Purification and properties of the soluble hydrogenase from Desulfovibrio desulfuricans (strain Norway 4)

A soluble hydrogenase has been isolated from Desulfovibrio desulfuricans (strain Norway 4) grown on Postgate's medium. The enzyme differs significantly from a membrane-bound hydrogenase previously purified from the same organism grown on Starkey's medium. The enzyme consisted of two subunits of 56 kDa and 29 kDa compared with masses of 60 kDa and 27 kDa for the membrane-bound enzyme. Analysis of preparations of the soluble enzyme by various methods gave values of 5-10 iron atoms, 6 labile sulphur atoms and 0.45-0.8 nickel atom per molecule. The enzyme was unusual in that it contained selenium, in quantities equivalent to nickel. The highly purified active enzyme produced no electron-spin-resonance (ESR) signals in the oxidized state. ESR signals due to a [3Fe-xS] cluster and nickel were observed only in some of the less active fractions of the enzyme, demonstrating that neither of these ESR-detectable components is a prerequisite for hydrogenase activity. Treatment of D. desulfuricans (Norway) cells with EDTA released a minor fraction with hydrogenase activity, which might indicate the presence of a periplasmic enzyme.     

Comparative studies of polyhemic cytochromes c isolated from Desulfovibrio vulgaris (Hildenborough) and Desulfovibrio desulfuricans (Norway)

Cytochrome c3 (Mr 26,000) has been characterized in Desulfovibrio vulgaris (Hildenborough) and its properties compared with polyhemic cytochromes c isolated from the same organism and from D. desulfuricans (Norway). It can be described as an octaheme cytochrome c3 constituted of two identical subunits. Absorption spectrum is similar to cytochrome c3 (Mr 13,000) and individual redox potentials have an average value of -180 mV.3 The N terminal sequence is compared with an homologous cytochrome isolated from D. desulfuricans Norway.     

Activation and deactivation of the membrane-bound hydrogenase from Desulfovibrio desulfuricans, Norway strain

The hydrogenase from D. desulfuricans, when isolated in air, had a low activity in the hydrogen-methyl viologen reductase assay, and no activity in the hydrogen-methylene blue reductase assay. The activity increased markedly during incubation under hydrogen. This process is interpreted in terms of conversion of the enzyme from a relatively inactive Unready state to the Active state. Oxidation by dichloro-indophenol caused conversion to a state in which the hydrogen-uptake activity to methyl viologen was preserved, but hydrogen-methylene blue activity was not. This form is termed the Ready state. This behaviour resembles that of the hydrogenase of Desulfovibrio gigas and thus may be a widespread property of this class of hydrogenases. The electron-spin-resonance spectra of the D. desulfuricans enzyme showed the presence of [3Fe-xS] and [4Fe-4S] clusters. Spectra were also observed in the various states of activation of the enzyme. In these respects, the hydrogenase of D. desulfuricans resembles that from D. gigas, although the latter may have an additional iron-sulphur cluster.     

A cobalt containing protein isolated from Desulfovibrio gigas, a sulfate reducer

A protein which contains a cobalt porphyrin was isolated from the sulfate reducer Desulfovibrio gigas. This protein has a molecular weight of approximately 16,700 daltons and is acidic, having an iso-electric point at 3.7. The N-terminal residue was shown to be threonine, and a cobalt analysis gave 0.8 cobalt atoms/molecule, suggesting the presence of a single prosthetic group. The protein has a violet color with absorption bands typical of a metal porphyrin center with maxima at 420 nm, 580 nm with a shoulder at 550 nm. The ratio $\text{A}{}_{420}\text{(γ)}\text{A}{}_{588}\text{(α)}$ is 2.1. The protein has no electron paramagnetic resonance (e.p.r.) spectrum, and as the visible spectrum suggests, it probably contains diamagnetic Co^III porphyrin. However the cobalt centre appears to be protected from reduction by sodium dithionite or sodium borohydride. Att$\text{e}$mpts at ligand substitution with strong nucleophiles such as CN, causes a slight spectral shift to higher wavelenghts. The cobalt porphyrin can be extracted from the protein with an acidified acetone solution, indicating that it is not covalently bound to the protein.     

Purification and some properties of cytochrome C553(550) isolated from Desulfovibrio desulfuricans Norway.

A new c-type cytochrome containing a single heme group, cytochrome c_553(550) has been purified from $\text{Desulfovibrio desulfuricans}$ (Norway strain) and some of its properties have been investigated. It has an isoelectric point of 6.6 and a higher redox potential than cytochrome c₃ isolated from the same bacteria. Its molecular weight was estimated to be 9,200 by gel filtration. The main absorption peaks are at 553, 522.5 and 417 nm in the reduced form and at 690, 529, 411, 357 and 280 nm in the oxidized form. The asymmetric α band of the reduced state is similar to the one reported for socalled “split α” cytochromes c. The cytochrome contains 86 amino acid residues with 5 methionine, two cysteine and two histidine residues. The N terminal sequence of $\text{D. desulfuricans}$ Norway cytochrome c_553(550) presents no evident homology with that of $\text{Desulfovibrio vulgaris}$ Hildenborough cytochrome c₅₅₃.     

Electrochemical study of the electron exchange between cytochrome c3 and hydrogenase from Desulfovibrio desulfuricans Norway

The kinetics of the reduction of the Desulfovibrio desulfuricans Norway cytochrome c3 by its physiological partner hydrogenase, in the presence of hydrogen, was investigated by an electrochemical method; from cyclic voltammetry experiments a value of 3 X 10(7) M-1 s-1 was obtained for the second-order rate constant. Results are discussed in terms of specific interactions between physiological partner proteins.     

Spectroscopic studies of the nature of the iron clusters in the soluble hydrogenase from Desulfovibrio desulfuricans (strain Norway 4)

57Fe-enriched samples of the soluble hydrogenase from Desulfovibrio desulfuricans (Norway) have been investigated in both the native (oxidized) and the dithionite-reduced states using M?ssbauer spectroscopy. The data clearly show that the iron in this enzyme is predominantly in the form of iron-sulphur clusters which are closely similar to the [4Fe-4S] clusters found in a large number of ferredoxins, such as that from Bacillus stearothermophilus. There appear to be two [4Fe-4S] clusters. The iron-sulphur clusters in the oxidized protein are virtually diamagnetic, as indicated by M?ssbauer, electron spin resonance and magnetic circular dichroic spectroscopy. On reduction by dithionite + methyl viologen, M?ssbauer spectroscopy showed that only 50% of the [4Fe-4S] clusters were reduced. Even reduction with hydrogen up to a pressure of 23 GPa did not reduce the iron-sulphur clusters completely. An ESR signal due to a rapidly relaxing species with g = 2.03, 1.89 was observed in the reduced protein, together with a weaker spectrum from a slower-relaxing species at g = 2.34, 2.12.     

Purification and properties of the membrane-bound by hydrogenase from Desulfovibrio desulfuricans.

The membrane-bound hydrogenase from the anaerobic sulphate-reducing bacterium Desulfovibrio desulfuricans (Norway strain) has been purified to homogeneity, with an overall 80-fold purification and a specific activity of 70 mumol of H2 evolved/min per mg of protein. The hydrogenase had a relative molecular mass of 58 000 as determined by gel filtration and was estimated to contain six iron atoms and six acid-labile sulphur groups per molecule. The absorption spectrum of the enzyme was characteristic of an iron-sulphur protein. The E400 and E280 were 28 500 and 109 000 M-1.cm-1 respectively. The e.s.r. of the oxidized protein indicated the presence of [4Fe-4S]3+ or [3Fe-3S]3+, and another paramagnetic centre, probably Ni(III). The hydrogenase was inhibited by heavy-metal salts, carbon monoxide and high ionic strength. However, it was resistant to inhibition by thiol-blocking and metal-complexing reagents. N-Bromosuccinimide totally inhibited the enzyme activity at low concentrations. The enzyme was stable to O2 over long periods and to high temperatures. It catalyses both H2-evolution and H2-uptake with a variety of artificial electron carriers. D. desulfuricans cytochrome C3, its natural electron carrier, had a high affinity for the enzyme (Km = 2 microns). Rate enhancement was observed when cytochrome C3 was added to Methyl Viologen in the H2-evolution assay. The pH optimum for H2-evolution was 6.5.     

Kinetic properties of hydrogenase isolated from Desulfovibrio vulgaris (Hildenborough)

Hydrogenase of Desulfovibrio vulgaris shows nonlinear kinetics in hydrogen production with both the natural electron carrier, cytochrome c3, and the artificial donor, methyl viologen semiquinone. Increasing concentrations of salt progressively inhibit the hydrogen production, as do increasing amounts of dimethylsulfoxide (Me2SO). Hydrogen consumption activity does not change up to 30% (v/v) of Me2SO. Preincubation in Me2SO up to 55% (v/v) does not affect the hydrogen uptake or production. The production activity of the enzyme shows an optimum around pH 6. When plotted as a function of redox potential the activity can be fitted to a Nernst equation with n = 1. Midpoint potentials calculated at various values follow approximately the hydrogen electrode to pH 6. Thereafter, there is a shift of about 40 mV to higher redox potentials.     

A four-iron ferredoxin from Desulfovibrio desulfuricans (Short Communication)

Ferredoxin from Desulfovibrio desulfuricans was isolated, purified and crystallized. It contains four iron atoms and four sulphido or ;acid-labile' sulphur atoms for a molecule of 6000 daltons. The absorption spectrum in the u.v.-visible region and the electron-paramagnetic-resonance signals of the reduced protein are similar to those observed for other four-iron ferredoxins. The amino acid composition is different from that of Desulfovibrio gigas ferredoxin. The redox potential of -0.33V at pH7.0 was determined by dye techniques.     

Kinetic studies of the electron exchange reaction between the octaheme cytochrome c3 (Mr 26000) and the hydrogenase from Desulfovibrio desulfuricans Norway.

The octaheme cytochrome c3 (Mr 26000) from Desulfovibrio desulfuricans Norway was studied using cyclic voltammetry at the pyrolytic graphite electrode. The kinetics of reduction of the octaheme cytochrome c3 (Mr 26000) from D. desulfuricans Norway by the Ni-Fe-Se hydrogenase purified from the same organism was investigated by an electrochemical method. From cyclic voltammetry experiments a value of 8.108M-1S-1 was obtained for the second order homogenous rate constant of the electron transfer between the two proteins. Results are compared with similar experiments performed on the electron exchange between the tetrahemic cytochrome c3 (Mr 13000) and hydrogenase.     

A new restriction endonuclease from the anaerobic bacterium, Desulfovibrio desulfuricans, Norway.

The purification and characterization of a new restriction endonuclease, Dde 1, from a sulfate-reducing, anaerobic bacterium, Desulfovibrio desulfuricans, Norway, is reported. The enzyme recognizes the sequence (see formula index) and cleaves at the position indicated by the arrows. The enzyme preparation obtained is suitable for restriction mapping an ligation.     

Amino acid sequence of the [4Fe-4S] ferredoxin isolated from Desulfovibrio desulfuricans Norway

The complete amino acid sequence of the [4Fe-4S] ferredoxin from Desulfovibrio desulfuricans Norway was determined by repetitive Edman degradation of the whole protein and peptides derived from tryptic digestion. The protein has 59 residues. Four of the six cysteine residues are involved in the binding of the [4Fe-4S] cluster in the same arrangement as in clostridial ferredoxins. This sequence is compared to various Desulfovibrio ferredoxin sequences and to the sequence and three-dimensional structure of Peptococcus aerogenes ferredoxin. Evidence of gene duplication is indicated. The requirement of some sequence features in the ferredoxin for an interaction process with its electron transfer partner, cytochrome c3, is postulated in the discussion.     

Sulfide Production from Cysteine by Desulfovibrio desulfuricans

Two rumen nitrate-reducing isolates of Desulfovibrio desulfuricans were found to hydrolyze cysteine with the production of sulfide and pyruvate. When cultured on agar medium containing yeast extract with nitrate as the primary electron acceptor and ferrous chloride as the indicator, blackening of colonies occurred. The blackening of colonies appeared sooner and was more intense when either cysteine or sulfate was added to the culture medium with nitrate present.     

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. Its amino acid composition suggests that it is homologous with the other Desulfovibrio ferredoxins. The rubredoxin is also an acidic protein of 6,000 molecular weight and contains one atom of iron and four cysteine residues per molecule. The amino acid composition and molecular weight of the cytochrome c3 from D. desulfuricans (strain Norway 4) are reported. Its spectral properties are very similar to those of the other cytochromes c3 (molecular weight, 13,000) of Desulfovibrio and show that it contains four hemes per molecule. This cytochrome has a very low redox potential and acts as a carrier in the coupling of hydrogenase and thiosulfate reductase in extracts of Desulfovibrio gigas and Desulfovibrio desulfuricans (Norway strain) in contrast to D. gigas cytochrome c3 (molecular weight, 13,000). A comparison of the activities of the cytochrome c3 (molecular weight, 13,000) of D. gigas and that of D. desulfuricans in this reaction suggests that these homologous proteins can have different specificity in the electron transfer chain of these bacteria.     

Primary structure of the two (4 Fe-4 S) clusters ferredoxin from Desulfovibrio desulfuricans (strain Norway 4)

The primary structure of a ferredoxin isolated from D. desulfuricans Norway strain, which we called ferredoxin II (Fd II) has been elucidated. This ferredoxin is a dimer constituted of two identical subunits of molecular weight 6000. In ferredoxin II two (4 Fe-4 S) centers are present per subunit instead of one (Fe-S) center as is the case for the other ferredoxins isolated from Desulfovibrio and for Fd I from the same organism. The comparison of amino-acid sequences shows that ferredoxin II presents more homologies with clostridial type ferredoxin than with the ferredoxins from D. gigas and D. africanus.     

Extraction of periplasmic hydrogenase from Desulfovibrio vulgaris (Hildenborough)

Abstract Periplasmic hydrogenase from Desulfovibrio vulgaris (Hildenborough) was extracted according to the method of van der Westen [8] and the effect of trace minerals on the extractability of this enzyme was investigated. The final growth yields in the presence or absence of trace minerals were the same; however, the growth was much faster and the amount of periplasmic hydrogenase extracted was significantly lower in the presence of trace minerals. Polyacrylamide gel electrophoresis showed the presence of 2 hydrogenases in D. vulgaris , one soluble and the other possibly membrane-bound.     

Carboxy-terminal processing of the large subunit of [Fe] hydrogenase from Desulfovibrio desulfuricans ATCC 7757

hydA and hydB, the genes encoding the large (46-kDa) and small (13. 5-kDa) subunits of the periplasmic [Fe] hydrogenase from Desulfovibrio desulfuricans ATCC 7757, have been cloned and sequenced. The deduced amino acid sequence of the genes product showed complete identity to the sequence of the well-characterized [Fe] hydrogenase from the closely related species Desulfovibrio vulgaris Hildenborough (G. Voordouw and S. Brenner, Eur. J. Biochem. 148:515-520, 1985). The data show that in addition to the well-known signal peptide preceding the NH2 terminus of the mature small subunit, the large subunit undergoes a carboxy-terminal processing involving the cleavage of a peptide of 24 residues, in agreement with the recently reported data on the three-dimensional structure of the enzyme (Y. Nicolet, C. Piras, P. Legrand, E. C. Hatchikian, and J. C. Fontecilla-Camps, Structure 7:13-23, 1999). We suggest that this C-terminal processing is involved in the export of the protein to the periplasm.     

Iron-sulphur cluster composition and redox properties of two ferredoxins from Desulfovibrio desulfuricans Norway strain

Two ferredoxins from Desulfovibrio desulfuricans, Norway Strain, were investigated by EPR spectroscopy. Ferredoxin I appears to be a conventional [4Fe-4S]2+;1+ ferredoxin, with a midpoint reduction potential of -374 mV at pH 8. Ferredoxin II when reduced, at first showed a more complex spectrum, indicating an interaction between two [4Fe-4S] clusters, and probably, has two clusters per protein subunit. Upon reductive titration ferredoxin II changed to give a spectrum in which no intercluster interaction was seen. The midpoint potentials of the native and modified ferredoxin at pH 8 were estimated to be -500 and -440 mV, respectively.