Could a diiron-containing four-helix-bundle protein have been a primitive oxygen reductase?
Explore the source record for details and available documents.
Amino Acid Sequence↗
Biomedical subjects
J Le Gall
Publications and source records attributed to J Le Gall.
[NiFe] hydrogenase from Desulfovibrio desulfuricans ATCC 27774: gene sequencing, three-dimensional structure determination and refinement at 1.8 A and modelling studies of its interaction with the tetrahaem cytochrome c3.
The primary and three-dimensional structures of a [NiFe] hydrogenase isolated from D. desulfitricans ATCC 27774 were determined, by nucleotide analysis and single-crystal X-ray crystallography. The three-dimensional structural model was refined to R=0.167 and Rfree=0.223 using data to 1.8 A resolution. Two unique structural features are observed: the [4Fe-4S] cluster nearest the [NiFe] centre has been modified [4Fe-3S-3O] by loss of one sulfur atom and inclusion of three oxygen atoms; a three-fold disorder was observed for Cys536 which binds to the nickel atom in the [NiFe] centre. Also, the bridging sulfur atom that caps the active site was found to have partial occupancy, thus corresponding to a partly activated enzyme. These structural features may have biological relevance. In particular, the two less-populated rotamers of Cys536 may be involved in the activation process of the enzyme, as well as in the catalytic cycle. Molecular modelling studies were carried out on the interaction between this [NiFe] hydrogenase and its physiological partner, the tetrahaem cytochrome c3 from the same organism. The lowest energy docking solutions were found to correspond to an interaction between the haem IV region in tetrahaem cytochrome c3 with the distal [4Fe-4S] cluster in [NiFe] hydrogenase. This interaction should correspond to efficient electron transfer and be physiologically relevant, given the proximity of the two redox centres and the fact that electron transfer decay coupling calculations show high coupling values and a short electron transfer pathway. On the other hand, other docking solutions have been found that, despite showing low electron transfer efficiency, may give clues on possible proton transfer mechanisms between the two molecules.
Amino Acid Sequence↗
A bacterioferritin from the strict anaerobe Desulfovibrio desulfuricans ATCC 27774.
A bacterioferritin was isolated from the anaerobic bacterium Desulfovibrio desulfuricans ATCC 27774, grown with nitrate as the terminal electron acceptor, which is the first example of a bacterioferritin from a strict anaerobic organism. This new bacterioferritin was isolated mainly as a 24-mer of 20 kDa identical subunits, containing 0.5 noncovalently bound heme and 2 iron atoms per monomer. Although its N-terminal sequence is significantly homologous with ferritins from other microorganisms and the ligands to the di-iron ferroxidase center are conserved, it is one of the most divergent bacterioferritins so far characterized. Also, in contrast to all other known bacterioferritins, its heme is not of the B type; its chromatographic behavior is identical to that of iron uroporphyrin. Thus, D. desulfuricans bacterioferritin appears to be the second example of a protein unexpectedly containing this heme cofactor, or a closely related porphyrin, after its finding in Desulfovibrio gigas rubredoxin:oxygen oxidoreductase ¿Timkovich, R., Burkhalter, R. S., Xavier, A. V., Chen, L., and Le Gall, J. (1994) Bioorg. Chem. 22, 284-293. The oxidized form of the protein has a visible spectrum characteristic of low-spin ferric hemes, exhibiting a weak absorption band at 715 nm, indicative of bis-methionine heme axial coordination; upon reduction, the alpha-band appears at 550 nm and a splitting of the Soret band occurs, with two maxima at 410 and 425 nm. The heme center has a reduction potential of 140 +/- 10 mV (pH 7.6), a value unusually high compared to that of other bacterioferritins (ca. -200 mV).
Amino Acid Sequence↗
Structure of a dioxygen reduction enzyme from Desulfovibrio gigas.
Desulfovibrio gigas is a strict anaerobe that contains a well-characterized metabolic pathway that enables it to survive transient contacts with oxygen. The terminal enzyme in this pathway, rubredoxin:oxygen oxidoreductase (ROO) reduces oxygen to water in a direct and safe way. The 2.5 A resolution crystal structure of ROO shows that each monomer of this homodimeric enzyme consists of a novel combination of two domains, a flavodoxin-like domain and a Zn-beta-lactamase-like domain that contains a di-iron center for dioxygen reduction. This is the first structure of a member of a superfamily of enzymes widespread in strict and facultative anaerobes, indicating its broad physiological significance.
Amino Acid Sequence↗
The superoxide dismutase activity of desulfoferrodoxin from Desulfovibrio desulfuricans ATCC 27774.
Desulfoferrodoxin (Dfx), a small iron protein containing two mononuclear iron centres (designated centre I and II), was shown to complement superoxide dismutase (SOD) deficient mutants of Escherichia coli [Pianzzola, M.J., Soubes M. & Touati, D. (1996) J. Bacteriol. 178, 6736-6742]. Furthermore, neelaredoxin, a protein from Desulfovibrio gigas containing an iron site similar to centre II of Dfx, was recently shown to have a significant SOD activity [Silva, G., Oliveira, S., Gomes, C.M., Pacheco, I., Liu, M.Y., Xavier, A.V., Teixeira, M., Le Gall, J. & Rodrigues-Pousada, C. (1999) Eur. J. Biochem. 259, 235-243]. Thus, the SOD activity of Dfx isolated from the sulphate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 was studied. The protein exhibits a SOD activity of 70 U x mg-1, which increases approximately 2.5-fold upon incubation with cyanide. Cyanide binds specifically to Dfx centre II, yielding a low-spin iron species with g-values at 2.27 (g perpendicular) and 1.96 (g parallel). Upon reaction of fully oxidized Dfx with the superoxide generating system xanthine/xanthine oxidase, Dfx centres I and II become partially reduced, suggesting that Dfx operates by a redox cycling mechanism, similar to those proposed for other SODs. Evidence for another SOD in D. desulfuricans is also presented - this enzyme is inhibited by cyanide, and N-terminal sequence data strongly indicates that it is an analogue to Cu,Zn-SODs isolated from other sources. This is the first indication that a Cu-containing protein may be present in a sulphate-reducing bacterium.
Amino Acid Sequence↗
Polymorphisms in the HFE gene.
Hereditary hemochromatosis is an autosomal recessive disease characterized by progressive iron overload. Recently, a candidate gene named HFE was isolated on the short arm of the chromosome 6 within which two mutations were identified: C282Y and H63D. To date, only homozygosity for the C282Y mutation is considered as a diagnostic criterion of hemochromatosis. 7.6% of the patients studied in our laboratory did not carry two copies of the C282Y mutation. On the other hand, a dysmetabolic iron overload syndrome has recently been described and the search for the C282Y and H63D mutations revealed that none of the patients was homozygous for C282Y while 67% exhibited one of the mutations. The possibility of a new mutation in the HFE gene has been raised to explain the disease in the remaining patients, as well as, in the few hemochromatotic patients without two copies of the C282Y mutation. The aim of this study was to search for new mutations in the HFE gene in 16 such patients. Direct sequencing of exons and 3 introns did not reveal any new mutation but identified a few polymorphisms.
Alleles↗
Microbial and plant metabolism of NO.
During microbial denitrification, NO is produced by reduction of nitrite by either the reduced high spin d1 hemes in a unique reductase (NIR) or at the expense of a blue copper protein that transfers electrons that move first to a type I copper and then to a type II copper in a unique trimeric NIR. This latter type of NIR is also produced by several denitrifying filamentous fungi. Reduction of NO is then carried out by either a specific cytochrome be complex NOR in denitrifying bacteria or a unique cytochrome P-450 in denitrifying filamentous fungi. NO is also produced by an anomalous reaction of a molybdoprotein, nitrate reductase (NAR), acting on an odd substrate, NO2-. NO is also reduced by a multiheme NIR that serves physiologically for reduction of NO2- to NH3. This type NIR reduces NO to either N2O, if only partially reduced, or NH3, if fully reduced, when it encounters NO. This multiheme NIR is very sensitive to cyanide. Transcription of the genes for NIR and NOR production in a denitrifier is activated by NO, a process that also requires the presence of the gene product, a transcriptional activator, NnrR.
Bacteria↗
Preliminary crystallographic analysis and further characterization of a dodecaheme cytochrome c from Desulfovibrio desulfuricans ATCC 27774.
Dodecaheme cytochrome c has been purified from Desulfovibrio (D.) desulfuricans ATCC 27774 cells grown under both nitrate and sulfate-respiring conditions. Therefore, it is likely to play a role in the electron-transfer system of both respiratory chains. Its molecular mass (37768 kDa) was determined by electrospray mass spectrometry. Its first 39 amino acids were sequenced and a motif was found between amino acids 32 and 37 that seems to exist in all the cytochromes of the c(3) type from sulfate-reducing bacteria sequenced at present. The midpoint redox potentials of this cytochrome were estimated to be -68, -120, -248 and -310 mV. Electron paramagnetic resonance spectroscopy of the oxidized cytochrome shows several low-spin components with a g(max) spreading from 3.254 to 2.983. Two crystalline forms were obtained by vapour diffusion from a solution containing 2% PEG 6000 and 0.25-0.75 M acetate buffer pH = 5.5. Both crystals belong to monoclinic space groups: one is P2(1), with a = 61.00, b = 106.19, c = 82.05 A, beta = 103.61 degrees, and the other is C2 with a = 152.17, b = 98.45, c = 89.24 A, beta = 119.18 degrees. Density measurements of the P2(1) crystals suggest that there are two independent molecules in the asymmetric unit. Self-rotation function calculations indicate, in both crystal forms, the presence of a non-crystallographic axis perpendicular to the crystallographic twofold axis. This result and the calculated values for the volume per unit molecular weight of the C2 crystals suggest the presence of two or four molecules in the asymmetric unit.
Journal Article↗
Primary structure of desulfoferrodoxin from Desulfovibrio desulfuricans ATCC 27774, a new class of non-heme iron proteins.
The primary structure of desulfoferrodoxin from Desulfovibrio desulfuricans ATCC 27774, a redox protein with two mononuclear iron sites, was determined by automatic Edman degradation and mass spectrometry of the composing peptides. It contains 125 amino acid residues of which five are cysteines. The first four, Cys-9, Cys-12, Cys-28 and Cys-29, are responsible for the binding of Center I which has a distorted tetrahedral sulfur coordination similar to that found in desulforedoxin from D. gigas. The remaining Cys-115 is proposed to be involved in the coordination of Center II, which is probably octahedrally coordinated with predominantly nitrogen/oxygen containing ligands as previously suggested by Mössbauer and Raman spectroscopy.
Amino Acid Sequence↗
Preliminary crystallographic analysis of the oxidized form of a two mono-nuclear iron centres protein from Desulfovibrio desulfuricans ATCC 27774.
Crystals of the fully oxidized form of desulfoferrodoxin were obtained by vapor diffusion from a solution containing 20% PEG 4000, 0.1 M HEPES buffer, pH 7.5, and 0.2 M CaCl2. Trigonal and/or rectangular prisms could be obtained, depending on the temperature used for the crystal growth. Trigonal prisms belong to the rhombohedral space group R32, with a = 112.5 A and c = 63.2 A; rectangular prisms belong to the monoclinic space group C2, with a = 77.7 A, b = 80.9 A, c = 53.9 A, and beta = 98.1 degrees. The crystallographic asymmetric unit of the rhombohedral crystal form contains one molecule. There are two molecules in the asymmetric unit of the monoclinic form, in agreement with the self-rotation function.
Crystallization↗
Anaerobes response to oxygen: the sulfate-reducing bacteria.
Explore the source record for details and available documents.
Journal Article↗
Siroamide: a prosthetic group isolated from sulfite reductases in the genus Desulfovibrio.
While isolating siroheme from enzymes or whole cells of Desulfovibrio species, it was discovered that the main product after metal removal and esterification was not the octamethyl ester derivative of sirohydrochlorin, but a monoamide, heptamethyl ester derivative. The structure of this derivative was established by mass spectrometry and NMR. Nuclear Overhauser enhancement measurements in combination with chemical shift analogy arguments indicate that the 2(1)-acetate has been stereospecifically amidated. Other cellular sources of siroheme were investigated, but only the octamethyl ester derivative was found, with no traces of the amide derivative. The results suggest that, in Desulfovibrio, the physiologically active prosthetic group may be an amidated form of siroheme.
Desulfovibrio↗
A blue non-heme iron protein from Desulfovibrio gigas.
A novel iron-containing blue protein, named neelaredoxin, was isolated from the sulfate-reducing bacterium Desulfovibrio gigas. It is a monomeric protein with a molecular mass of 15 kDa containing two iron atoms/molecule. The N-terminal sequence of neelaredoxin has similarity to the second domain of desulfoferrodoxin, a protein purified from Desulfovibrio vulgaris Hildenborough. This finding supports the hypothesis that the gene coding for desulfoferrodoxin (rbo) might have arisen from a gene fusion [Brumlik, M. J., Leroy, G., Bruschi, M. & Voordouw, G. (1990) J. Bacteriol. 172, 7289-7292]. The visible spectrum exhibits a single band at 666 nm, responsible for the blue color of the protein, which is completely bleached upon reduction with sodium ascorbate. In the oxidized state the EPR spectrum is complex, exhibiting well-resolved features at g = 7.6, 7.0, 5.9, and 5.8 which are assigned to two high-spin (S = 5/2) mononuclear-iron (III) centers with different rhombic distortions (E/D approximately 0.05 and approximately 0.08). The two iron atoms contribute identically to the visible spectrum as judged from visible redox titrations, from which a reduction potential of +190 mV was determined for both iron sites at pH 7.5. At high pH the visible and the EPR spectra become pH-dependent with a pKa above 9: the 666-nm band shifts to 590 nm and the EPR signals are converted into a signal with gmax approximately 4.7. Neelaredoxin is readily reduced both by H2/hydrogenase/cytochrome c3 and by NADH/NADH-rubredoxin oxidoreductase.
Amino Acid Sequence↗
Purification, characterization and properties of an NADH oxidase from Desulfovibrio vulgaris (Hildenborough) and its coupling to adenylyl phosphosulfate reductase.
An NADH oxidase was purified from Desulfovibrio vulgaris. This FMN-containing enzyme reacts with oxygen forming hydrogen peroxide with a specific activity of 0.21 mumoles.min-1.mg-1. The molecular weight of the protein was determined to be 65 kDa on 12.5% SDS/PAGE. It shows very low NADH: rubredoxin oxidoreductase activity specifically towards the rubredoxin from the same organism. However, adenylyl phosphosulfate reductase can be fully reduced by NADH with the purified enzyme, suggesting that NADH could be an electron donor for respiratory sulfate reduction.
Amino Acid Sequence↗
Isolation and characterization of a high molecular weight cytochrome from the sulfate reducing bacterium Desulfovibrio gigas.
A high molecular weight c-type cytochrome (Hmc) was purified and characterized from Desulfovibrio gigas. The molecular weight was estimated to be 67 kDa by SDS-PAGE and its N-terminus is homologous to those of the 16 hemes containing high molecular weight cytochrome c from Desulfovibrio vulgaris strains Hildenborough and Miyazaki. The purified hemoprotein shows c-type cytochrome absorption spectrum with e533 (red) = 368 mM-1.cm-1. A band at 640 nm, characteristic of high-spin hemes, was detected. The EPR spectra show the presence of two high-spin heme species, plus several non-equivalent low-spin hemes. The heme reduction potentials, at pH 7.6, range from -50 mV to -315 mV. In contrast to what has been described for D. vulgaris Hmc, the protein isolated from D. gigas directly accepts electrons from hydrogenase and further reduces other redox proteins.
Amino Acid Sequence↗
Localization and specificity of cytochromes and other electron transfer proteins from sulfate-reducing bacteria.
Recently data have accumulated concerning the electron transfer chains of sulfate-reducing bacteria in general and of the genus Desulfovibrio in particular. Because of the ever growing number of newly discovered individual redox proteins, it has become essential to try to assign them to physiologically relevant chains. This work presents some new data concerning the localization of these proteins within the bacterial cell and the specificity of electron transfer between the three types of hydrogenases which have been found so far in Desulfovibrio, namely the iron-only, the iron-nickel and the iron-nickel-selenium enzymes. The iron-only hydrogenase reduces cytochromes which have bis-histidinyl heme ligation or histidinyl-methionyl heme ligation. In contrast, the iron-nickel and iron-nickel-selenium hydrogenases cannot reduce cytochromes having a His-Met heme ligation, but are very active toward the cytochromes having a bis-histidinyl ligand. This observation has been used to demonstrate that the tetraheme cytochrome c3 can exchange electrons with the monoheme cytochrome c553. No clear specificity has been established for the reaction of hydrogenases toward the hexadecaheme cytochromes from either D vulgaris or D gigas.
Amino Acid Sequence↗
Voltammetric studies of the catalytic electron-transfer process between the Desulfovibrio gigas hydrogenase and small proteins isolated from the same genus.
The kinetics of electron transfer between the Desulfovibrio gigas hydrogenase and several electron-transfer proteins from Desulfovibrio species were investigated by cyclic voltammetry, square-wave voltammetry and chronoamperometry. The cytochrome c3 from Desulfovibrio vulgaris (Hildenborough), Desulfovibrio desulfuricans (Norway 4), Desulfovibrio desulfuricans (American Type Culture Collection 27774) and D. gigas (NCIB 9332) were used as redox carriers. They differ in their redox potentials and isoelectric point. Depending on the pH, all the reduced forms of these cytochromes were effective in electron exchange with hydrogenase. Other small electron-transfer proteins such as ferredoxin I, ferredoxin II and rubredoxin from D. gigas were tentatively used as redox carriers. Only ferredoxin II was effective in mediating electron exchange between hydrogenase and the working electrode. The second-order rate constants k for the reaction between reduced proteins and hydrogenase were calculated based on the theory of the simplest electrocatalytic mechanism [Moreno, C., Costa, C., Moura, I., Le Gall, J., Liu, M. Y., Payne, W. J., van Dijk, C. & Moura, J. J. G. (1993) Eur. J. Biochem. 212, 79-86] and the results obtained by cyclic voltammetry were compared with those obtained by chronoamperometry. Values for k of 10(5)-10(6) M-1 s-1 (cytochrome c3 as electron carrier) and 10(4) M-1 s-1 (ferredoxin II as the electron carrier) were determined. The rate-constant values are discussed in terms of the existence of an electrostatic interaction between the electrode surface and the redox carrier and between the redox carrier and a positively charged part of the enzyme.
Catalysis↗
Electrochemical studies of the hexaheme nitrite reductase from Desulfovibrio desulfuricans ATCC 27774.
The electron-transfer kinetics between three different mediators and the hexahemic enzyme nitrite reductase isolated from Desulfovibrio desulfuricans (ATCC 27774) were investigated by cyclic voltammetry and by chronoamperometry. The mediators, methyl viologen, Desulfovibrio vulgaris (Hildenborough) cytochrome c3 and D. desulfuricans (ATCC 27774) cytochrome c3 differ in structure, redox potential and charge. The reduced form of each mediator exchanged electrons with nitrite reductase. Second-order rate constants, k, were calculated on the basis of the theory for a simple catalytic mechanism and the results, obtained by cyclic voltammetry, were compared with those obtained by chronoamperometry. Values for k are in the range 10(6)-10(8) M-1 s-1 and increase in the direction D. desulfuricans cytochrome c3-->D. vulgaris cytochrome c3-->methyl viologen. An explanation is advanced on the basis of electrostatic interactions and relative orientation between the partners involved. Chronoamperometry (computer controlled) offers advantages over cyclic voltammetry in the determination of homogeneous rate constants (faster, more accurate and better reproducibility). Direct, unmediated electrochemical responses of the hexaheme nitrite reductase were also reported.
Cytochrome c Group↗