Molecular study and partial characterization of iron-only hydrogenase in Desulfovibrio fructosovorans.

An iron-only hydrogenase was partially purified and characterized from Desulfovibrio fructosovorans wild-type strain. The enzyme exhibits a molecular mass of 56 kDa and is composed of two distinct subunits HydA and HydB (46 and 13 kDa, respectively). The N-terminal amino acid sequences of the two subunits of the enzyme were determined with the aim of designing degenerate oligonucleotides.

The cytochrome c3-[Fe]-hydrogenase electron-transfer complex: structural model by NMR restrained docking.

Cytochrome c(3) (M(r) 13000) is a low redox potential cytochrome specific of the anaerobic metabolism in sulfate-reducing bacteria. This tetrahemic cytochrome is an intermediate between the [Fe]-hydrogenase and the cytochrome Hmc in Desulfovibrio vulgaris Hildenborough strain. The present work describes the structural model of the cytochrome c(3)-[Fe]-hydrogenase complex obtained by nuclear magnetic resonance restrained docking.

Hydrogenases in the "active" state: determination of g-matrix axes and electron spin distribution at the active site by 1H ENDOR spectroscopy.

Hydrons and electrons are substrates for the enzyme hydrogenase, but cannot be observed in X-ray crystal structures. High-resolution 1H electron nuclear double resonance (ENDOR) spectroscopy offers a means to detect the distribution of protons and unpaired electrons. ENDOR spectra were recorded from frozen solutions of the nickel-iron hydrogenases of Desulfovibrio gigas and Desulfomicrobium baculatum, in the "active" state ("Ni-C" EPR signal) and analyzed by orientationally selective simulation methods.

Structural model of the Fe-hydrogenase/cytochrome c553 complex combining transverse relaxation-optimized spectroscopy experiments and soft docking calculations.

Fe-hydrogenase is a 54-kDa iron-sulfur enzyme essential for hydrogen cycling in sulfate-reducing bacteria. The x-ray structure of Desulfovibrio desulfuricans Fe-hydrogenase has recently been solved, but structural information on the recognition of its redox partners is essential to understand the structure-function relationships of the enzyme. In the present work, we have obtained a structural model of the complex of Fe-hydrogenase with its redox partner, the cytochrome c(553), combining docking calculations and NMR experiments.

Crystal structure of the oxidised and reduced acidic cytochrome c3from Desulfovibrio africanus.

Unique among sulphate-reducing bacteria, Desulfovibrio africanus has two periplasmic tetraheme cytochromes c3, one with an acidic isoelectric point which exhibits an unusually low reactivity towards hydrogenase, and another with a basic isoelectric point which shows the usual cytochrome c3reactivity. The crystal structure of the oxidised acidic cytochrome c3of Desulfovibrio africanus (Dva.a) was solved by the multiple anomalous diffraction (MAD) method and refined to 1.

Desulfovibrio desulfuricans iron hydrogenase: the structure shows unusual coordination to an active site Fe binuclear center.

Background: Many microorganisms have the ability to either oxidize molecular hydrogen to generate reducing power or to produce hydrogen in order to remove low-potential electrons. These reactions are catalyzed by two unrelated enzymes: the Ni-Fe hydrogenases and the Fe-only hydrogenases.

Results: We report here the structure of the heterodimeric Fe-only hydrogenase from Desulfovibrio desulfuricans - the first for this class of enzymes.

Crystallization and preliminary crystallographic analysis of the pyruvate-ferredoxin oxidoreductase from Desulfovibrio africanus.

For the first time, crystals of a pyruvate-ferredoxin oxidoreductase (PFOR) suitable for X-ray analysis have been obtained. This enzyme catalyzes, in anaerobic organisms, the crucial energy-yielding reaction of pyruvate decarboxylation to acetylCoA. Polyethylene glycol and divalent metal cations have been used to crystallize the PFOR from the sulfate-reducing bacterium Desulfovibrio africanus.

Hydrogenase: a hydrogen-metabolizing enzyme. What do the crystal structures tell us about its mode of action?

Hydrogenases are proteins which metabolize the most simple of chemical compounds, molecular hydrogen, according to the reaction H2<-->2H+ + 2e-. These enzymes are found in many microorganisms of great biotechnological interest such as methanogenic, acetogenic, nitrogen fixing, photosynthetic or sulfate-reducing bacteria. The X-ray structure of a dimeric [NiFe] hydrogenase together with a wealth of biophysical, biochemical and genetic studies have revealed that the large subunit contains the bimetallic [Ni-Fe] active site, with biologically uncommon CO and CN ligands to the iron, whereas the small subunit contains three iron-sulfur cluster.

Crystal structure of the ferredoxin I from Desulfovibrio africanus at 2.3 A resolution.

The crystal structure of the ferredoxin I from the sulfate-reducing bacterium Desulfovibrio africanus (DaFdI) has been solved and refined by X-ray diffraction. The crystals are orthorhombic with a = 96.6 A, b = 58.

MCD and 1H-NMR spectroscopic studies of Desulfovibrio africanus ferredoxin I: revised amino-acid sequence and identification of secondary structure.

Desulfovibrio africanus ferredoxin I was studied by magnetic circular dichroism and 1H-NMR spectroscopies. These showed the presence of histidine and tryptophan, in contrast to the previously reported amino-acid sequence (Bruschi and Hatchikian (1982) Biochimie 64, 503-507). This was redetermined and the revised sequence shown to contain both histidine and tryptophan, as well as four other corrections (Sery et al.

Cloning and sequencing of the locus encoding the large and small subunit genes of the periplasmic [NiFe]hydrogenase from Desulfovibrio fructosovorans.

The genetic locus encoding the periplasmic [NiFe]hydrogenase (Hyd) from Desulfovibrio fructosovorans was cloned and sequenced. The genes of this two-subunit enzyme have an operon organization in which the 0.94-kb gene encoding the small subunit precedes the 1.

Characterization of the nickel-iron periplasmic hydrogenase from Desulfovibrio fructosovorans.

The periplasmic hydrogenase from Desulfovibrio fructosovorans grown on fructose/sulfate medium was purified to homogeneity. It exhibits a molecular mass of 88 kDa and is composed of two different subunits of 60 kDa and 28.5 kDa.

A pulsed EPR study of redox-dependent hyperfine interactions for the nickel centre of Desulfovibrio gigas hydrogenase.

The nickel centre of hydrogenase from Desulfovibrio gigas was studied by electron spin echo envelope modulation (ESEEM) spectroscopy in the oxidized, unready (Ni-A) and H2-reduced active (Ni-C) states, both in H2O and 2H2O solutions. Fourier transforms of the 3-pulse ESEEM, taken at 8.7 GHz, for Ni-A and Ni-C in H2O contained similar peaks with narrow linewidths at frequencies of 0.

Isolation, amino acid analysis and N-terminal sequence determination of the two subunits of the nickel-containing hydrogenase of Desulfovibrio gigas.

The two subunits of the nickel-iron hydrogenase from Desulfovibrio gigas have been purified by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis and their amino acid compositions have been determined. The N-terminal sequences for 15 residues of the large subunit (Mr 62,000) and 25 residues of the small subunit (Mr 26,000), respectively, were established. The occurrence of several cysteine residues in the small subunit is discussed in relation with their possible role in the binding of the redox centers of the enzyme.

Crystallization, preliminary X-ray study and crystal activity of the hydrogenase from Desulfovibrio gigas.

Hydrogenase (EC 1.12) from Desulfovibrio gigas is a dimeric enzyme (26 and 62 (X 10(3) Mr) that catalyzes the reversible oxidation of molecular hydrogen. Single crystals of hydrogenase have been produced using the hanging drop method, with either PEG (polyethylene glycol) 6000 or ammonium sulfate as precipitants at pH 6.

Energetics of Growth of a Defined Mixed Culture of Desulfovibrio vulgaris and Methanosarcina barkeri: Interspecies Hydrogen Transfer in Batch and Continuous Cultures.

Interspecies hydrogen transfer was studied in Desulfovibrio vulgaris-Methanosarcina barkeri mixed cultures. Experiments were performed under batch and continuous growth culture conditions. Lactate or pyruvate was used as an energy source.

Energetics of growth of a defined mixed culture of Desulfovibrio vulgaris and Methanosarcina barkeri: maintenance energy coefficient of the sulfate-reducing organism in the absence and presence of its partner.

The maintenance energy coefficient of Desulfovibrio vulgaris was studied by using a chemostat, with Methanosarcina barkeri or sulfate as the electron acceptor; lithium lactate or sodium pyruvate served as the electron donor. The experiments showed that the growth energetics of D. vulgaris or M.

Microcalorimetric studies of the growth of sulfate-reducing bacteria: comparison of the growth parameters of some Desulfovibrio species.

We performed a comparative study of the growth energetics of some species of Desulfovibrio by measuring microcalorimetric and molar growth yield values. Lactate and pyruvate were used as energy sources for sulfate reduction. On lactate-sulfate media Desulfovibrio desulfuricans Norway, Desulfovibrio gigas, and Desulfovibrio africanus exhibited molar growth yields of 4.

Microcalorimetric studies of the growth of sulfate-reducing bacteria: energetics of Desulfovibrio vulgaris growth.

The metabolism of Desulfovibrio vulgaris Hildenborough grown on medium containing lactate or pyruvate plus a high concentration of sulfate (36 mM) was studied. Molecular growth yields were 6.7 +/- 1.

Isolation and characterization of a rubredoxin and two ferredoxins from Desulfovibrio africanus.

Rubredoxin and two distinct ferredoxins have been purified from Desulfovibrio africanus. The rubredoxin has a molecular weight of 6000 while the ferredoxins appear to be dimers of identical subunits of approximately 6000 to 7000 molecular weight. Rubredoxin contains one iron atom, no acid-labile sulfide and four cysteine residues per molecule.

Purification and characterization of cytochrome c3, ferredoxin, and rubredoxin isolated from Desulfovibrio desulfuricans Norway.

Different electron carriers of the non-desulfoviridin-containing, sulfate-reducing bacterium Desulfovibrio desulfuricans (Norway strain) have been studied. Two nonheme iron proteins, ferredoxin and rubredoxin, have been purified. This ferredoxin contains four atoms of non-heme iron and acid-labile sulfur and six residues of cysteine per molecule.

Purification, characterization and biological activity of three forms of ferredoxin from the sulfate-reducing bacterium Desulfovibrio gigas.

Three forms of ferredoxin FdI, FdI', and FdII have been isolated from Desulfovibrio gigas, a sulfate reducer. They are separated by a combination of DEAE-cellulose and gel filtration chromatographic procedures. FdI and FdI' present a slight difference in isoelectric point which enables the separation of the two forms over DEAE-cellulose, while FdII is easily separated from the two other forms by gel filtration.