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ABSTRACT: Nitric oxide (NO) production by mammalian NO synthase (NOS) is believed to be regulated by the docking of the flavin mononucleotide (FMN) domain in one subunit of the dimer onto the heme domain of the adjacent subunit. Glu546, a conserved charged surface residue of the FMN domain in human inducible NOS (iNOS), is proposed to participate in the interdomain FMN/heme interactions [Sempombe et al. Inorg. Chem.2011, 50, 6869-6861]. In the present work, we further investigated the role of the E546 residue in the FMN-heme interdomain electron transfer (IET), a catalytically essential step in the NOS enzymes. Laser flash photolysis was employed to directly measure the FMN-heme IET kinetics for the E546N mutant of human iNOS oxygenase/FMN (oxyFMN) construct. The temperature dependence of the IET kinetics was also measured over the temperature range of 283-304 K to determine changes in the IET activation parameters. The E546N mutation was found to retard the IET by significantly raising the activation entropic barrier. Moreover, pulsed electron paramagnetic resonance data showed that the geometry of the docked FMN/heme complex in the mutant is basically the same as in the wild type construct, whereas the probability of formation of such a complex is about twice lower. These results indicate that the retarded IET in the E546N mutant is not caused by an altered conformation of the docked FMN/heme complex, but by a lower population of the IET-active conformation. In addition, the negative activation entropy of the mutant is still substantially lower than that of the holoenzyme. This supports a mechanism by which the FMN domain can modify the IET through altering probability of the docked state formation.
Inorganic Chemistry 04/2013; · 4.60 Impact Factor
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ABSTRACT: Nitric oxide synthase (NOS) catalyzes nitric oxide (NO) synthesis via a two-step process: L-arginine (L-Arg)→N-hydroxy-L-arginine (NOHA)→citrulline+NO. In the active site, the heme is coordinated by a thiolate ligand, which accepts a H-bond from a nearby tryptophan residue, W188. Mutation of W188 to histidine in murine inducible NOS was shown to retard NO synthesis and allow for transient accumulation of a new intermediate with a Soret maximum at 420 nm during the L-Arg hydroxylation reaction [Tejero et al. J. Biol. Chem. 283, 33498-507 (2008)]. However, crystallographic data showed that the mutation did not perturb the overall structure of the enzyme. To understand how the proximal mutation affects the oxygen chemistry, we carried out biophysical studies of the W188H mutant. Our stopped-flow data showed that the 420 nm intermediate was not only populated during the L-Arg reaction, but also during the NOHA reaction. Spectroscopic data and structural analysis demonstrated that the 420 nm intermediate is a hydroxide bound ferric heme species, which is stabilized by an out-of-plane distortion of the heme macrocycle and a cation radical centered on the tetrahydrobiopterin cofactor. The current data add important new insights into the previously proposed catalytic mechanism of NOS [Li et al, J. Am. Chem. Soc. 129, 6943-6951 (2007)].
Journal of Biological Chemistry 12/2012; · 4.77 Impact Factor
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ABSTRACT: Being an obligate aerobe, Mycobacterium tuberculosis faces a number of energetic challenges when it encounters hypoxia and environmental stress during intracellular infection. Consequently, it has evolved innovative strategies to cope with these unfavorable conditions. Here, we report a novel flavohemoglobin (MtbFHb) from M. tuberculosis that exhibits unique features within its heme and reductase domains distinct from conventional FHbs, including the absence of the characteristic hydrogen bonding interactions within the proximal heme pocket and mutations in the FAD and NADH binding regions of the reductase domain. In contrast to conventional FHbs, it has a hexacoordinate low-spin heme with a proximal histidine ligand lacking imidazolate character and a distal heme pocket with a relatively low electrostatic potential. Additionally, MtbFHb carries a new FAD binding site in its reductase domain similar to that of D-lactate dehydrogenase (D-LDH). When overexpressed in Escherichia coli or Mycobacterium smegmatis, MtbFHb remained associated with the cell membrane and exhibited D-lactate:phenazine methosulfate reductase activity and oxidized D-lactate into pyruvate by converting the heme iron from Fe(3+) to Fe(2+) in a FAD-dependent manner, indicating electron transfer from D-lactate to the heme via FAD cofactor. Under oxidative stress, MtbFHb-expressing cells exhibited growth advantage with reduced levels of lipid peroxidation. Given the fact that D-lactate is a byproduct of lipid peroxidation and that M. tuberculosis lacks the gene encoding D-LDH, we propose that the novel D-lactate metabolizing activity of MtbFHb uniquely equips M. tuberculosis to balance the stress level by protecting the cell membrane from oxidative damage via cycling between the Fe(3+)/Fe(2+) redox states.
Journal of Biological Chemistry 03/2012; 287(20):16435-46. · 4.77 Impact Factor
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ABSTRACT: The critical role of the ferryl intermediate in catalyzing the oxygen chemistry of monooxygenases, oxidases, or peroxidases has been known for decades. In contrast, its involvement in heme-based dioxygenases, such as human indoleamine 2,3-dioxygenase (hIDO), was not recognized until recently. In this study, H(2)O(2) was used as a surrogate to generate the ferryl intermediate of hIDO. Spectroscopic data demonstrate that the ferryl species is capable of oxidizing azinobis(3-ethylbenzothiazoline-6-sulfonic acid) but not L-Trp. Kinetic studies reveal that the conversion of the ferric enzyme to the ferryl intermediate facilitates the L-Trp binding rate by >400-fold; conversely, L-Trp binding to the enzyme retards the peroxide reaction rate by ∼9-fold, because of the significant elevation of the entropic barrier. The unfavorable entropic factor for the peroxide reaction highlights the scenario that the structure of hIDO is not optimized for utilizing H(2)O(2) as a co-substrate for oxidizing L-Trp. Titration studies show that the ferryl intermediate possesses two substrate-binding sites with a K(d) of 0.3 and 440 μM and that the electronic properties of the ferryl moiety are sensitive to the occupancy of the two substrate-binding sites. The implications of the data are discussed in the context of the structural and functional relationships of the enzyme.
Journal of Biological Chemistry 06/2011; 286(24):21220-30. · 4.77 Impact Factor
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ABSTRACT: Oxygen affinity in heme-containing proteins is determined by a number of factors, such as the nature and conformation of the distal residues that stabilize the heme bound-oxygen via hydrogen-bonding interactions. The truncated hemoglobin III from Campylobacter jejuni (Ctb) contains three potential hydrogen-bond donors in the distal site: TyrB10, TrpG8, and HisE7. Previous studies suggested that Ctb exhibits an extremely slow oxygen dissociation rate due to an interlaced hydrogen-bonding network involving the three distal residues. Here we have studied the structural and kinetic properties of the G8(WF) mutant of Ctb and employed state-of-the-art computer simulation methods to investigate the properties of the O(2) adduct of the G8(WF) mutant, with respect to those of the wild-type protein and the previously studied E7(HL) and/or B10(YF) mutants. Our data indicate that the unique oxygen binding properties of Ctb are determined by the interplay of hydrogen-bonding interactions between the heme-bound ligand and the surrounding TyrB10, TrpG8, and HisE7 residues.
Biochemistry 05/2011; 50(19):3946-56. · 3.42 Impact Factor
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ABSTRACT: Flavohemoglobins (flavoHbs) constitute a distinct class of chimeric hemoglobins in which a globin domain is coupled with a ferredoxin reductase such as FAD- and NADH-binding modules. Structural features and active site of heme and reductase domains are highly conserved in various flavoHbs. A new class of flavoHbs, displaying crucial differences in functionally conserved regions of heme and reductase domains, have been identified in mycobacteria. Mining of microbial genome data indicated that the occurrence of such flavoHbs might be restricted to a small group of microbes unlike conventional flavoHbs that are widespread among prokaryotes and lower eukaryotes. One of the representative flavoHbs of this class, encoded by Rv0385 gene (MtbFHb) of Mycobacterium tuberculosis, has been cloned, expressed, and characterized. The ferric and deoxy spectra of MtbFHb displayed a hexacoordinate state indicating that its distal site may be occupied by an intrinsic amino acid or an external ligand and it may not be involved in nitric oxide detoxification. Phylogenetic analysis revealed that mycobacterial flavoHbs constitute a separate cluster distinct from conventional flavoHbs and may have novel function(s).
International Union of Biochemistry and Molecular Biology Life 05/2011; 63(5):337-45. · 3.51 Impact Factor
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ABSTRACT: Human indoleamine 2,3-dioxygenase (hIDO), a monomeric heme enzyme, catalyzes the oxidative degradation of L-tryptophan (L-Trp) and other indoleamine derivatives. Its activity follows typical Michaelis-Menten behavior only for L-Trp concentrations up to 50 μM; a further increase in the concentration of L-Trp causes a decrease in the activity. This substrate inhibition of hIDO is a result of the binding of a second L-Trp molecule in an inhibitory substrate binding site of the enzyme. The molecular details of the reaction and the inhibition are not yet known. In the following, we summarize the present knowledge about this heme enzyme.
International Union of Biochemistry and Molecular Biology Life 03/2011; 63(3):153-9. · 3.51 Impact Factor
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ABSTRACT: Human indoleamine 2,3-dioxygenase (hIDO) is an intracellular heme-containing enzyme, which catalyzes the initial and rate-determining step of l-tryptophan (l-Trp) metabolism via the kynurenine pathway in nonhepatic tissues. Steady-state kinetic data showed that hIDO exhibits substrate inhibition behavior, implying the existence of a second substrate binding site in the enzyme, although so far there is no direct evidence supporting it. The kinetic data also revealed that the K(m) of l-Trp (15 microM) is approximately 27-fold lower than the K(d) of l-Trp (0.4 mM) for the ligand-free ferrous enzyme, suggesting that O(2) binding proceeds l-Trp binding during the catalytic cycle. With cyanide as a structural probe, we have investigated the thermodynamic and kinetic parameters associated with ligand and substrate binding to hIDO. Equilibrium titration studies show that the cyanide adduct is capable of binding two l-Trp molecules, with K(d) values of 18 microM and 26 mM. The data offer the first direct evidence of the second substrate binding site in hIDO. Kinetic studies demonstrate that prebinding of l-Trp to the enzyme retards cyanide binding by approximately 13-fold, while prebinding of cyanide to the enzyme facilitates l-Trp binding by approximately 22-fold. The data support the view that during the active turnover of the enzyme it is kinetically more favored to bind O(2) prior to l-Trp.
Biochemistry 06/2010; 49(24):5028-34. · 3.42 Impact Factor
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Mark Shepherd,
Vladimir Barynin, Changyuan Lu,
Paul V. Bernhardt,
Guanghui Wu,
Syun-Ru Yeh,
Tsuyoshi Egawa,
Svetlana E. Sedelnikova,
David W. Rice,
Jayne Louise Wilson,
Robert K. Poole
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ABSTRACT: The food-borne pathogen Campylobacter jejuni possesses a single-domain globin (Cgb) whose role in detoxifying nitric oxide has been unequivocally demonstrated through
genetic and molecular approaches. The x-ray structure of cyanide-bound Cgb has been solved to a resolution of 1.35 Å. The
overall fold is a classic three-on-three α-helical globin fold, similar to that of myoglobin and Vgb from Vitreoscilla stercoraria. However, the D region (defined according to the standard globin fold nomenclature) of Cgb adopts a highly ordered α-helical
conformation unlike any previously characterized members of this globin family, and the GlnE7 residue has an unexpected role
in modulating the interaction between the ligand and the TyrB10 residue. The proximal hydrogen bonding network in Cgb demonstrates
that the heme cofactor is ligated by an imidazolate, a characteristic of peroxidase-like proteins. Mutation of either proximal
hydrogen-bonding residue (GluH23 or TyrG5) results in the loss of the high frequency νFe-His stretching mode (251 cm−1), indicating that both residues are important for maintaining the anionic character of the proximal histidine ligand. Cyanide
binding kinetics for these proximal mutants demonstrate for the first time that proximal hydrogen bonding in globins can modulate
ligand binding kinetics at the distal site. A low redox midpoint for the ferrous/ferric couple (−134 mV versus normal hydrogen electrode at pH 7) is consistent with the peroxidase-like character of the Cgb active site. These data provide
a new insight into the mechanism via which Campylobacter may survive host-derived nitrosative stress.
Journal of Biological Chemistry 04/2010; 285(17):12747-12754. · 4.77 Impact Factor
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ABSTRACT: Cytochrome c oxidase (CcO), the terminal enzyme in the mitochondrial respiratory chain, catalyzes the four-electron reduction of dioxygen to water in a binuclear center comprised of a high-spin heme (heme a(3)) and a copper atom (Cu(B)) coordinated by three histidine residues. As a minimum model for CcO, a mutant of sperm whale myoglobin, named Cu(B)Mb, has been engineered, in which a copper atom is held in the distal heme pocket by the native E7 histidine and two nonnative histidine residues. In this work, the role of the copper in regulating ligand binding in Cu(B)Mb was investigated. Resonance Raman studies show that the presence of copper in CO-bound Cu(B)Mb leads to a CcO-like distal heme pocket. Stopped-flow data show that, upon the initiation of the CO binding reaction, the ligand first binds to the Cu(+); it subsequently transfers from Cu(+) to Fe(2+) in an intramolecular process, similar to that reported for CcO. The high CO affinity toward Cu(+) and the slow intramolecular CO transfer rate between Cu(+) and Fe(2+) in the Cu(B)Mb/Cu(+) complex are analogous to those in Thermus thermophilus CcO (TtCcO) but distinct from those in bovine CcO (bCcO). Additional kinetic studies show that, upon photolysis of the NO-bound Cu(B)Mb/Cu(+) complex, the photolyzed ligand transiently binds to Cu(+) and subsequently rebinds to Fe(2+), accounting for the 100% geminate recombination yield, similar to that found in TtCcO. The data demonstrate that the Cu(B)Mb/Cu(+) complex reproduces essential structural and kinetic features of CcO and that the complex is more akin to TtCcO than to bCcO.
Journal of the American Chemical Society 02/2010; 132(5):1598-605. · 9.91 Impact Factor
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Mark Shepherd,
Vladimir Barynin, Changyuan Lu,
Paul V Bernhardt,
Guanghui Wu,
Syun-Ru Yeh,
Tsuyoshi Egawa,
Svetlana E Sedelnikova,
David W Rice,
Jayne Louise Wilson,
Robert K Poole
[show abstract]
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ABSTRACT: The food-borne pathogen Campylobacter jejuni possesses a single-domain globin (Cgb) whose role in detoxifying nitric oxide has been unequivocally demonstrated through genetic and molecular approaches. The x-ray structure of cyanide-bound Cgb has been solved to a resolution of 1.35 A. The overall fold is a classic three-on-three alpha-helical globin fold, similar to that of myoglobin and Vgb from Vitreoscilla stercoraria. However, the D region (defined according to the standard globin fold nomenclature) of Cgb adopts a highly ordered alpha-helical conformation unlike any previously characterized members of this globin family, and the GlnE7 residue has an unexpected role in modulating the interaction between the ligand and the TyrB10 residue. The proximal hydrogen bonding network in Cgb demonstrates that the heme cofactor is ligated by an imidazolate, a characteristic of peroxidase-like proteins. Mutation of either proximal hydrogen-bonding residue (GluH23 or TyrG5) results in the loss of the high frequency nu(Fe-His) stretching mode (251 cm(-1)), indicating that both residues are important for maintaining the anionic character of the proximal histidine ligand. Cyanide binding kinetics for these proximal mutants demonstrate for the first time that proximal hydrogen bonding in globins can modulate ligand binding kinetics at the distal site. A low redox midpoint for the ferrous/ferric couple (-134 mV versus normal hydrogen electrode at pH 7) is consistent with the peroxidase-like character of the Cgb active site. These data provide a new insight into the mechanism via which Campylobacter may survive host-derived nitrosative stress.
Journal of Biological Chemistry 02/2010; 285(17):12747-54. · 4.77 Impact Factor
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ABSTRACT: Human indoleamine 2,3-dioxygenase (hIDO) is an intracellular heme-containing enzyme, which catalyzes the initial and rate-determining step of L-tryptophan (L-Trp) metabolism via the kynurenine pathway. Due to its immunosuppressive function, hIDO has been recognized as an important drug target for cancer. Here we report evidence supporting the presence of an inhibitory substrate binding site (S(i)) in hIDO that is capable of binding molecules with a wide variety of structures, including substrates (L-Trp and 1-methyl-L-tryptophan), an effector (3-indole ethanol), and an uncompetitive inhibitor (Mitomycin C). The data offer useful guidelines for future development of more potent hIDO inhibitors; they also call for the re-evaluation of the action mechanism of Mitomycin C (MtoC), a widely used antitumor chemotherapeutic agent.
Journal of the American Chemical Society 10/2009; 131(36):12866-7. · 9.91 Impact Factor
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ABSTRACT: In contrast to the wide spectrum of cytochrome P450 monooxygenases, there are only 2 heme-based dioxygenases in humans: tryptophan dioxygenase (hTDO) and indoleamine 2,3-dioxygenase (hIDO). hTDO and hIDO catalyze the same oxidative ring cleavage reaction of L-tryptophan to N-formyl kynurenine, the initial and rate-limiting step of the kynurenine pathway. Despite immense interest, the mechanism by which the 2 enzymes execute the dioxygenase reaction remains elusive. Here, we report experimental evidence for a key ferryl intermediate of hIDO that supports a mechanism in which the 2 atoms of dioxygen are inserted into the substrate via a consecutive 2-step reaction. This finding introduces a paradigm shift in our understanding of the heme-based dioxygenase chemistry, which was previously believed to proceed via simultaneous incorporation of both atoms of dioxygen into the substrate. The ferryl intermediate is not observable during the hTDO reaction, highlighting the structural differences between the 2 dioxygenases, as well as the importance of stereoelectronic factors in modulating the reactions.
Proceedings of the National Academy of Sciences 09/2009; 106(41):17371-6. · 9.68 Impact Factor
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ABSTRACT: Human indoleamine 2,3-dioxygenase (hIDO), a monomeric heme enzyme, catalyzes the oxidative degradation of L-Trp and other indoleamine derivatives. Using Fourier transform infrared and optical absorption spectroscopy, we have investigated the interplay between ferrous hIDO, the ligand analog CO, and the physiological substrate L-Trp. These data provide the long sought evidence for two distinct L-Trp binding sites. Upon photodissociation from the heme iron at T > 200 K, CO escapes into the solvent. Concomitantly, L-Trp exits the active site and, depending on the l-Trp concentration, migrates to a secondary binding site or into the solvent. Although L-Trp is spectroscopically silent at this site, it is still noticeable due to its pronounced effect on the CO association kinetics, which are significantly slower than those of L-Trp-free hIDO. L-Trp returns to its initial site only after CO has rebound to the heme iron.
Journal of Biological Chemistry 09/2009; 284(46):31548-54. · 4.77 Impact Factor
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ABSTRACT: Resonance Raman studies show that the heme-bound CO in trHbO, a truncated-II hemoglobin from Mycobacterium tuberculosis, is exposed to an environment with a positive electrostatic potential. The mutation of Trp(G8), an absolutely conserved residue in group II and III truncated hemoglobins, to Phe introduces two new Fe-CO conformers, both of which exhibit reduced electrostatic potentials. Computer simulations reveal that the structural perturbation is a result of the increased flexibility of the Tyr(CD1) and Leu(E11) side chains due to the reduction of the size of the G8 residue. Laser flash photolysis studies show that the G8 mutation induces 1) the presence of two new geminate recombination phases, one with a rate faster than the time resolution of our instrument and the other with a rate 13-fold slower than that of the wild type protein, and 2) the reduction of the total geminate recombination yield from 86 to 62% and the increase in the bimolecular recombination rate by a factor of 530. Computer simulations uncover that the photodissociated ligand migrates between three distal temporary docking sites before it subsequently rebinds to the heme iron or ultimately escapes into the solvent via a hydrophobic tunnel. The calculated energy profiles associated with the ligand migration processes are in good agreement with the experimental observations. The results highlight the importance of the Trp(G8) in regulating ligand migration in trHbO, underscoring its pivotal role in the structural and functional properties of the group II and III truncated hemoglobins.
Journal of Biological Chemistry 12/2008; 284(5):3106-16. · 4.77 Impact Factor
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Journal of the American Chemical Society 10/2008; · 9.91 Impact Factor
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ABSTRACT: Femtosecond coherence spectroscopy is used to probe the low-frequency (20-200 cm(-1)) vibrational modes of heme proteins in solution. Horseradish peroxidase (HRP), myoglobin (Mb), and Campylobacter jejuni globin (Cgb) are compared and significant differences in the coherence spectra are revealed. It is concluded that hydrogen bonding and ligand charge do not strongly affect the low-frequency coherence spectra and that protein-specific deformations of the heme group lower its symmetry and control the relative spectral intensities. Such deformations potentially provide a means for proteins to tune heme reaction coordinates, so that they can perform a broad array of specific functions. Native HRP displays complex spectral behavior above approximately 50 cm(-1) and very weak activity below approximately 50 cm(-1). Binding of the substrate analog, benzhydroxamic acid, leads to distinct changes in the coherence and Raman spectra of HRP that are consistent with the stabilization of a heme water ligand. The CN derivatives of the three proteins are studied to make comparisons under conditions of uniform heme coordination and spin-state. MbCN is dominated by a doming mode near 40 cm(-1), while HRPCN displays a strong oscillation at higher frequency (96 cm(-1)) that can be correlated with the saddling distortion observed in the X-ray structure. In contrast, CgbCN displays low-frequency coherence spectra that contain strong modes near 30 and 80 cm(-1), probably associated with a combination of heme doming and ruffling. HRPNO displays a strong doming mode near 40 cm(-1) that is activated by photolysis. The damping of the coherent motions is significantly reduced when the heme is shielded from solvent fluctuations by the protein material and reduced still further when T approximately < 50 K, as pure dephasing processes due to the protein-solvent phonon bath are frozen out.
Journal of the American Chemical Society 04/2008; 130(15):5231-44. · 9.91 Impact Factor
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ABSTRACT: Three groups of hemoglobins (Hbs) have been identified in unicellular organisms: (1) the truncated Hbs (trHb) that display a novel two-over-two alpha-helical structure, (2) the flavohemoglobins (FHb) that comprise a Hb domain with a classical three-over-three alpha-helical structure and a covalently attached flavin-containing reductase domain, and (3) the single-domain Hbs (sdHb) that exhibit high sequence homology and structural similarity to the Hb domain of FHb. On the basis of phylogenetic analysis, the trHbs can be further divided into three subgroups: TrHb-I, TrHb-II, and TrHb-III. The various classes of Hbs may coexist in the same organism, suggesting distinct functions for each class of Hb. This chapter reviews the structural and functional properties of a TrHb-I (trHbN) and a TrHb-II (trHbO) from Mycobacterium tuberculosis, as well as a TrHb-III (trCtb) and a sdHb (Cgb) from Campylobacter jejuni on the basis of resonance Raman spectroscopic studies.
Methods in Enzymology 02/2008; 437:255-86. · 2.04 Impact Factor
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ABSTRACT: Campylobacter jejuni contains two globins, a truncated hemoglobin, Ctb, and a single domain hemoglobin, Cgb. The physiological function of Ctb remains unclear, whereas Cgb has been linked to NO detoxification. With resonance Raman scattering, the iron-histidine stretching mode of Cgb was identified at 251 cm(-1). This frequency is unusually high, suggesting an imidazolate character of the proximal histidine as a result of the H-bonding network linking the catalytic triad involving the F8His, H23Glu, and G5Tyr residues. In the CO-complex, two conformers were identified with the nuC-O/nuFe-CO at 529/1914 cm(-1) and 492/1963 cm(-1). The former is assigned to a "closed" conformation, in which the heme-bound CO is stabilized by the H-bond(s) donated from the B10Tyr-E7Gln residues, whereas the latter is assigned to an "open" conformer, in which the H-bonding interaction is absent. The presence of the two alternative conformations demonstrates the plasticity of the protein matrix. In the O2-complex, the iron-O2 stretching frequency was identified at 554 cm(-1), which is unusually low, indicating that the heme-bound O2 is stabilized by strong H-bond(s) donated by the B10Tyr-E7Gln residues. This scenario is consistent with its low O2 off-rate (0.87 s(-1)). Taken together the data suggest that the NO-detoxifying activity of Cgb is facilitated by the imidazolate character of the proximal F8His and the distal positive polar environment provided by the B10Tyr-E7Gln. They may offer electronic "push" and "pull," respectively, for the O-O bond cleavage reaction required for the isomerization of the presumed peroxynitrite intermediate to the product, nitrate.
Journal of Biological Chemistry 09/2007; 282(35):25917-28. · 4.77 Impact Factor
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ABSTRACT: Campylobacter jejuni contains two hemoglobins, Cgb and Ctb. Cgb has been suggested to perform an NO detoxification reaction to protect the bacterium against NO attack. On the other hand, the physiological function of Ctb, a class III truncated hemoglobin, remains unclear. By using CO as a structural probe, resonance Raman data show that the distal heme pocket of Ctb exhibits a positive electrostatic potential. In addition, two ligand-related vibrational modes, nu(Fe-O(2)) and nu(O-O), were identified in the oxy derivative, with frequencies at 542 and 1132 cm(-1), respectively, suggesting the presence of an intertwined H-bonding network surrounding the heme-bound ligand, which accounts for its unusually high oxygen affinity (222 microm(-1)). Mutagenesis studies of various distal mutants suggest that the heme-bound dioxygen is stabilized by H-bonds donated from the Tyr(B10) and Trp(G8) residues, which are highly conserved in the class III truncated hemoglobins; furthermore, an additional H-bond donated from the His(E7) to the Tyr(B10) further regulates these H-bonding interactions by restricting the conformational freedom of the phenolic side chain of the Tyr(B10). Taken together, the data suggest that it is the intricate balance of the H-bonding interactions that determines the unique ligand binding properties of Ctb. The extremely high oxygen affinity of Ctb makes it unlikely to function as an oxygen transporter; on the other hand, the distal heme environment of Ctb is surprisingly similar to that of cytochrome c peroxidase, suggesting a role of Ctb in performing a peroxidase or P450-type of oxygen chemistry.
Journal of Biological Chemistry 06/2007; 282(18):13627-36. · 4.77 Impact Factor
