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PubMed1996

Factors influencing redox thermodynamics and electron self-exchange for the [Fe4S4] cluster in Chromatium vinosum high potential iron protein: the role of core aromatic residues in defining cluster redox chemistry.

The roles of aromatic core residues in regulating the reduction potential, the enthalpy and entropy of reduction, and the self-exchange rate constants for electron-transfer reactions for the prosthetic [Fe4S4]3+/2+ cluster of Chromatium vinosum high potential iron protein (HiPIP) have been addressed by a combination of site-directed mutagenesis, high field NMR (EXSY) experiments, and variable temperature spectrochemical redox titration measurements. Minimal changes are observed following nonconservative mutation of residues Tyr19, Phe48, and Phe66. Apparently these hydrophobic residues play only a minor role in defining the electronic properties of the cluster. These data support a model, first defined from results obtained on Tyr19 mutant HiPIP's [Agarwal, A., Li, D., & Cowan, J.A. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 9440-9444], in which the aromatic core restricts solvent accessibility and thereby stabilizes the oxidized [Fe4S4]3+ cluster.

Bacterial Proteins↗
PubMed1998

Crystal structure and possible dimerization of the high-potential iron-sulfur protein from Chromatium purpuratum.

The crystal structure of the high-potential iron-sulfur protein (HiPIP) isolated from Chromatium purpuratum is reported at 2.7 A resolution. The three HiPIP molecules in the asymmetric unit of the crystals form one and one-half dimers. Two molecules are related by a noncrystallographic symmetry rotation of approximately 175 degrees with negligible translation along the dyad axis. The third molecule in the asymmetric unit also forms a dimer with a second HiPIP molecule across the crystallographic 2-fold symmetry axis. The Fe4S4 clusters in both the crystallographic and noncrystallographic dimers are separated by approximately 13.0 A. Solution studies give mixed results regarding the oligomeric state of the C. purpuratum HiPIP. A comparison with crystal structures of HiPIPs from other species shows that HiPIP tends to associate rather nonspecifically about a conserved, relatively hydrophobic surface patch to form dimers.

Amino Acid Sequence↗
PubMed1999

PHA synthase from chromatium vinosum: cysteine 149 is involved in covalent catalysis.

Polyhydroxyalkanoate synthase (PHA) from Chromatium vinosum catalyzes the conversion of 3-hydroxybutyryl-CoA (HB-CoA) to polyhydroxybutyrate (PHB) and CoA. The synthase is composed of a approximately 1:1 mixture of two subunits, PhaC and PhaE. Size-exclusion chromatography indicates that in solution PhaC and PhaE exist as large molecular weight aggregates. The holo-enzyme, PhaEC, has a specific activity of 150 units/mg. Each subunit was cloned, expressed, and purified as a (His)6-tagged construct. The PhaC-(His)6 protein catalyzed polymerization with a specific activity of 0.9 unit/mg; the PhaE-(His)6 protein was inactive (specific activity <0.001 unit/mg). Addition of PhaE-(His)6 to PhaC-(His)6 increased the activity several 100-fold. To investigate the priming step of the polymerization process, the PhaEC was incubated with a trimer of HB-CoA in which the terminal hydroxyl was replaced with tritium ([3H]-sT-CoA). After Sephadex G50 chromatography, the synthase contained approximately 0.25 equiv of the labile label per PhaC. Incubation of [3H]-sT-synthase with HB-CoA resulted in production of [3H]-polymer. Digestion of [3H]-sT-synthase with trypsin and HPLC analysis resulted in isolation of three labeled peptides. Sequencing by ion trap mass spectrometry showed that they were identical and that they each contained an altered cysteine (C149). One peptide contained the [3H]-sT while the other two contained, in addition to the [3H]-sT, one and two additional monomeric HBs, respectively. Mutation of C149 to alanine gave inactive synthase. The remaining two cysteines of PhaC, 292 and 130, were also mutated to alanine. The former had wild-type (wt) activity, while the latter had 0.004 wt % activity and was capable of making polymer. A mechanism is proposed in which PhaC contains all the elements essential for catalysis and the polymerization proceeds by covalent catalysis using C149 and potentially C130.

Acyltransferases↗
PubMed1999

Unusual NMR, EPR, and Mössbauer properties of Chromatium vinosum 2[4Fe-4S] ferredoxin.

The ferredoxin from Chromatium vinosum (CvFd) exhibits sequence and structure peculiarities. Its two Fe4S4(SCys)4 clusters have unusually low potential transitions that have been unambiguously assigned here through NMR, EPR, and Mössbauer spectroscopy in combination with site-directed mutagenesis. The [4Fe-4S]2+/1+ cluster (cluster II) whose coordination sphere includes a two-turn loop between cysteines 40 and 49 was reduced by dithionite with an E degrees ' of -460 mV. Its S = 1/2 EPR signal was fast relaxing and severely broadened by g-strain, and its Mössbauer spectra were broad and unresolved. These spectroscopic features were sensitive to small perturbations of the coordination environment, and they were associated with the particular structural elements of CvFd, including the two-turn loop between two ligands and the C-terminal alpha-helix. Bulk reduction of cluster I (E degrees ' = -660 mV) was not possible for spectroscopic studies, but the full reduction of the protein was achieved by replacing valine 13 with glycine due to an approximately 60 mV positive shift of the potential. At low temperatures, the EPR spectrum of the fully reduced protein was typical of two interacting S = 1/2 [4Fe-4S]1+ centers, but because the electronic relaxation of cluster I is much slower than that of cluster II, the resolved signal of cluster I was observed at temperatures above 20 K. Contact-shifted NMR resonances of beta-CH2 protons were detected in all combinations of redox states. These results establish that electron transfer reactions involving CvFd are quantitatively different from similar reactions in isopotential 2[4Fe-4S] ferredoxins. However, the reduced clusters of CvFd have electronic distributions that are similar to those of clusters coordinated by the CysIxxCysIIxxCysIII.CysIVP sequence motif found in other ferredoxins with different biochemical properties. In all these cases, the electron added to the oxidized clusters is mainly accommodated in the pair of iron ions coordinated by CysII and CysIV.

Bacterial Proteins↗
PubMed1999

Catalytic electron transport in Chromatium vinosum [NiFe]-hydrogenase: application of voltammetry in detecting redox-active centers and establishing that hydrogen oxidation is very fast even at potentials close to the reversible H+/H2 value.

The nickel-iron hydrogenase from Chromatium vinosum adsorbs at a pyrolytic graphite edge-plane (PGE) electrode and catalyzes rapid interconversion of H(+)((aq)) and H(2) at potentials expected for the half-cell reaction 2H(+) right arrow over left arrow H(2), i.e., without the need for overpotentials. The voltammetry mirrors characteristics determined by conventional methods, while affording the capabilities for exquisite control and measurement of potential-dependent activities and substrate-product mass transport. Oxidation of H(2) is extremely rapid; at 10% partial pressure H(2), mass transport control persists even at the highest electrode rotation rates. The turnover number for H(2) oxidation lies in the range of 1500-9000 s(-)(1) at 30 degrees C (pH 5-8), which is significantly higher than that observed using methylene blue as the electron acceptor. By contrast, proton reduction is slower and controlled by processes occurring in the enzyme. Carbon monoxide, which binds reversibly to the NiFe site in the active form, inhibits electrocatalysis and allows improved definition of signals that can be attributed to the reversible (non-turnover) oxidation and reduction of redox centers. One signal, at -30 mV vs SHE (pH 7.0, 30 degrees C), is assigned to the [3Fe-4S](+/0) cluster on the basis of potentiometric measurements. The second, at -301 mV and having a 1. 5-2.5-fold greater amplitude, is tentatively assigned to the two [4Fe-4S](2+/+) clusters with similar reduction potentials. No other redox couples are observed, suggesting that these two sets of centers are the only ones in CO-inhibited hydrogenase capable of undergoing simple rapid cycling of their redox states. With the buried NiFe active site very unlikely to undergo direct electron exchange with the electrode, at least one and more likely each of the three iron-sulfur clusters must serve as relay sites. The fact that H(2) oxidation is rapid even at potentials nearly 300 mV more negative than the reduction potential of the [3Fe-4S](+/0) cluster shows that its singularly high equilibrium reduction potential does not compromise catalytic efficiency.

Bacterial Proteins↗
PubMed2000

Lipases provide a new mechanistic model for polyhydroxybutyrate (PHB) synthases: characterization of the functional residues in Chromatium vinosum PHB synthase.

Polyhydroxybutyrate (PHB) synthases catalyze the conversion of beta-hydroxybutyryl coenzyme A (HBCoA) to PHB. These enzymes require an active site cysteine nucleophile for covalent catalysis. A protein BLASTp search using the Class III Chromatium vinosum synthase sequence reveals high homology to prokaryotic lipases whose crystal structures are known. The homology is very convincing in the alpha-beta-elbow (with the active site nucleophile)-alpha-beta structure, residues 131-175 of the synthase. A conserved histidine of the Class III PHB synthases aligns with the active site histidine of the lipases using the ClustalW algorithm. This is intriguing as this histidine is approximately 200 amino acids removed in sequence space from the catalytic nucleophile. Different threading algorithms suggest that the Class III synthases belong to the alpha/beta hydrolase superfamily which includes prokaryotic lipases. Mutagenesis studies were carried out on C. vinosum synthase C149, H331, H303, D302, and C130 residues. These studies reveal that H331 is the general base catalyst that activates the nucleophile, C149, for covalent catalysis. The model indicates that C130 is not involved in catalysis as previously proposed [Müh, U., Sinskey, A. J., Kirby, D. P., Lane, W. S., and Stubbe, J. (1999) Biochemistry 38, 826-837]. Studies with D302 mutants suggest D302 functions as a general base catalyst in activation of the 3-hydroxyl of HBCoA (or a hydroxybutyrate acyl enzyme) for nucleophilic attack on the covalently linked thiol ester intermediate. The relationship of the lipase model to previous models based on fatty acid synthases is discussed.

Acyltransferases↗
PubMed2003

Reconstitution of carotenoids into the light-harvesting complex B800-850 of Chromatium minutissimum.

Chromatophores and peripheral light-harvesting complexes B800-850 with a trace of carotenoids were isolated from Chromatium minutissimum cells in which carotenoid biosynthesis was inhibited by diphenylamine. Three methods previously used for the reconstitution of carotenoids into either the light-harvesting (LH1) type complexes or reaction centers (RC) of carotenoidless mutants were examined for the possibility of carotenoid reconstitution into the carotenoid depleted chromatophores. All these methods were found to be unsuitable because carotenoid depleted complex B800-850 from Chr. minutissimum is characterized by high lability. We have developed a novel method maintaining the native structure of the complexes and allowing reconstitution of up to 80% of the carotenoids as compared to the control. The reconstituted complex has a similar CD spectrum in the carotenoid region as the control, and its structure restores its stability. These data give direct proof for the structural role of carotenoids in bacterial photosynthesis.

Bacterial Proteins↗
PubMed1972

Coproporphyrinogenase activities in extracts of Rhodopseudomonas spheroides and Chromatium strain D.

1. The anaerobic coproporphyrinogenase activity in an extract of Rhodopseudomonas spheroides is inhibited by 1,10-phenanthroline, alphaalpha'-bipyridyl, flavins, 2,4-dinitrophenol and 1,4-naphthaquinone. These compounds have no effect on the aerobic coproporphyrinogenase activity. 2. On removal of small-molecular-weight material from a crude extract, the anaerobic system becomes very unstable; it can be stabilized by adding succinate. Now nicotinamide nucleotides, in addition to Mg(2+), ATP and methionine, are required for protoporphyrin to be formed. 3. A mechanism for the anaerobic reaction is proposed, based on the cofactor requirements and the effect of inhibitors. 4. The enzyme responsible for aerobic activity has been partially purified and some of its properties are reported. 5. A crude extract of Chromatium strain D also exhibits coproporphyrinogenase activity under anaerobic conditions in the presence of S-adenosylmethionine or ATP plus methionine. The requirement for other cofactors is variable.

Adenosine Triphosphate↗
PubMed1974

Mössbauer effect in the high-potential iron-sulphur protein from Chromatium. Evidence for the state of the iron atoms.

1. The previous Mössbauer work on Chromatium high-potential iron-sulphur protein by Moss et al. (1968) and Evans et al. (1970) was extended to high applied magnetic fields. 2. Measurements of the reduced protein confirm that it is non-magnetic. 3. Spectra of the oxidized protein in applied magnetic fields clearly indicate that some iron atoms have a positive hyperfine field, which is evidence for antiferromagnetic coupling. 4. The spectra can be interpreted in terms of two types of iron atom with positive and negative hyperfine fields of 9 and 12T respectively. 5. A consideration of the chemical shifts and other evidence suggests formal valences of two Fe(3+) and two Fe(2+) atoms in the non-magnetic reduced state, and three Fe(3+) atoms and one Fe(2+) atom in the oxidized state. 6. However, no separate Fe(3+) and Fe(2+) spectra are seen, suggesting that the d electrons are not localized on particular iron atoms.

Bacterial Proteins↗
PubMed1974

Mössbauer effect in the 'super-reduced' form of the high-potential iron-sulphur protein from Chromatium.

Mössbauer-effect studies of the super-reduced form of Chromatium high-potential iron-sulphur protein indicate that the iron atoms are in a similar valency state to those in reduced ferredoxin from Clostridium pasteurianum, with possibly some inequivalence between the iron atoms within the four-iron centre. Mössbauer spectroscopy also shows magnetic differences between the four-iron centres in the two proteins.

Bacterial Proteins↗
PubMed1979

The amino acid sequence of cytochrome c' from the purple sulphur bacterium Chromatium vinosum.

An amino acid sequence is proposed for the cytochrome c' from the photosynthetic purple sulphur bacterium Chromatium vinosum strain D. It is single polypeptide chain of 131 residues, with haem-attachment cysteine residues at positions 121 and 124. The results discredit an earlier report [Dus, Bartsch & Kamen (1962) J. Biol. Chem 237, 3083--3093] of a di-haem peptide sequence from this protein. The sequence belongs to the same class as the published Alcaligenes and Rhodospirillum rubrum cytochrome c' squences, but the resemblance is not close. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50,093 (15 pp.) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1978) 169, 5.

Amino Acid Sequence↗
PubMed1999

Pre-steady-state kinetics of the reactions of [NiFe]-hydrogenase from Chromatium vinosum with H2 and CO.

Results are presented of the first rapid-mixing/rapid-freezing studies with a [NiFe]-hydrogenase. The enzyme from Chromatium vinosum was used. In particular the reactions of active enzyme with H2 and CO were monitored. The conversion from fully reduced, active hydrogenase (Nia-SR state) to the Nia-C* state was completed in less than 8 ms, a rate consistent with the H2-evolution activity of the enzyme. The reaction of CO with fully reduced enzyme was followed from 8 to 200 ms. The Nia-SR state did not react with CO. It was discovered, contrary to expectations, that the Nia-C* state did not react with CO when reactions were performed in the dark. When H2 was replaced by CO, a Nia-C* EPR signal appeared within 11 ms; this was also the case when H2 was replaced by Ar. With CO, however, the Nia-C* state decayed within 40 ms, due to the generation of the Nia-S.CO state (the EPR-silent state of the enzyme with bound CO). The Nia-C* state, induced after 11 ms by replacing H2 by CO in the dark, could be converted, in the frozen enzyme, into the EPR-detectable state with CO bound to nickel (Nia*.CO) by illumination at 30 K (evoking the Nia-L* state), followed by dark adaptation at 200 K. This can be explained by assuming that the Nia-C* state represents a formally trivalent state of nickel, which is unable to bind CO, whereas nickel in the Nia-L* and the Nia*.CO states is formally monovalent.

Bacterial Proteins↗
PubMed1977

Dependence of the rates of dissolution of the Fe4S4 clusters of Chromatium vinosum high-potential iron protein and ferredoxin on cluster oxidation state.

The influence of oxidation state on the pH dependence of the dissolution of the Fe(4)S(4) clusters of Chromatium vinosum ferredoxin and high-potential iron protein (HIPIP) has been studied. The first-order rate constants (k(obs)) for dissolution of both the Fe(4)S(4)(S-Cys)(4) (2-) and Fe(4)S(4)(S-Cys)(4) (3-) clusters of the ferredoxin follow the same overall kinetic equation but with differing specific rate and equilibrium constants. The dependence of rate and equilibrium constants upon oxidation state may be rationalized on the basis of the accompanying change in electrostatic affinity of a cluster toward H(+) and HO(-). A more drastic change in the pH dependence of the kinetics of dissolution of the Fe(4)S(4) cluster of the HIPIP accompanies its change in oxidation state. Whereas the values of k(obs) for dissolution of HIPIP containing the Fe(4)S(4)(S-Cys)(4) (2-) cluster are strictly second order to [H(+)] and [HO(-)], the pH dependence for dissolution of the HIPIP Fe(4)S(4)(S-Cys)(4) (1-) cluster indicates a first-order dependence upon [H(+)], a second-order dependence upon [HO(-)], and a spontaneous or water rate. These reactivity differences may be related to changes in cluster charge density. Mechanisms of dissolution involve preequilibrium protonation at acidic pH and preequilibrium ligand exchange at basic pH.

Chromatium↗
PubMed1977

Chromatium hydrogenase.

Previous reports of a relatively air-stable soluble hydrogenase from the photosynthetic anaerobe, Chromatium vinosum, have been substantiated. The properties of this enzyme, as seen in highly purified samples prepared by procedures that permit improved yields, are described. A possible role for flavin mediation is noted.

Chromatium↗
PubMed1995

Role of aromatic residues in stabilization of the [Fe4S4] cluster in high-potential iron proteins (HiPIPs): physical characterization and stability studies of Tyr-19 mutants of Chromatium vinosum HiPIP.

The functional role of residue Tyr-19 of Chromatium vinosum HiPIP has been evaluated by site-directed mutagenesis experiments. The stability of the [Fe4S4] cluster prosthetic center is sensitive to side-chain replacements. Polar residues result in significant instability, while nonpolar residues (especially with aromatic side chains) maintain cluster stability. Two-dimensional NMR data of native and mutant HiPIPs are consistent with a model where Tyr-19 serves to preserve the structural rigidity of the polypeptide backbone, thereby maintaining a hydrophobic barrier for exclusion of water from the cluster cavity. Solvent accessibility results in more facile oxidation of the cluster by atmospheric oxygen, with subsequent rapid hydrolysis of the [Fe4S4]3+ core.

Aerobiosis↗
PubMed1998

The two [4Fe-4S] clusters in Chromatium vinosum ferredoxin have largely different reduction potentials. Structural origin and functional consequences.

The 2[4Fe-4S] ferredoxin from Chromatium vinosum arises as one prominent member of a recently defined family of proteins found in very diverse bacteria. The potentiometric circular dichroism titrations of the protein and of several molecular variants generated by site-directed mutagenesis have established that the reduction potentials of the two clusters differ widely by almost 200 mV. This large difference has been confirmed by electrochemical methods, and each redox transition has been assigned to one of the clusters. The unusually low potential center is surprisingly the one that displays a conventional CX1X2CX3X4C (Xn, variable amino acid) binding motif and a structural environment similar to that of clusters having less negative potentials. A comparison with other ferredoxins has highlighted factors contributing to the reduction potential of [4Fe-4S] clusters in proteins. (i) The loop between the coordinating cysteines 40 and 49 and the C terminus alpha-helix of C. vinosum ferredoxin cause a negative, but relatively moderate, shift of approximately 60 mV for the nearby cluster. (ii) Very negative potentials, below -600 mV, correlate with the presence of a bulky side chain in position X4 of the coordinating triad of cysteines. These findings set the framework in which previous observations on ferredoxins can be better understood. They also shed light onto the possible occurrence and properties of very low potential [4Fe-4S] clusters in less well characterized proteins.

Chromatium↗
PubMed2001

Characterization of glutathione amide reductase from Chromatium gracile. Identification of a novel thiol peroxidase (Prx/Grx) fueled by glutathione amide redox cycling.

Among the Chromatiaceae, the glutathione derivative gamma-l-glutamyl-l-cysteinylglycine amide, or glutathione amide, was reported to be present in facultative aerobic as well as in strictly anaerobic species. The gene (garB) encoding the central enzyme in glutathione amide cycling, glutathione amide reductase (GAR), has been isolated from Chromatium gracile, and its genomic organization has been examined. The garB gene is immediately preceded by an open reading frame encoding a novel 27.5-kDa chimeric enzyme composed of one N-terminal peroxiredoxin-like domain followed by a glutaredoxin-like C terminus. The 27.5-kDa enzyme was established in vitro to be a glutathione amide-dependent peroxidase, being the first example of a prokaryotic low molecular mass thiol-dependent peroxidase. Amino acid sequence alignment of GAR with the functionally homologous glutathione and trypanothione reductases emphasizes the conservation of the catalytically important redox-active disulfide and of regions involved in binding the FAD prosthetic group and the substrates glutathione amide disulfide and NADH. By establishing Michaelis constants of 97 and 13.2 microm for glutathione amide disulfide and NADH, respectively (in contrast to K(m) values of 6.9 mm for glutathione disulfide and 1.98 mm for NADPH), the exclusive substrate specificities of GAR have been documented. Specificity for the amidated disulfide cofactor partly can be explained by the substitution of Arg-37, shown by x-ray crystallographic data of the human glutathione reductase to hydrogen-bond one of the glutathione glycyl carboxylates, by the negatively charged Glu-21. On the other hand, the preference for the unusual electron donor, to some extent, has to rely on the substitution of the basic residues Arg-218, His-219, and Arg-224, which have been shown to interact in the human enzyme with the NADPH 2'-phosphate group, by Leu-197, Glu-198, and Phe-203. We suggest GAR to be the newest member of the class I flavoprotein disulfide reductase family of oxidoreductases.

Amino Acid Sequence↗