Further characterization of the [Fe]-hydrogenase from Desulfovibrio desulfuricans ATCC 7757.

The properties of the periplasmic hydrogenase from Desulfovibrio desulfuricans ATCC 7757, previously reported to be a single-subunit protein [Glick, B. R., Martin, W. G., and Martin, S. M. (1980) Can. J. Microbiol. 26, 1214-1223] were reinvestigated. The pure enzyme exhibited a molecular mass of 53.5 kDa as measured by analytical ultracentrifugation and was found to comprise two different subunits of 42.5 kDa and 11 kDa, with serine and alanine as N-terminal residues, respectively. The N-terminal amino acid sequences of its large and small subunits, determined up to 25 residues, were identical to those of the Desulfovibrio vulgaris Hildenborough [Fe]-hydrogenase. D. desulfuricans ATCC 7757 hydrogenase was free of nickel and contained 14.0 atoms of iron and 14.4 atoms of acid-labile sulfur/molecule and had E400, 52.5 mM-1.cm-1. The purified hydrogenase showed a specific activity of 62 kU/mg of protein in the H2-uptake assay, and the H2-uptake activity was higher than H2-evolution activity. The enzyme isolated under aerobic conditions required incubation under reducing conditions to express its maximum activity both in the H2-uptake and 2H2/1H2 exchange reaction. The ratio of the activity of activated to as-isolated hydrogenase was approximately 3. EPR studies allowed the identification of two ferredoxin-type [4Fe-4S]1+ clusters in hydrogenase samples reduced by hydrogen. In addition, an atypical cluster exhibiting a rhombic signal (g values 2.10, 2.038, 1.994) assigned to the H2-activating site in other [Fe]-hydrogenases was detected in partially reduced samples. Molecular properties, EPR spectroscopy, catalytic activities with different substrates and sensitivity to hydrogenase inhibitors indicated that D. desulfuricans ATCC 7757 periplasmic hydrogenase is a [Fe]-hydrogenase, similar in most respects to the well characterized [Fe]-hydrogenase from D. vulgaris Hildenborough.     

Electron transfer mechanism and interaction studies between cytochrome C3 and ferredoxin

Ferredoxin, cytochrome c3 and hydrogenase are specific partners of the sulfate reduction pathway of Desulfovibrio desulfuricans Norway and might be exemplary for electron exchange mechanism studies. Cytochrome c3 contains four low redox potential haems for 13 000 molecular weight. Two ferredoxins isolated from the same bacteria are dimers of 6 000 molecular weight per subunit (Ferredoxin I: one (4 Fe-4S) cluster per subunit, ferredoxin II: two (4 Fe-4 S) clusters per subunit). The amino acid sequence of ferredoxin I is reported and compared to the ferredoxin II sequence. The structural characteristics of ferredoxins and cytochrome c3 should allow a discussion on the nature of the interaction. 1H-NMR spectra of ferredoxin I and cytochrome c3 in the absence and presence of ferredoxin are presented.     

The interaction of polymeric viologens with hydrogenases from Desulfovibrio desulfuricans and Clostridium pasteurianum.

The interaction between hydrogenases from either Desulfovibrio desulfuricans or Clostridium pasteurianum and electron donors methyl viologen or polymeric viologens was examined. Extracts from each organism contained a single gel electophoretic band of active hydrogenase. The hydrogenase of D. desulfuricans was much more stable than that of Cl. pasteurianum. With methyl viologen apparent Km and Vm values were 0.5 mM and 0.62 mumole H2/min per milligram protein for the Cl. pasteurianum and 0.7 and 6.2 mumole H2/min per milligram protein, respectively, for the D. desulfuricans enzyme. The hydrogenases bound the polymeric viologens more tightly than methyl viologen, more so for the enzyme of D. desulfuricans than for Cl. pasteurianum. Maximal rate of hydrogen production was less with the polymeric than with methyl viologen. The results suggest that the D. desulfuricans enzyme in conjunction wiion than that from Cl. pasteurianum.     

Purification and catalytic properties of a CO-oxidizing:H2-evolving enzyme complex from Carboxydothermus hydrogenoformans.

From the membrane fraction of the Gram-positive bacterium Carboxydothermus hydrogenoformans, an enzyme complex catalyzing the conversion of CO to CO2 and H2 was purified. The enzyme complex showed maximal CO-oxidizing:H2-evolving enzyme activity with 5% CO in the headspace (450 U per mg protein). Higher CO concentrations inhibited the hydrogenase present in the enzyme complex. For maximal activity, the enzyme complex had to be activated by either CO or strong reductants. The enzyme complex also catalyzed the CO- or H2-dependent reduction of methylviologen at 5900 and 180 U per mg protein, respectively. The complex was found to be composed of six hydrophilic and two hydrophobic polypeptides. The amino-terminal sequences of the six hydrophilic subunits were determined allowing the identification of the encoding genes in the preliminary genome sequence of C. hydrogenoformans. From the sequence analysis it was deduced that the enzyme complex is formed by a Ni-containing carbon monoxide dehydrogenase (CooS), an electron transfer protein containing four [4Fe-4S] clusters (CooF) and a membrane bound [NiFe] hydrogenase composed of four hydrophilic subunits and two membrane integral subunits. The hydrogenase part of the complex shows high sequence similarity to members of a small group of [NiFe] hydrogenases with sequence similarity to energy conserving NADH:quinone oxidoreductases. The data support a model in which the enzyme complex is composed of two catalytic sites, a CO-oxidizing site and a H2-forming site, which are connected via a different iron-sulfur cluster containing electron transfer subunits. The exergonic redox reaction catalyzed by the enzyme complex in vivo has to be coupled to energy conservation, most likely via the generation of a proton motive force.     

Purification and catalytic properties of Ech hydrogenase from Methanosarcina barkeri.

Methanosarcina barkeri has recently been shown to produce a multisubunit membrane-bound [NiFe] hydrogenase designated Ech (Escherichia coli hydrogenase 3) hydrogenase. In the present study Ech hydrogenase was purified to apparent homogeneity in a high yield. The enzyme preparation obtained only contained the six polypeptides which had previously been shown to be encoded by the ech operon. The purified enzyme was found to contain 0.9 mol of Ni, 11.3 mol of nonheme-iron and 10.8 mol of acid-labile sulfur per mol of enzyme. Using the purified enzyme the kinetic parameters were determined. The enzyme catalyzed the H2 dependent reduction of a M. barkeri 2[4Fe-4S] ferredoxin with a specific activity of 50 U x mg protein-1 at pH 7.0 and exhibited an apparent Km for the ferredoxin of 1 microM. The enzyme also catalyzed hydrogen formation with the reduced ferredoxin as electron donor at a rate of 90 U x mg protein-1 at pH 7.0. The apparent Km for the reduced ferredoxin was 7.5 microM. Reduction or oxidation of the ferredoxin proceeded at similar rates as the reduction or oxidation of oxidized or reduced methylviologen, respectively. The apparent Km for H2 was 5 microM. The kinetic data strongly indicate that the ferredoxin is the physiological electron donor or acceptor of Ech hydrogenase. Ech hydrogenase amounts to about 3% of the total cell protein in acetate-grown, methanol-grown or H2/CO2-grown cells of M. barkeri, as calculated from quantitative Western blot experiments. The function of Ech hydrogenase is ascribed to ferredoxin-linked H2 production coupled to the oxidation of the carbonyl-group of acetyl-CoA to CO2 during growth on acetate, and to ferredoxin-linked H2 uptake coupled to the reduction of CO2 to the redox state of CO during growth on H2/CO2 or methanol.     

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.     

Microbial reduction of technetium by Escherichia coli and Desulfovibrio desulfuricans: enhancement via the use of high-activity strains and effect of process parameters

Escherichia coli and Desulfovibrio desulfuricans reduce Tc(VII) (TcO(4)(-)) with formate or hydrogen as electron donors. The reaction is catalyzed by the hydrogenase component of the formate hydrogenlyase complex (FHL) of E. coli and is associated with a periplasmic hydrogenase activity in D. desulfuricans. Tc(VII) reduction in E. coli by H(2) and formate was either inhibited or repressed by 10 mM nitrate. By contrast, Tc(VII) reduction catalyzed by D. desulfuricans was less sensitive to nitrate when formate was the electron donor, and unaffected by 10 mM or 100 mM nitrate when H(2) was the electron donor. The optimum pH for Tc(VII) reduction by both organisms was 5.5 and the optimum temperature was 40 degrees C and 20 degrees C for E. coli and D. desulfuricans, respectively. Both strains had an apparent K(m) for Tc(VII) of 0.5 mM, but Tc(VII) was removed from a solution of 300 nM TcO(4)(-) within 30 h by D. desulfuricans at the expense of H(2). The greater bioprocess potential of D. desulfuricans was shown also by the K(s) for formate (>25 mM and 0.5 mM for E. coli and D. desulfuricans, respectively), attributable to the more accessible, periplasmic localization of the enzyme in the latter. The relative rates of Tc(VII) reduction for E. coli and D. desulfuricans (with H(2)) were 12.5 and 800 micromol Tc(VII) reduced/g biomass/h, but the use of an E. coli HycA mutant (which upregulates FHL activities by approx. 50%) had a similarly enhancing effect on the rate of Tc reduction. The more rapid reduction of Tc(VII) by D. desulfuricans compared with the E. coli strains was also shown using cells immobilized in a hollow-fiber reactor, in which the flow residence times sustaining steady-state removal of 80% of the radionuclide were 24.3 h for the wild-type E. coli, 4.25 h for the upregulated mutant, and 1.5 h for D. desulfuricans.     

Nonaheme cytochrome c, a new physiological electron acceptor for [Ni,Fe] hydrogenase in the sulfate-reducing bacterium Desulfovibrio desulfuricans Essex: primary sequence, molecular parameters, and redox properties

A nonaheme cytochrome c was purified to homogeneity from the soluble and the membrane fractions of the sulfate-reducing bacterium Desulfovibrio desulfuricans Essex. The gene encoding for the protein was cloned and sequenced. The primary structure of the multiheme protein was highly homologous to that of the nonaheme cytochrome c from D. desulfuricans ATCC 27774 and to that of the 16-heme HmcA protein from Desulfovibrio vulgaris Hildenborough. The analysis of the sequence downstream of the gene encoding for the nonaheme cytochrome c from D. desulfuricans Essex revealed an open reading frame encoding for an HmcB homologue. This operon structure indicated the presence of an Hmc complex in D. desulfuricans Essex, with the nonaheme cytochrome c replacing the 16-heme HmcA protein found in D. vulgaris. The molecular and spectroscopic parameters of nonaheme cytochrome c from D. desulfuricans Essex in the oxidized and reduced states were analyzed. Upon reduction, the pI of the protein changed significantly from 8.25 to 5.0 when going from the Fe(III) to the Fe(II) state. Such redox-induced changes in pI have not been reported for cytochromes thus far; most likely they are the result of a conformational rearrangement of the protein structure, which was confirmed by CD spectroscopy. The reactivity of the nonaheme cytochrome c toward [Ni,Fe] hydrogenase was compared with that of the tetraheme cytochrome c(3); both the cytochrome c(3) and the periplasmic [Ni,Fe] hydrogenase originated from D. desulfuricans Essex. The nonaheme protein displayed an affinity and reactivity toward [Ni,Fe] hydrogenase [K(M) = 20.5 +/- 0.9 microM; v(max) = 660 +/- 20 nmol of reduced cytochrome min(-1) (nmol of hydrogenase)(-1)] similar to that of cytochrome c(3) [K(M) = 12.6 +/- 0.7 microM; v(max) = 790 +/- 30 nmol of reduced cytochrome min(-1) (nmol of hydrogenase)(-1)]. This shows that nonaheme cytochrome c is a competent physiological electron acceptor for [Ni,Fe] hydrogenase.     

Hydrogenase activities in cyanobacteria

In the unicellular Anacystis nidulans, the expression of both the H2-uptake (with phenazine methosulfate or methylene blue as the electron acceptor) and H2-evolution (with methyl viologen reduced by Na2S2O4) was dependent on Ni in the culture medium. In extracts from Anacystis and Anabaena 7119, H2-evolution and uptake activities were strongly inhibited by Cu2+, p-chloromercuribenzoate and HgCl2 suggesting that at least one functional SH-group is involved in catalysis by hydrogenase. Extracts from the N2-fixing Anabaena 7119 contained two different hydrogenase fractions which could be separated by chromatography on DE-52 cellulose using a linear NaCl concentration gradient. The fraction eluting with 0.13 M NaCl from the column catalyzed only the uptake of H2 with methylene blue as the electron acceptor but virtually not the evolution of H2 ("uptake" hydrogenase fraction). The fraction eluting at a NaCl strength of 0.195 M catalyzed both H2-uptake with methylene blue and H2-evolution with reduced methyl viologen ("reversible" hydrogenase fraction). Growth under anaerobic conditions drastically enhanced the activity levels of the "reversible" but not of the "uptake" hydrogenase fraction. The "uptake" hydrogenase but not the "reversible" protein was activated by reduced thioredoxin. It is suggested that thioredoxin activates the H2-uptake by the membrane-bound "uptake" hydrogenase also in intact cells. The occurrence of the number of hydrogenases in cyanobacteria will be reevaluated.     

Characterization of a native subunit of the NAD-linked hydrogenase isolated from a mutant of Alcaligenes eutrophus H16

The cytoplasmic, NAD-linked hydrogenase of Alcaligenes eutrophus H16 consists of four non-identical subunits. From the mutant strain HF14, defective in this enzyme, a protein was isolated that reacted with specific antibodies raised against the wild-type hydrogenase; the reaction type was of partial identity. The same protein was also tested with specific antibodies raised against each of the four denatured subunits of the wild-type hydrogenase and was found to be completely identical with the second largest subunit; it reacted weakly with the antibody against the largest subunit and not at all with the antibody against the small subunits. In SDS-polyacrylamide gel electrophoresis the protein of the mutant migrated as a single polypeptide and corresponded to the second largest subunit of soluble hydrogenase with Mr = 56,000. The mutant enzyme strongly differed from the wild-type hydrogenase in its binding behaviour to chromatographic gels. It had pronounced hydrophobic properties and bound strongly to phenyl-Sepharose; it had high affinity to triazin dye gels. Enzymatically the polypeptide was totally inactive with NAD as electron acceptor, but showed weak residual activities with methylene blue, ferricyanide and cytochrome c. The protein also contained nickel; however, because of the instability of this polypeptide the amount varied between 0.2-1.4 nickel per enzyme molecule. As shown by ESR studies, the iron is organized in a [4Fe-4S] cluster but is partially present also in the 3Fe-form. No nickel signal could be detected. The role of the polypeptide subunit for hydrogen activation in the intact hydrogenase is discussed.     

Periplasmic oxygen reduction by Desulfovibrio species

Washed cells of Desulfovibrio vulgaris strain Marburg (DSM 2119) reduced oxygen to water with H(2) as electron donor at a mean rate of 253 nmol O(2) min(-1) (mg protein)(-1). After separating the periplasm from the cells, more than 60% of the cytochrome c activity and 90% of the oxygen-reducing activity were found in the periplasmic fraction. Oxygen reduction and the reduction of cytochrome c with H(2) were inhibited by CuCl(2). After further separation of the periplasm by ultrafiltration (exclusion sizes 30, 50, and 100 kDa), oxygen reduction with H(2) occurred with the retentates only. Ascorbate plus tetramethyl-p-phenylenediamine (TMPD), however, were also oxidized by the filtrates. The stoichiometry of 1 mol O(2) reduced per 2 mol ascorbate oxidized indicated the formation of water. Our experiments present evidence that in D. vulgaris periplasmic hydrogenase and cytochrome c play a major role in oxygen reduction. Preliminary studies with other Desulfovibrio species indicated a similar function of periplasmic c-type cytochromes in D. desulfuricans CSN and D. termitidis KH1.     

Purification and properties of the membrane-bound hydrogenase from N2-fixing Alcaligenes latus

The nitrogen-fixing, aerobic hydrogen-oxidizing bacterium Alcaligenes latus forms hydrogenase when growing lithoautotrophically with hydrogen as electron donor and carbon dioxide as sole carbon source or when growing heterotrophically with N2 as sole nitrogen source. The hydrogenase is membrane-bound and relatively oxygen-sensitive. The enzymes formed under both conditions are identical on the basis of the following criteria: molecular mass, mobility in polyacrylamide gel electrophoresis, Km value for hydrogen (methylene blue reduction), stability properties, localization, and cross-reactivity to antibodies raised against the 'autotrophic' hydrogenase. The hydrogenase was solubilized by Triton X-100 and deoxycholate treatment and purified by ammonium sulfate precipitation and chromatography on Phenyl-Sepharose C1-4B, DEAE-Sephacel and Matrix Gel Red A under hydrogen to homogeneity to a specific activity of 113 mumol H2 oxidized/min per mg protein (methylene blue reduction). SDS gel electrophoresis revealed two nonidentical subunits of molecular weights of 67 000 and 34 000, corresponding to a total molecular weight of 101 000. The pure enzyme was able to reduce FAD, FMN, riboflavin, flavodoxin isolated from Megasphaera elsdenii, menadione and horse heart cytochrome c as well as various artificial electron acceptors. The reversibility of the hydrogenase function was demonstrated by H2 evolution from reduced methyl viologen.     

The iron-sulphur centres of soluble hydrogenase from Alcaligenes eutrophus.

The soluble hydrogenase (hydrogen:NAD+ oxidoreductase (EC 1.12.1.2) from Alcaligenes eutrophus has been purified to homogeneity by an improved procedure, which includes preparative electrophoresis as final step. The specific activity of 57 mumol H2 oxidized/min per mg protein was achieved and the yield of pure enzyme from 200 g cells (wet weight) was about 16 mg/purification. After removal of non-functional iron, analysis of iron and acid-labile sulphur yielded average values of 11.5 and 12.9 atoms/molecule of enzyme, respectively. p-Chloromercuribenzoate was a strong inhibitor of hydrogenase and apparently competed with NAD not with H2. Chelating agents, CO and O2 failed to inhibit enzyme activity. The oxidized hydrogenase showed an EPR spectrum with a small signal at g = 2.02. On reduction the appearance of a high temperature (50--77 K) signal at g = 2.04, 1.95 and a more complex low temperature (less than 30 K) spectrum at g = 2.04, 2.0, 1.95, 1.93, 1.86 was observed. The pronounced temperature dependence and characteristic lineshape of the signals obtained with hydrogenase in 80--85% dimethylsulphoxide demonstrated that iron-sulphur centres of both the [2Fe-2S] and [4Fe-4S] types are present in the enzyme. Quantitation of the EPR signals indicated the existence of two identical centres each of the [4Fe-4S] and of the [2Fe-2S] type. The midpoint redox potentials of the [4Fe-4S] and the [2Fe-2S] centres were determined to be -445 mV and -325 mV, respectively. Spin coupling between two centres, indicated by the split feature of the low temperature spectrum of the native hydrogenase around g = 1.95, 1.93, has been established by power saturation studies. On reduction of the [Fe-4S] centres, the electron spin relaxation rate of the [2Fe-2S] centres was considerably increased. Treatment of hydrogenase with CO caused no change in EPR spectra.     

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.     

Purification and characterization of ferredoxin from Desulfovibrio vulgaris Miyazaki

Two ferredoxins, Fd I and Fd II, were isolated and purified from Desulfovibrio vulgaris Miyazaki. The major component, Fd I, is an iron-sulfur protein of Mr 12,000, composed of two identical subunits. The absorption spectra of Fd I and Fd II have a broad absorption shoulder near 400 nm characteristic of iron-sulfur proteins. The purity index, A400/A280, of Fd I is 0.69, and its millimolar absorption coefficient at 400 nm is 3.73 per Fe. It contains two redox centers with discrete redox behaviors. The amino acid composition and the N-terminal sequence of Fd I are similar to those of Fd III of Desulfovibrio africanus Benghazi and Fd II of Desulfovibrio desulfuricans Norway. Fd I does not serve as an electron carrier for the hydrogenase of D. vulgaris Miyazaki, but it serves as a carrier for pyruvate dehydrogenase of this bacterium. The evolution of H2 from pyruvate was observed by a reconstructed system containing purified hydrogenase, cytochrome C3, Fd I, partially purified pyruvate dehydrogenase, and CoA. The H2-sulfite reducing system can be reconstructed from the purified hydrogenase, cytochrome C3, Fd I and desulfoviridin (sulfite reductase), but the reaction rate is very slow compared to that of the crude extract at the same molar ratio of the components.     

Purification of the membrane-bound hydrogenase of Escherichia coli.

The membrane-bound hydrogenase (EC class 1.12) of aerobically grown Escherichia coli cells was solubilized by treatment with deoxycholate and pancreatin. The enzyme was further purified to electrophoretic homogeneity by chromoatographic methods, including hydrophobic-interaction chromatography, with a yield of 10% as judged by activity and an overall purification of 2140-fold. The hydrogenase was a dimer of identical subunits with a mol.wt. of 113,000 and contained 12 iron and 12 acid-labile sulphur atoms per molecule. The epsilon 400 was 49,000M-1 . cm-1. The hydrogenase catalysed both H2 evolution and H2 uptake with a variety of artificial electron carriers, but would not interact with flavodoxin, ferredoxin or nicotinamide and flavin nucleotides. We were unable to identify any physiological electron carrier for the hydrogenase. With Methyl Viologen as the electron carrier, the pH optimum for H2 evolution and H2 uptake was 6.5 and 8.5 respectively. The enzyme was stable for long periods at neutral pH, low temperatures and under anaerobic conditions. The half-life of the hydrogenase under air at room temperature was about 12 h, but it could be stabilized by Methyl Viologen and Benzyl Viologen, both of which are electron carriers for the enzyme, and by bovine serum albumin. The hydrogenase was strongly inhibited by carbon monoxide (Ki = 1870Pa), heavy-metal salts and high concentrations of buffers, but was resistant to inhibition by thiol-blocking and metal-complexing reagents. These aerobically grown E. coli cells lacked formate hydrogenlyase activity and cytochrome c552.     

Comparison of the properties of two hydrogenases from the photosynthetic bacterium Chromatium vinosum

Chromatium vinosum hydrogenases I and II were purified to specific activities of 9.6 and 28.0 units/mg protein, respectively. They have the same isoelectric point (pI = 4.1), and their visible spectra are typical of iron-sulfur proteins. Hydrogenase II in general was more stable than hydrogenase I. Both enzymes lost their activities slowly during storage in air, and this inactivation was more apparent in preparations of hydrogenase I. Bovine serum albumin helped to stabilize hydrogenase I against thermal and storage inactivation. The pH optima of H2-evolution activity of hydrogenases I and II were 7.4 and 5.4, respectively. Neither enzyme was able to evolve H2 from reduced ferredoxins as the sole electron carrier, but ferredoxins had an effect on the activity with methyl viologen as carrier to hydrogenase I. None of the natural compounds tested was able to serve as a physiological donor for H2 production. Hydrogenase I was more susceptible than hydrogenase II to inhibition by heavy metal ions and other enzyme inhibitors. Both enzymes were reversibly inhibited by CO with Ki values of 12 and 6 Torr for hydrogenase I and II, respectively. Hydrogenase I was more sensitive to denaturation by urea and guanidinium chloride while hydrogenase II was more susceptible to sodium dodecyl sulfate. Both enzymes were rapidly and irreversibly inactivated by dimethyl sulfoxide. Hydrogenase I evolved H2 from methyl viologen and ferredoxin photoreduced by chloroplasts. The enzymes differed in their iron and acid-labile sulfur contents.     

Characterization of the Desulfovibrio desulfuricans ATCC 27774 DsrMKJOP complex--a membrane-bound redox complex involved in the sulfate respiratory pathway

Sulfate-reducing organisms use sulfate as an electron acceptor in an anaerobic respiratory process. Despite their ubiquitous occurrence, sulfate respiration is still poorly characterized. Genome analysis of sulfate-reducing organisms sequenced to date permitted the identification of only two strictly conserved membrane complexes. We report here the purification and characterization of one of these complexes, DsrMKJOP, from Desulfovibrio desulfuricans ATCC 27774. The complex has hemes of the c and b types and several iron-sulfur centers. The corresponding genes in the genome of Desulfovibrio vulgaris were analyzed. dsrM encodes an integral membrane cytochrome b; dsrK encodes a protein homologous to the HdrD subunit of heterodisulfide reductase; dsrJ encodes a triheme periplasmic cytochrome c; dsrO encodes a periplasmic FeS protein; and dsrM encodes another integral membrane protein. Sequence analysis and EPR studies indicate that DsrJ belongs to a novel family of multiheme cytochromes c and that its three hemes have different types of coordination, one bis-His, one His/Met, and the third a very unusual His/Cys coordination. The His/Cys-coordinated heme is only partially reduced by dithionite. About 40% of the hemes are reduced by menadiol, but no reduction is observed upon treatment with H2 and hydrogenase, irrespective of the presence of cytochrome c3. The aerobically isolated Dsr complex displays an EPR signal with similar characteristics to the catalytic [4Fe-4S]3+ species observed in heterodisulfide reductases. Further five different [4Fe-4S](2+/1+) centers are observed during a redox titration followed by EPR. The role of the DsrMKJOP complex in the sulfate respiratory chain of Desulfovibrio spp. is discussed.     

Spectroscopic investigation and determination of reactivity and structure of the tetraheme cytochrome c3 from Desulfovibrio desulfuricans Essex 6.

Cytochrome c3, a small (14-kDa) soluble tetraheme protein was isolated from the periplasmic fraction of Desulfovibrio desulfuricans strain Essex 6. Its major physiological function appears to be that of an electron carrier for the periplasmic hydrogenase. It has been also shown to interact with the high-molecular-mass cytochrome complex in the cytoplasmic membrane, which eventually feeds electrons into the membraneous quinone pool, as well as with the membrane-associated dissimilatory sulfite reductase. The EPR spectra show features of four different low-spin Fe(III) hemes. Orthorhombic crystals of cytochrome c3 were obtained and X-ray diffraction data were collected to below 2 A resolution. The structure was solved by molecular replacement using cytochrome c3 from D. desulfuricans ATCC 27774 as a search model.     

Purification and properties of soluble hydrogenase from Alcaligenes eutrophus H 16.

The soluble hydrogenase (hydrogen: NAD+ oxidoreductase, EC 1.12.1.2) from Alcaligenes eutrophus H 16 was purified 68-fold with a yield of 20% and a final specific activity (NAD reduction) of about 54 mumol H2 oxidized/min per mg protein. The enzyme was shown to be homogenous by polyacrylamide gel electrophoresis. Its molecular weight and isoelectric point were determined to be 205 000 and 4.85 respectively. The oxidized hydrogenase, as purified under aerobic conditions, was of high stability but not reactive. Reductive activation of the enzyme by H2, in the presence of catalytic amounts of NADH, or by reducing agents caused the hydrogenase to become unstable. The purified enzyme, in its active state, was able to reduce NAD, FMN, FAD, menaquinone, ubiquinone, cytochrome c, methylene blue, methyl viologen, benzyl viologen, phenazine methosulfate, janus green, 2,6-dichlorophenoloindophenol, ferricyanide and even oxygen. In addition to hydrogenase activitiy, the enzyme exhibited also diaphorase and NAD(P)H oxidase activity. The reversibility of hydrogenase function (i.e. H2 evolution from NADH, methyl viologen and benzyl viologen) was demonstrated. With respect to H2 as substrate, hydrogenase showed negative cooperativity; the Hill coefficient was n = 0.4. The apparent Km value for H2 was found to be 0.037 mM. The absorption spectrum of hydrogenase was typical for non-heme iron proteins, showing maxima (shoulders) at 380 and 420 nm. A flavin component could be extracted from native hydrogenase characterized by its absorption bands at 375 and 447 nm and a strong fluorescense at 526 nm.