 (I 0)-degrees. The zinc atom in 4 sits on a crystallographic inversion center, so the isomer found here is similar to one of those in 3. The EPR spectrum of Mn doped in the crystals of 2 reveals the presence of the anticipated geometry at Mn, and this spectrum has been very successfully simulated by diagonalization of the energy matrix. The EPR spectrum of Mn doped in the crystals of 3 demonstrates that, at low concentrations, the manganese atoms evidently occupy only the sites of C2 symmetry, the resulting spectrum being very similar to that of Mn doped in 2; this spectrum has also been precisely simulated. The C-13 NMR spectra of the complexes demonstrate that NMR techniques will be of value in distinguishing between the various isomers in this and other diamagnetic zinc and cadmium series.
High magnetic-field electron paramagnetic resonance (HFEPR) has been extensively applied to the study of photosynthetic reaction
centers. HFEPR experiments have provided accurate measurements ofg-values of radicals in photosystems I and II. Theg-values not only reflect the structure of the radical, but also its immediate environment. Hence, valuable information about
radicalprotein interactions can be obtained fromg-values. Data on tyrosine-, quinone-, pheophytin- and chlorophyll-based radicals are reviewed. Experiments dealing with spin
pairs in photosystem I and II are examined. The spectrometer with which numerous experiments on photosystems have been carried
out is described, as well as the 10 T magnet around which the spectrometer is based.
As a facultative aerobe with a high iron requirement and a highly active aerobic respiratory chain, Neisseria gonorrhoeae requires defence systems to respond to toxic oxygen species such as superoxide. It has been shown that supplementation of media with 100 microM Mn(II) considerably enhanced the resistance of this bacterium to oxidative killing by superoxide. This protection was not associated with the superoxide dismutase enzymes of N. gonorrhoeae. In contrast to previous studies, which suggested that some strains of N. gonorrhoeae might not contain a superoxide dismutase, we identified a sodB gene by genome analysis and confirmed its presence in all strains examined by Southern blotting, but found no evidence for sodA or sodC. A sodB mutant showed very similar susceptibility to superoxide killing to that of wild-type cells, indicating that the Fe-dependent SOD B did not have a major role in resistance to oxidative killing under the conditions tested. The absence of a sodA gene indicated that the Mn-dependent protection against oxidative killing was independent of Mn-dependent SOD A. As a sodB mutant also showed Mn-dependent resistance to oxidative killing, then it is concluded that this resistance is independent of superoxide dismutase enzymes. Resistance to oxidative killing was correlated with accumulation of Mn(II) by the bacterium. We hypothesize that this bacterium uses Mn(II) as a chemical quenching agent in a similar way to the already established process in Lactobacillus plantarum. A search for putative Mn(II) uptake systems identified an ABC cassette-type system (MntABC) with a periplasmic-binding protein (MntC). An mntC mutant was shown to have lowered accumulation of Mn(II) and was also highly susceptible to oxidative killing, even in the presence of added Mn(II). Taken together, these data show that N. gonorrhoeae possesses a Mn(II) uptake system that is critical for resistance to oxidative stress.
Oxalate oxidase catalyzes the oxidation of oxalate to carbon dioxide and hydrogen peroxide, making it useful for clinical analysis of oxalate in biological fluids. An artificial gene for barley oxalate oxidase has been used to produce functional recombinant enzyme in a Pichia
pastoris heterologous expression system, yielding 250 mg of purified oxalate oxidase from 5 L of fermentation medium. The recombinant oxalate oxidase was expressed as a soluble, hexameric 140 kDa glycoprotein containing 0.2 g-atom Mn/monomer with a specific activity of 10 U/mg, similar to the properties reported for enzyme isolated from barley. No superoxide dismutase activity was detected in the recombinant oxalate oxidase. EPR spectra indicate that the majority of the manganese in the protein is present as Mn(II), and are consistent with the six-coordinate metal center reported in the recent X-ray crystal structure for barley oxalate oxidase. The EPR spectra change when bulky anions such as iodide bind, indicating conversion to a five-coordinate complex. Addition of oxalate perturbs the EPR spectrum of the Mn(II) sites, providing the first characterization of the substrate complex. The optical absorption spectrum of the concentrated protein contains features associated with a minor six-coordinate Mn(III) species, which disappears on addition of oxalate. EPR spin-trapping experiments indicate that carboxylate free radicals (CO2•–) are transiently produced by the enzyme in the presence of oxalate, most likely during reduction of the Mn(III) sites. These features are incorporated into a turnover mechanism for oxalate oxidase.
FosA is a manganese metalloglutathione transferase that confers resistance to the broad-spectrum antibiotic fosfomycin, (1R,2S)-epoxypropylphosphonic acid. The reaction catalyzed by FosA involves the attack by glutathione on fosfomycin to yield the product 1-(S-glutathionyl)-2-hydroxypropylphosphonic acid. The enzyme is a dimer of 16 kDa subunits, each of which harbors one mononuclear Mn(II) site. The coordination environment of the Mn(II) in the FosA x Mn(2+) complex is composed of a glutamate and two histidine ligands and three water molecules. Here we report EPR spectroscopic studies on FosA, in which EPR spectra were obtained at 35 GHz and 2 K using dispersion-detection rapid-passage techniques. This approach provides an absorption envelope line shape, in contrast to the conventional (slow-passage) derivative line shape, and is a more reliable way to collect spectra from Mn(II) centers with large zero-field splitting. We obtain excellent spectra of FosA bound with substrate, substrate analogue phosphate ion, and product, whereas these states cannot be studied by X-band, slow-passage methods. Simulation of the EPR spectra shows that binding of substrate or analogue causes a profound change in the electronic parameters of the Mn(II) ion. The axial zero-field splitting changes from [D] = 0.06 cm(-1) for substrate-free enzyme to 0.23 cm(-1) for fosfomycin-bound enzyme, 0.28 (1) cm(-1) for FosA with phosphate, and 0.27 (1) cm(-1) with product. Such a large zero-field splitting is uncommon for Mn(II). A simple ligand field analysis of this change indicates that binding of the phosphonate/phosphate group of substrate or analogue changes the electronic energy levels of the Mn(II) 3d orbitals by several thousand cm(-1), indicative of a significant change in the Mn(II) coordination sphere. Comparison with related enzymes (glyoxalase I and MnSOD) suggests that the change in the coordination environment on substrate binding may correspond to loss of the glutamate ligand.
Oxalate decarboxylase is a manganese-dependent enzyme that catalyzes the conversion of oxalate to formate and carbon dioxide. We have determined the structure of oxalate decarboxylase from Bacillus subtilis at 1.75 A resolution in the presence of formate. The structure reveals a hexamer with 32-point symmetry in which each monomer belongs to the cupin family of proteins. Oxalate decarboxylase is further classified as a bicupin because it contains two cupin folds, possibly resulting from gene duplication. Each oxalate decarboxylase cupin domain contains one manganese binding site. Each of the oxalate decarboxylase domains is structurally similar to oxalate oxidase, which catalyzes the manganese-dependent oxidative decarboxylation of oxalate to carbon dioxide and hydrogen peroxide. Amino acid side chains in the two metal binding sites of oxalate decarboxylase and the metal binding site of oxalate oxidase are very similar. Four manganese binding residues (three histidines and one glutamate) are conserved as well as a number of hydrophobic residues. The most notable difference is the presence of Glu333 in the metal binding site of the second cupin domain of oxalate decarboxylase. We postulate that this domain is responsible for the decarboxylase activity and that Glu333 serves as a proton donor in the production of formate. Mutation of Glu333 to alanine reduces the catalytic activity by a factor of 25. The function of the other domain in oxalate decarboxylase is not yet known.
Oxalate decarboxylase (OxDC) catalyzes a remarkable transformation in which the C-C bond in oxalate is cleaved to give carbon dioxide and formate. Like the native OxDC isolated from Aspergillus niger, the recombinant, bacterial OxDC from Bacillus subtilis contains Mn(II) in its resting state and requires catalytic dioxygen for activity. The most likely mechanism for OxDC-catalyzed C-C bond cleavage involves the participation of free radical intermediates, although this hypothesis remains to be unequivocally demonstrated. Efforts to delineate the catalytic mechanism have been placed on a firm foundation by the high-resolution crystal structure of recombinant, wild type B. subtilis OxDC (Anand et al., Biochemistry 2002, 41, 7659-7669). We now report the results of heavy-atom kinetic isotope effect measurements for the OxDC-catalyzed decarboxylation of oxalate, in what appear to be the first detailed studies of the mechanism employed by OxDC. At pH 4.2, the OxDC-catalyzed formation of formate and CO(2) have normal (13)C isotope effects of 1.5% +/- 0.1% and 0.5% +/- 0.1%, respectively, while the (18)O isotope effect on the formation of formate is 1.1% +/- 0.2% normal. Similarly at pH 5.7, the production of formate and CO(2) exhibits normal (13)C isotope effects of 1.9% +/- 0.1% and 0.8% +/- 0.1%, respectively, and the (18)O isotope effect on the formation of formate is 1.0% +/- 0.2% normal. The (18)O isotope effect on the formation of CO(2), however, 0.7% +/- 0.2%, is inverse at pH 5.7. These results are consistent with a multistep model in which a reversible, proton-coupled, electron transfer from bound oxalate to the Mn-enzyme gives an oxalate radical, which decarboxylates to yield a formate radical anion. Subsequent reduction and protonation of this intermediate then gives formate.
Superoxide dismutase protects organisms from potentially damaging oxygen radicals by catalyzing the disproportionation of superoxide to oxygen and hydrogen peroxide. We report the use of cryogenic temperatures to kinetically capture the sixth ligand bound to the active site of manganese superoxide dismutase (MnSOD). Synchrotron X-ray diffraction data was collected from Escherichia coli MnSOD crystals grown at pH 8.5 and cryocooled to 100 K. Structural refinement to 1.55 A resolution and close inspection of the active site revealed electron density for a sixth ligand that was interpreted to be a hydroxide ligand. The six-coordinate, distorted-octahedral geometry assumed during inhibition by hydroxide is compared to the room temperature, five-coordinate, trigonal bipyramidal active site determined with crystals grown from practically identical conditions. The gateway residues Tyr34, His30 and a tightly bound water molecule are implicated in closing-off the active site and blocking the escape route of the sixth ligand.
The cupin superfamily of proteins, named on the basis of a conserved beta-barrel fold ('cupa' is the Latin term for a small barrel), was originally discovered using a conserved motif found within germin and germin-like proteins from higher plants. Previous analysis of cupins had identified some 18 different functional classes that range from single-domain bacterial enzymes such as isomerases and epimerases involved in the modification of cell wall carbohydrates, through to two-domain bicupins such as the desiccation-tolerant seed storage globulins, and multidomain transcription factors including one linked to the nodulation response in legumes. Recent advances in comparative genomics, and the resolution of many more 3-D structures have now revealed that the largest subset of the cupin superfamily is the 2-oxyglutarate-Fe(2+) dependent dioxygenases. The substrates for this subclass of enzyme are many and varied and in total amount to probably 50-100 different biochemical reactions, including several involved in plant growth and development. Although the majority of enzymatic cupins contain iron as an active site metal, other members contain either copper, zinc, cobalt, nickel or manganese ions as a cofactor, with each cofactor allowing a different type of chemistry to occur within the conserved tertiary structure. This review discusses the range of structures and functions found in this most diverse of superfamilies.
The Mn(II) high-magnetic-field electron paramagnetic resonance (HFEPR) spectra of five different superoxide dismutases (SODs) were measured at 190 and 285 GHz. The native E. coli manganese SOD was found to be distinct from the other SODs by virtue of its large zero-field E-value. The two wild-type cambialistic proteins from Porphyromonas gingivalis and Rhodobacter capsulatus were also distinct. However, the Gly155Thr mutant of the P. gingivalis SOD changed the Mn(II) spectrum so that it closely resembled the spectrum of manganese reconstituted E. coli iron SOD. This observation paralleled enzyme activity measurements that show that this mutation causes the loss of activity with manganese and enhanced activity with iron indicating a conversion from a cambialistic to an iron-specific protein. The Mn(II) magnetic parameters were determined by simultaneously fitting the multifrequency data. Simulations were carried out by numerically diagonalizing the spin Hamiltonian and explicitly calculating all possible transition probabilities. The relationship between the Mn(II) zero-field interaction and structure of the metal binding site is also discussed.
Two different temperature dependences of the manganese(II) high-field electron paramagnetic resonance spectrum of manganese superoxide dismutase from E. coli were observed. In the 25-200 K range, the zero-field interaction steadily decreased with increasing temperature. This was likely due to the thermal expansion of the protein. From these results, it was possible to deduce an approximately r(-)(2.5) dependence of Mn(II) zero-field interaction on ligand-metal distance. At temperatures above 240 K, a distinct six-line component was detected, the amplitude of which decreased with increasing temperature. On the basis of similarities to the six-line spectrum observed for the azide-complexed E. coli manganese superoxide dismutase, the newly detected six-line spectrum was assigned to a hexacoordinate Mn(II) center resulting from the coordination of a nearby water molecule to the normally five-coordinate center. The changes in enthalpy and entropy characterizing the hexacoordinate-pentacoordinate equilibrium in the 240-268 K range were -5 kcal/mol and -24 cal/mol.K, respectively. The structural implications of the zero-field parameters of the newly found hexacoordinate form in comparison to those of the Mn(II) centers in concanavalin-A and manganese-containing R. spheroides photosynthetic reaction centers and the values predicted by the superposition model are discussed.
The properties of the Mn2+ site in the protein concanavalin A were investigated by single crystal W-band EPR/ENDOR (electron-nuclear double resonance) measurements. Initially, room temperature EPR measurements were carried out, one type of Mn2+ was identified and its zero-field splitting (ZFS) tensor was determined. In contrast, low temperature EPR measurements showed that two chemically inequivalent Mn2+ are present, Mn(A)2+ and Mn(B)2+, differing in their ZFS tensors. Variable temperature measurements revealed a two-site exchange between the two types. Although the dynamic process has been characterized by its rate and activation energy, just from the EPR measurements it was not possible to assign it to a specific residue. 1H ENDOR measurements of the water and imidazole protons, which are the main contributors to the ENDOR spectra, showed only one type of signals, namely, they were not sensitive to the differences between Mn(A)2+ and Mn(B)2+. 55Mn ENDOR spectra, which are dominated by the 55Mn isotropic hyperfine, a(iso), and the nuclear quadrupole interaction did sense the differences. Analysis of the spectra recorded with the magnetic field along the crystallographic axes showed that the two have the same a(iso) but different quadrupole tensors.
The effect of the substrate analogues azide and fluoride on the manganese(II) zero-field interactions of different manganese-containing superoxide dismutases (SOD) was measured using high-field electron paramagnetic resonance spectroscopy. Two cambialistic types, proteins that are active with manganese or iron, were studied along with two that were only active with iron and another that was only active with manganese. It was found that azide was able to coordinate directly to the pentacoordinated Mn(II) site of only the MnSOD from Escherichia coli and the cambialistic SOD from Rhodobacter capsulatus. The formation of a hexacoordinate azide-bound center was characterized by a large reduction in the Mn(II) zero-field interaction. In contrast, all five SODs were affected by fluoride, but no evidence for hexacoordinate Mn(II) formation was detected. For both azide and fluoride, the extent of binding was no more than 50%, implying either that a second binding site was present or that binding was self-limiting. Only the Mn(II) zero-field interactions of the two SODs that had little or no activity with manganese were found to be significantly affected by pH, the manganese-substituted iron superoxide dismutase from E. coli and the Gly155Thr mutant of the cambialistic SOD from Porphyromonas gingivalis. A model for anion binding and the observed pK involving tyrosine-34 is presented.
Phosphoenolpyruvate carboxykinase catalyzes the reversible decarboxylation of oxaloacetic acid with the concomitant transfer of the gamma-phosphate of GTP to form PEP and GDP as the first committed step of gluconeogenesis and glyceroneogenesis. The three structures of the mitochondrial isoform of PEPCK reported are complexed with Mn2+, Mn2+-PEP, or Mn2+-malonate-Mn2+ GDP and provide the first observations of the structure of the mitochondrial isoform and insight into the mechanism of catalysis mediated by this enzyme. The structures show the involvement of the hyper-reactive cysteine (C307) in the coordination of the active site Mn2+. Upon formation of the PEPCK-Mn2+-PEP or PEPCK-Mn2+-malonate-Mn2+ GDP complexes, C307 coordination is lost as the P-loop in which it resides adopts a different conformation. The structures suggest that stabilization of the cysteine-coordinated metal geometry holds the enzyme as a catalytically incompetent metal complex and may represent a previously unappreciated mechanism of regulation. A third conformation of the mobile P-loop in the PEPCK-Mn2+-malonate-Mn2+ GDP complex demonstrates the participation of a previously unrecognized, conserved serine residue (S305) in mediating phosphoryl transfer. The ordering of the mobile active site lid in the PEPCK-Mn2+-malonate-Mn2+ GDP complex yields the first observation of this structural feature and provides additional insight into the mechanism of phosphoryl transfer.
Superoxide dismutases (SOD) are important anti-oxidant enzymes that guard against superoxide toxicity. Various SOD enzymes have been characterized that employ either a copper, manganese, iron or nickel co-factor to carry out the disproportionation of superoxide. This review focuses on the copper and manganese forms, with particular emphasis on how the metal is inserted in vivo into the active site of SOD. Copper and manganese SODs diverge greatly in sequence and also in the metal insertion process. The intracellular copper SODs of eukaryotes (SOD1) can obtain copper post-translationally, by way of interactions with the CCS copper chaperone. CCS also oxidizes an intrasubunit disulfide in SOD1. Adventitious oxidation of the disulfide can lead to gross misfolding of immature forms of SOD1, particularly with SOD1 mutants linked to amyotrophic lateral sclerosis. In the case of mitochondrial MnSOD of eukaryotes (SOD2), metal insertion cannot occur post-translationally, but requires new synthesis and mitochondrial import of the SOD2 polypeptide. SOD2 can also bind iron in vivo, but is inactive with iron. Such metal ion mis-incorporation with SOD2 can become prevalent upon disruption of mitochondrial metal homeostasis. Accurate and regulated metallation of copper and manganese SOD molecules is vital to cell survival in an oxygenated environment.
Oxalate decarboxylase from Bacillus subtilis is composed of two cupin domains, each of which contains a Mn(II) ion coordinated by four identical conserved residues. The similarity between the two Mn(II) sites has precluded previous attempts to distinguish them spectroscopically and complicated efforts to understand the catalytic mechanism. A multifrequency cw-EPR approach has now enabled us to show that the two Mn ions can be distinguished on the basis of their differing fine structure parameters and to observe that acetate and formate bind to Mn(II) in only one of the two sites. The EPR evidence is consistent with the hypothesis that this Mn-binding site is located in the N-terminal domain, in agreement with predictions based on a recent X-ray structure of the enzyme.
Oxalate decarboxylase (OxDC) catalyzes the conversion of oxalate into CO(2) and formate using a catalytic mechanism that remains poorly understood. The Bacillus subtilis enzyme is composed of two cupin domains, each of which contains Mn(II) coordinated by four conserved residues. We have measured heavy atom isotope effects for a series of Bacillus subtilis OxDC mutants in which Arg-92, Arg-270, Glu-162, and Glu-333 are conservatively substituted in an effort to define the functional roles of these residues. This strategy has the advantage that observed isotope effects report directly on OxDC molecules in which the active site manganese center(s) is (are) catalytically active. Our results support the proposal that the N-terminal Mn-binding site can mediate catalysis, and confirm the importance of Arg-92 in catalytic activity. On the other hand, substitution of Arg-270 and Glu-333 affects both Mn(II) incorporation and the ability of Mn to bind to the OxDC mutants, thereby precluding any definitive assessment of whether the metal center in the C-terminal domain can also mediate catalysis. New evidence for the importance of Glu-162 in controlling metal reactivity has been provided by the unexpected observation that the E162Q OxDC mutant exhibits a significantly increased oxalate oxidase and a concomitant reduction in decarboxylase activities relative to wild type OxDC. Hence the reaction specificity of a catalytically active Mn center in OxDC can be perturbed by relatively small changes in local protein environment, in agreement with a proposal based on prior computational studies.
The synthesis, structural characterization, and electronic properties of a new series of high-spin six-coordinate dihalide mononuclear MnII complexes [Mn(tpa)X2] (tpa=tris-2-picolylamine; X=I (1), Br (2), and Cl (3)) are reported. The analysis of the crystallographic data shows that in all investigated complexes the manganese ion lies in the center of a distorted octahedron with a cis configuration of the halides imposed by the tpa ligand. By a multifrequency high-field electron paramagnetic resonance investigation (95-285 GHz), the electronic properties of 1-3 were determined (DI=-0.600, DBr=-0.360, DCl=+0.115 cm-1), revealing the important effect of (i) the nature of the halide and (ii) the configuration (cis/trans) of the two halides on the magnitude of D. The spin Hamiltonian parameters obtained by density functional theory calculations initiated from the crystal structure of 1-3 are in reasonable agreement with the experimental values. The absolute value of D is consistently overestimated, but the sign and the trend over the chemical series is well reproduced. Theoretical models (cis- and trans-[Mn(NH3)4X2], X=I, Br, Cl and F) have been used to investigate the different contributions to D and also to understand the origin of the experimentally observed changes in D within the series reported here. This study reveals that the spin-spin coupling contributions to the D tensor are non-negligible for the lighter halides (F, Cl) but become insignificant for the heavier halides (I, Br). The four different types of excitations involved in the spin-orbit coupling (SOC) part of the D tensor contribute with comparable magnitudes and opposing signs. The general trend observed for halide MnII complexes (DI>DBr>DCl) can be explained by the fact that the halide SOC dominates the D value in these systems with a major contribution arising from interference between metal- and halide-SOC contributions, which are proportional to the product of the SOC constants of Mn and X.
Superoxide dismutases (SODs) are proteins specialized in the depletion of superoxide from the cell through disproportionation of this anion into oxygen and hydrogen peroxide. We have used high-field electron paramagnetic resonance (HFEPR) to test a two-site binding model for the interaction of manganese-SODs with small anions. Because tyrosine-34 was thought to act as a gate between these two sites in this model, a tyrosine to phenylalanine mutant of the superoxide dismutase from R. capsulatus was constructed. Although the replacement slightly reduced activity, HFEPR measurements demonstrated that the electronic structure of the Mn(II) center was unaffected by the mutation. In contrast, the mutation had a profound effect on the interactions of fluoride and azide with the Mn(II) center. It was concluded that the absence of tyrosine-34 prevented the close approach of these anions to the metal ion. This mutation also enhanced the formation of a hexacoordinated water-Mn(II)SOD complex at low temperatures. Together, these results showed that the role of Y34 is unlikely to involve redox tuning but rather is important in regulating the equilibria between the anionic substrate in solution and the two binding sites near the metal. These observations further supported the originally proposed mutually exclusive two-binding-site model.
Oxalate decarboxylases and oxalate oxidases are members of the cupin superfamily of proteins that have many common features: a manganese ion with a common ligand set, the substrate oxalate, and dioxygen (as either a unique cofactor or a substrate). We have hypothesized that these enzymes share common catalytic steps that diverge when a carboxylate radical intermediate becomes protonated. The Bacillus subtilis decarboxylase has two manganese binding sites, and we proposed that Glu162 on a flexible lid is the site 1 general acid. We now demonstrate that a decarboxylase can be converted into an oxidase by mutating amino acids of the lid that include Glu162 with specificity switches of 282,000 (SEN161-3DAS), 275,000 (SENS161-4DSSN), and 225,000 (SENS161-4DASN). The structure of the SENS161-4DSSN mutant showed that site 2 was not affected. The requirement for substitutions other than of Glu162 was, at least in part, due to the need to decrease the Km for dioxygen for the oxidase reaction. Reversion of decarboxylase activity could be achieved by reintroducing Glu162 to the SENS161-4DASN mutant to give a relative specificity switch of 25,600. This provides compelling evidence for the crucial role of Glu162 in the decarboxylase reaction consistent with it being the general acid, for the role of the lid in controlling the Km for dioxygen, and for site 1 being the sole catalytically active site. We also report the trapping of carboxylate radicals produced during turnover of the mutant with the highest oxidase activity. Such radicals were also observed with the wild-type decarboxylase.
The Mn(II)/Mn(III) redox potentials and Mn(II) zero-field interactions were measured for a series of Mn(II)(4'-X-terpy)2 complexes as a function of the electron-donating properties of the 4'-substituent. The two properties were found to be linearly related to each other. Density functional calculations demonstrated that the variation of both the redox potential and zero-field interaction depended on the charge on the center nitrogen of the terpy ligand that was modulated by the 4'-substituent. A similar relationship was found for Mn(II) complexes formed with the N,N -bis(2-ethyl-5-methylimidazol-4-ylmethyl)aminopropane ligand, indicating that the relationship between the Mn(II) zero-field interaction and the Mn(II)/Mn(III) redox potential was likely to be general.
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