Role of hydrogenases 1 and 2 in the hydrogen dependent nitrate respiration by Escherichia coli

The study of Escherichia coli mutants synthesizing either hydrogenase 1 (HDK203) or hydrogenase 2 (HDK103) showed that the nitrate-dependent uptake of hydrogen by E. coli cells can be accomplished through the action of either of these hydrogenases. The capability of the cells for hydrogen-dependent nitrate respiration was found to be dependent on the growth conditions. E. coli cells grown anaerobically without nitrate in the presence of glucose were potentially capable of nitrate-dependent hydrogen consumption. The cells grown anaerobically in the presence of nitrate exhibited a much lower capability for nitrate-dependent hydrogen consumption. The inhibitory effect of nitrate on this capability of bacterial cells was either weak (the mutant HDK203) or almost absent (the mutant HDK103) when the cells were grown in the presence of peptone and hydrogen. Hydrogen stimulated the growth of the wild-type strain and the mutant HDK103 (but not the mutant HDK203) cultivated in the medium with nitrate and peptone. These data suggest that hydrogenase 2 is much more active in catalyzing the nitrate-dependent hydrogen consumption than is hydrogenase 1.     

The Involvement of Hydrogenases 1 and 2 in the Hydrogen-Dependent Nitrate Respiration of Escherichia coli

The study of Escherichia coli mutants synthesizing either hydrogenase 1 (HDK203) or hydrogenase 2 (HDK103) showed that the nitrate-dependent uptake of hydrogen by E. coli cells can be accomplished through the action of either of these hydrogenases. The capability of the cells for hydrogen-dependent nitrate respiration was found to depend on the growth conditions. E. coli cells grown anaerobically without nitrate in the presence of glucose were potentially capable of nitrate-dependent hydrogen consumption. The cells grown anaerobically in the presence of nitrate exhibited a much lower capability for nitrate-dependent hydrogen consumption. The inhibitory effect of nitrate on this capability of bacterial cells was either weak (the mutant HDK203) or almost absent (the mutant HDK103) when the cells were grown in the presence of peptone and hydrogen. Hydrogen stimulated the growth of the wild-type strain and the mutant HDK103 (but not the mutant HDK203) cultivated in the medium with nitrate and peptone. These data suggest that hydrogenase 2 is much more active in catalyzing nitrate-dependent hydrogen consumption than hydrogenase 1.     

H2 consumption by Escherichia coli coupled via hydrogenase 1 or hydrogenase 2 to different terminal electron acceptors

Hydrogen uptake in the presence of various terminal electron acceptors was examined in Escherichia coli mutants synthesizing either hydrogenase 1 or hydrogenase 2. Both hydrogenases mediated nitrate-dependent H2 consumption but neither of them was coupled with nitrite. Unlike hydrogenase 2, hydrogenase 1 demonstrated poor activity with electron acceptors of low midpoint redox potential. Oxygen-linked H2 uptake via hydrogenase 1 was observed over a wide range of air concentrations. Hydrogenase 2 catalyzed this reaction only at low air concentrations. Thus, hydrogenase 1 works in cells at higher redox potential, being more tolerant to oxygen than hydrogenase 2.     

The mechanisms of H2 activation and CO binding by hydrogenase I and hydrogenase II of Clostridium pasteurianum

Hydrogenase I (bidirectional) and hydrogenase II (uptake) of Clostridium pasteurianum have been investigated by electron paramagnetic resonance (EPR) spectroscopy, in the presence and absence of the inhibitor, CO. These hydrogenases contain both a novel type of iron-sulfur cluster (H), which is the proposed site of H2 catalysis, and ferredoxin-type [4Fe-4S] clusters (F). The results show that the H clusters of these two hydrogenases have very different properties. The H cluster of oxidized hydrogenase II (Hox-II) exhibits three distinct EPR signals, two of which are pH-dependent. Hox-II binds CO reversibly to give a single, pH-independent species with a novel, rhombic EPR spectrum. The H cluster of reduced hydrogenase II (Hred-II) does not react with CO. In contrast, the EPR spectrum of Hox-I appears homogeneous and independent of pH. Hox-I has a much lower affinity for CO than Hox-II, and binds CO irreversibly to give an axial EPR signal. Hred-I also binds CO irreversibly. The EPR spectra of Fred-I and Fred-II show little or no change after CO treatment. Prior exposure to CO does not affect the catalytic activity of the reduced or oxidized hydrogenases when assayed in the absence of CO, but both enzymes are irreversibly inactivated if CO is present during catalysis. Mechanisms for H2 activation by hydrogenase I and hydrogenase II are proposed from the determined midpoint potentials (Em, pH 8.0) of H-I and H-II (Em approximately -400 mV, -CO; approximately -360 mV, +CO), F-I (Em = -420 mV, +/- CO), and F-II (Em = -180 mV, +/- CO). These allow one to rationalize the different modes of CO binding to the two hydrogenases and suggest why hydrogenase II preferentially catalyzes H2 oxidation. The results are discussed in light of recent spectroscopic data on the structures of the two H clusters.     

Characterization of catalytic properties of hydrogenase isolated from the unicellular cyanobacterium Gloeocapsa alpicola CALU 743

The main catalytic properties of the Hox type hydrogenase isolated from the Gloeocapsa alpicola cells have been studied. The enzyme effectively catalyzes reactions of oxidation and evolution of H2 in the presence of methyl viologen (MV) and benzyl viologen (BV). The rates of these reactions in the interaction with the physiological electron donor/acceptor NADH/NAD+ are only 3-8% of the MV(BV)-dependent values. The enzyme interacts with NADP+ and NADPH, but is more specific to NAD+ and NADH. Purification of the hydrogenase was accompanied by destruction of its multimeric structure and the loss of ability to interact with pyridine nucleotides with retained activity of the hydrogenase component (HoxYH). To show the catalytic activity, the enzyme requires reductive activation, which occurs in the presence of H2, and NADH accelerates this process. The final hydrogenase activity depends on the redox potential of the activation medium (E(h)). At pH 7.0, the enzyme activity in the MV-dependent oxidation of H2 increased with a decrease in E(h) from -350 mV and reached the maximum at E(h) of about -390 mV. However, the rate of H2 oxidation in the presence of NAD+ in the E(h) range under study was virtually constant and equal to 7-8% of the maximal rate of H2 oxidation in the presence of MV.     

Characterization of the CO-induced, CO-tolerant hydrogenase from Rhodospirillum rubrum and the gene encoding the large subunit of the enzyme.

In the presence of carbon monoxide, the photosynthetic bacterium Rhodospirillum rubrum induces expression of proteins which allow the organism to metabolize carbon monoxide in the net reaction CO + H2O --> CO2 + H2. These proteins include the enzymes carbon monoxide dehydrogenase (CODH) and a CO-tolerant hydrogenase. In this paper, we present the complete amino acid sequence for the large subunit of this hydrogenase and describe the properties of the crude enzyme in relation to other known hydrogenases. The amino acid sequence deduced from the CO-induced hydrogenase large-subunit gene (cooH) shows significant similarity to large subunits of other Ni-Fe hydrogenases. The closest similarity is with HycE (58% similarity and 37% identity) from Escherichia coli, which is the large subunit of an Ni-Fe hydrogenase (isoenzyme 3). The properties of the CO-induced hydrogenase are unique. It is exceptionally resistant to inhibition by carbon monoxide. It also exhibits a very high ratio of H2 evolution to H2 uptake activity compared with other known hydrogenases. The CO-induced hydrogenase is tightly membrane bound, and its inhibition by nonionic detergents is described. Finally, the presence of nickel in the hydrogenase is addressed. Analysis of wild-type R. rubrum grown on nickel-depleted medium indicates a requirement for nickel for hydrogenase activity. However, analysis of strain UR294 (cooC insertion mutant defective in nickel insertion into CODH) shows that independent nickel insertion mechanisms are utilized by hydrogenase and CODH. CooH lacks the C-terminal peptide that is found in other Ni-Fe hydrogenases; in other systems, this peptide is cleaved during Ni processing.     

Regulation of hydrogenase activity in vegetative cells of Anabaena variabilis.

Heterocyst-free (NH4+-grown) cultures of the cyanobacterium Anabaena variabilis produce a hydrogenase which is reversibly inhibited by light and O2. White or red light at an intensity of 5,000 lx inhibited greater than 95% of the activity. Oxygen at concentrations as low as 0.5% inhibited more than 85% of the hydrogenase in the vegetative cells of CO2-NH4+-grown cultures. The vegatative cell hydrogenase is also sensitive to strong oxidants like ferricyanide. In the presence of strong reductants like S2O4(2-), hydrogenase activity was not inhibited by light. However, hydrogenase activity in the heterocysts was insensitive to both light (greater than 5,000 lx) and O2 (10%). Heterocysts and light-insensitive hydrogenase activity appear simultaneously during differentiation of the vegetative cells into heterocysts (an NH4+-grown culture transferred to NH4+-free, N2-containing medium). This light-insensitive hydrogenase activity was detected several hours before the induction of nitrogenase activity. These results suggest a mode of regulation of hydrogenase in the vegetative cells of A. variabilis that is similar to "redox control" of hydrogenase and other "anaerobic" proteins in enteric bacteria like Escherichia coli.     

Characterization and physiological roles of membrane-bound hydrogenase isoenzymes from Salmonella typhimurium.

We found that Salmonella typhimurium strain LT2 (Z) possessed two immunologically distinct, membrane-bound hydrogenase isoenzymes, which were similar in electrophoretic mobilities and apoprotein contents to hydrogenase isoenzymes 1 and 2 of Escherichia coli. The S. typhimurium enzymes cross-reacted with antibodies raised to the respective hydrogenase isoenzymes of E. coli. As for E. coli, an additional membrane-bound hydrogenase activity (termed hydrogenase 3), which did not cross-react with antibodies raised against either hydrogenase 1 or 2, was also present in detergent-dispersed membrane preparations. The physiological role of each of the three isoenzymes in E. coli has remained unclear owing to the lack of mutants specifically defective for individual isoenzymes. However, analysis of two additional wild-type isolates of S. typhimurium revealed specific defects in their hydrogenase isoenzyme contents. S. typhimurium LT2 (A) lacked isoenzyme 2 but possessed normal levels of hydrogenases 1 and 3. S. typhimurium LT7 lacked both isoenzymes 1 and 2 but retained normal hydrogenase 3 activity. Characterization of hydrogen metabolism by these hydrogenase-defective isolates allowed us to identify the physiological role of each of the three isoenzymes. Hydrogenase 3 activity correlated closely with formate hydrogenlyase-dependent hydrogen evolution, whereas isoenzyme 2 catalyzed hydrogen uptake (oxidation) during anaerobic, respiration-dependent growth. Isoenzyme 1 also functioned as an uptake hydrogenase but only during fermentative growth. We postulate that this enzyme functions in a hydrogen-recycling reaction which operates during fermentative growth.     

Measuring the pH dependence of hydrogenase activities

The pH dependences of activities of homogenous hydrogenases of Thiocapsa roseopersicina and Desulfomicrobium baculatum in the reaction of hydrogen uptake in solution in the presence of benzyl viologen and the pH dependences of catalytic currents of hydrogen oxidation by electrodes on which these hydrogenases were immobilized were compared. Maximal activities of the hydrogenases from T. roseopersicina and D. baculatum in the reaction hydrogen uptake in solution were observed at pH 9.5 and 8.5, respectively. However, the steady-state current caused by catalytic uptake of hydrogen was maximal for the T. roseopersicina hydrogenase-containing electrode at pH 5.5-6.5 under overvoltage of 30-60 mV, whereas for electrodes with D. baculatum hydrogenase it was maximal at pH 6.0-6.5. Analysis of these data suggests that pH-dependent changes in the hydrogenase activities in solution during hydrogen uptake are due not only to the effect of proton concentration on the enzyme conformation or protonation of certain groups of the enzyme active center, but they are rather indicative of changes in free energy of the reaction accompanying changes in pH.     

The role of Hox hydrogenase in the H2 metabolism of Thiocapsa roseopersicina

The purple sulfur phototrophic bacterium Thiocapsa roseopersicina BBS synthesizes at least three NiFe hydrogenases (Hox, Hup, Hyn). We characterized the physiological H(2) consumption/evolution reactions in mutants having deletions of the structural genes of two hydrogenases in various combinations. This made possible the separation of the functionally distinct roles of the three hydrogenases. Data showed that Hox hydrogenase (unlike the Hup and Hyn hydrogenases) catalyzed the dark fermentative H(2) evolution and the light-dependent H(2) production in the presence of thiosulfate. Both Hox(+) and Hup(+) mutants demonstrated light-dependent H(2) uptake stimulated by CO(2) but only the Hup(+) mutant was able to mediate O(2)-dependent H(2) consumption in the dark. The ability of the Hox(+) mutant to evolve or consume hydrogen was found to depend on a number of interplaying factors including both growth and reaction conditions (availability of glucose, sulfur compounds, CO(2), H(2), light). The study of the redox properties of Hox hydrogenase supported the reversibility of its action. Based on the results a scheme is suggested to describe the role of Hox hydrogenase in light-dependent and dark hydrogen metabolism in T. roseopersicina BBS.     

Distinct redox behaviour of prosthetic groups in ready and unready hydrogenase from Chromatium vinosum.

The redox behaviour of the Ni(III)/Ni(II) transition in hydrogenase from Chromatium vinosum is described and compared with the redox behaviour of the nickel ion in the F420-nonreducing hydrogenase from Methanobacterium thermoautotrophicum. Analogous to the situation in the oxidised hydrogenase of Desulfovibrio gigas (Fernandez, V.M., Hatchikian, E.C., Patil, D.S. and Cammack, R. (1986) Biochim. Biophys. Acta 883, 145-154), the C. vinosum enzyme can also exist in two forms: the 'unready' form (EPR characteristics of Ni(III): gx,y,z = 2.32, 2.24, 2.01) and the 'ready' form (EPR characteristics Ni(III): gx,y,z = 2.34, 2.16, 2.01). Like in the oxidised enzyme of M. thermoautotrophicum the Ni(III)/Ni(II) transition for the unready form titrated completely reversible (both at pH 6.0 and pH 8.0). In contrast, the reversibility of the Ni(III)/Ni(II) transition in the ready enzyme was strongly dependent on pH and temperature. At pH 6.0 and 2 degrees C reduction of Ni(III) in ready enzyme was completely irreversible, whereas at pH 8.0 and 30 degrees C Ni(III) in both ready and unready enzyme titrated with E0' = -115 mV (n = 1). Hampered redox equilibration between the ready enzyme and the mediating dyes is interpreted in terms of an obstruction of the electron transfer from nickel at the active site to the artificial electron acceptors in solution. The origin of this obstruction might be related to possible changes in the protein structure induced by the activation process. The E0'-value of the Ni(III)/Ni(II) equilibrium was pH sensitive (-60 mV/delta pH) indicating that reduction of nickel is coupled to a protonation. A similar pH-dependence was observed for the titration of the spin-spin interaction of Ni(III) and a special form of the [3Fe-4S]+ cluster (E0' = +150 mV, pH 8.0, 30 degrees C). Redox equilibration of this coupling was extremely sensitive to pH and temperature. The uncoupled [3Fe-4S]+ cluster titrated pH-independently with E0' = -10 mV (pH 8.0, 30 degrees C).     

Comparative characterization of two distinct hydrogenases from Anabaena sp. strain 7120

Two distinct hydrogenases, hereafter referred to as "uptake" and "reversible" hydrogenase, were extracted from Anabaena sp. strain 7120 and partially purified. The properties of the two enzymes were compared in cell-free extracts. Uptake hydrogenase was largely particulate, and although membrane bound, it could catalyze an oxyhydrogen reaction. Particulate and solubilized uptake hydrogenase could catalyze H2 uptake with a variety of artificial electron acceptors which had midpoint potentials above 0 mV. Reversible hydrogenase was soluble, could donate electrons rapidly to electron acceptors of both positive and negative midpoint potential, and could evolve H2 rapidly when provided with reduced methyl viologen. Uptake hydrogenase was irreversibly inactivated by O2, whereas reversible hydrogenase was reversibly inactivated and could be reactivated by exposure to dithionite or H2. Reversible hydrogenase was stable to heating at 70 degrees C, but uptake hydrogenase was inactivated with a half-life of 12 min at this temperature. Uptake hydrogenase was eluted from Sephadex G-200 in a single peak of molecular weight 56,000, whereas reversible hydrogenase was eluted in two peaks with molecular weights of 165,000 and 113,000. CO was competitive with H2 for each enzyme; the Ki's for CO were 0.0095 atm for reversible hydrogenase and 0.039 atm for uptake hydrogenase. The pH optima for H2 evolution and H2 uptake by reversible hydrogenase were 6 and 9, respectively. Uptake hydrogenase existed in two forms with pH optima of 6 and 8.5. Both enzymes had very low Km's for H2, and neither was inhibited by C2H2.     

Aerobic hydrogenase activity in Anacystis nidulans. The oxyhydrogen reaction.

1. The oxyhydrogen reaction of Anacystis nidulans was studied manometrically and polarographically in whole cells and in cell-free preparations; the activity was found to be associated with the particulate fraction. 2. Besides O2, the isolated membranes reduced artificial electron acceptors of positive redox potential; the reactions were unaffected by O2 levels less than 10--15%; aerobically the artificial acceptors were reduced simultaneously with O2. 3. H2-supported O2 uptake was inhibited by CO, KCN and 2-n-heptyl-8-hydroxyquinoline-N-oxide. Inhibition by CO was partly reversed by strong light. Uncouplers stimulated the oxyhydrogen reaction. 4. The kinetic properties of O2 uptake by isolated membranes were the same in presence of H2 and of other respiratory substrates. 5. Low rates of H2 evolution by the membrane preparations were found in presence of dithionite; methyl viologen stimulated the reaction. 6. The results indicate that under certain growth conditions Anacystis synthesizes a membrane-bound hydrogenase which appears to be involved in phosphorylative electron flow from H2 to O2 through the respiratory chain.     

Reversible activation of hydrogenase from Escherichia coli

Hydrogenase from Escherichia coli exhibited low activity when assayed for hydrogen:methyl viologen reductase activity and no activity when assayed for hydrogen-uptake activity with acceptors of high redox potential (dichloroindophenol, methylene blue). Nor did the enzyme as isolated catalyse proton-tritium exchange activity. Incubation under hydrogen resulted in an increase in hydrogen-uptake activity with methyl viologen and the appearance of hydrogen-uptake activity with dichloroindophenol and methylene blue. Following such treatment, the enzyme also readily catalysed isotope exchange. This process is interpreted as the conversion of the hydrogenase from an inactive 'unready' state to an 'active' state. Oxidation of active hydrogenase with dichloroindophenol caused conversion to a state resembling that of the enzyme as isolated but capable of more rapid activation under reducing conditions. This form is termed the 'ready' state. Such interconversions have been reported for hydrogenases from Desulfovibrio gigas and D. desulfuricans, and the possibility that they constitute a regulatory mechanism suggested.     

Hydrogenases of phototrophic microorganisms

This review surveys recent work done in the laboratory of the author and related laboratories on the properties and possible practical applications of hydrogenases of phototrophic microorganisms. Homogeneous hydrogenase preparations were obtained from purple non-sulfur (Rhodospirillum rubrum S1, Rhodobacter capsulatus B10) and purple sulfur (Chromatium vinosum D, Thiocapsa roseopersicina BBS) bacteria, and from the green sulfur bacterium Chlorobium limicola forma thiosulfatophilum L; highly purified hydrogenase samples were prepared from the cyanobacterium Anabaena cylindrica and from the green alga Chlamydomonas reinhardii. It was shown that hydrogenases of R. capsulatus and T. roseopersicina contain Ni and Fe-S cluster. The cytochromes of the c or b type serve as native electron acceptors for the hydrogenases of the purple bacteria and cyanobacteria; rubredoxin or cytochrome c for the hydrogenase of the green sulfur bacterium; and ferredoxin for Ch. reinhardii hydrogenase. The hydrogenase of T. roseopersicina BBS reversibly activates H2 at Eh less than -290 mV (pH 7), whereas those from R. capsulatus and from C. limicola f. thiosulfatophilum exhibit their maximum activity at Eh greater than -300 mV and are thus favourable only for the H2 uptake. Hydrogenase synthesis in different phototrophs depends on pO2, H2 concentrations and organic substrates. Organic compounds, which serve as electron donors and carbon sources, repress hydrogenase synthesis in R. rubrum, R. capsulatus and in Ectothiorhodospira shaposhnikovii when present at high concentrations. The synthesis of T. roseopersicina hydrogenase is constitutive. H2 notably stimulates hydrogenase activity in R. capsulatus. The synthesis of hydrogenase in R. sphaeroides 2R occurs only in the presence of H2 and does not depend on the presence of organic compounds in the medium.     

Anaerobic hydrogenase activity in Anacystis nidulans. H2-dependent photoreduction and related reactions.

1. Anaerobic hydrogenase activity in whole cells and cell-free preparations of H2-induced Anacystis was studied both manometrically and spectrophotometrically in presence of physiological and artificial electron acceptors. 2. Up to 90% of the activity measured in crude extracts were recovered in the chlorophyll-containing membrane fraction after centrifugation (144 000 X g, 3 h). 3. Reduction of methyl viologen, diquat, ferredoxin, nitrite and NADP by the membranes was light dependent while oxidants of more positive redox potential were reduced also in the dark. 4. Evolution of H2 by the membranes was obtained with dithionite and with reduced methyl viologen; the reaction was stimulated by detergents. 5. Both uptake and evolution of H2 were sensitive to O2, CO, and thiolblocking agents. The H2-dependent reductions were inhibited also by the plastoquinone antagonist dibromothymoquinone, while the ferredoxin inhibitor disalicylidenepropanediamine affected the photoreduction of nitrite and NADP only. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea did not inhibit any one of the H2-dependent reactions. 6. The results present evidence for a membrane-bound 'photoreduction' hydrogenase in H2-induced Anacystis. The enzyme apparently initiates a light-driven electron flow from H2 to various low-potential acceptors including endogenous ferredoxin.     

Purification and properties of membrane-bound hydrogenase from Azotobacter vinelandii

Uptake hydrogenase (EC 1.12) from Azotobacter vinelandii has been purified 250-fold from membrane preparations. Purification involved selective solubilization of the enzyme from the membranes, followed by successive chromatography on DEAE-cellulose, Sephadex G-100, and hydroxylapatite. Freshly isolated hydrogenase showed a specific activity of 110 mumol of H2 uptake (min X mg of protein)-1. The purified hydrogenase still contained two minor contaminants that ran near the front on sodium dodecyl sulfate-polyacrylamide gels. The enzyme appears to be a monomer of molecular weight near 60,000 +/- 3,000. The pI of the protein is 5.8 +/- 0.2. With methylene blue or ferricyanide as the electron acceptor (dyes such as methyl or benzyl viologen with negative midpoint potentials did not function), the enzyme had pH optima at pH 9.0 or 6.0, respectively, It has a temperature optimum at 65 to 70 degrees C, and the measured half-life for irreversible inactivation at 22 degrees C by 20% O2 was 20 min. The enzyme oxidizes H2 in the presence of an electron acceptor and also catalyzes the evolution of H2 from reduced methyl viologen; at the optimal pH of 3.5, 3.4 mumol of H2 was evolved (min X mg of protein)-1. The uptake hydrogenase catalyzes a slow deuterium-water exchange in the absence of an electron acceptor, and the highest rate was observed at pH 6.0. The Km values varied widely for different electron acceptors, whereas the Km for H2 remained virtually constant near 1 to 2 microM, independent of the electron acceptors.     

Partial characterization of an electrophoretically labile hydrogenase activity of Escherichia coli K-12

A mutant of Escherichia coli K-12 is described that is specifically impaired in only one hydrogenase isoenzyme. By means of Tn5-mediated insertional mutagenesis, a class of mutants was isolated (class I) that had retained 20% of the overall hydrogenase activity. As determined by neutral polyacrylamide gel electrophoresis, the mutant contained normal amounts of the hydrogenase isoenzymes 1 and 2. Therefore, the hydrogenase activity affected seemed to be electrophoretically labile and was called hydrogenase L. The presence of such an activity was recently suggested in various papers and was called isoenzyme 3. Hydrogenase L might be identical or part of the latter isoenzyme. By DEAE ion-exchange chromatography it could be separated from hydrogenases 1 and 2. Hydrogenase activity in the parent strain HB101, determined manometrically with cell-free preparations and methylviologen as the electron acceptor, immediately showed maximal activity. However, class I mutants showed a lag phase which was dependent on the protein concentration utilized in the assay. This suggested that the fast initial activity of HB101 was due to hydrogenase L. The enzyme or enzyme complex showed an Mr around 300,000 and a pH optimum between 7 and 8. Strong indications about its physiological role were provided by the finding that in class I mutants H2 production by the formate-hydrogen lyase pathway was unimpaired, whereas fumarate-dependent H2 uptake was essentially zero. Complementation with F-prime factor F'116 but not with F'143 and coconjugation and cotransduction experiments localized the mutation (hydL) close to metC at approximately 64.8 min.     

The role of Hox hydrogenase in the H2 metabolism of Thiocapsa roseopersicina

The purple sulfur phototrophic bacterium Thiocapsa roseopersicina BBS synthesizes at least three NiFe hydrogenases (Hox, Hup, Hyn). We characterized the physiological H₂ consumption/evolution reactions in mutants having deletions of the structural genes of two hydrogenases in various combinations. This made possible the separation of the functionally distinct roles of the three hydrogenases. Data showed that Hox hydrogenase (unlike the Hup and Hyn hydrogenases) catalyzed the dark fermentative H₂ evolution and the light-dependent H₂ production in the presence of thiosulfate. Both Hox⁺ and Hup⁺ mutants demonstrated light-dependent H₂ uptake stimulated by CO₂ but only the Hup⁺ mutant was able to mediate O₂-dependent H₂ consumption in the dark. The ability of the Hox⁺ mutant to evolve or consume hydrogen was found to depend on a number of interplaying factors including both growth and reaction conditions (availability of glucose, sulfur compounds, CO₂, H₂, light). The study of the redox properties of Hox hydrogenase supported the reversibility of its action. Based on the results a scheme is suggested to describe the role of Hox hydrogenase in light-dependent and dark hydrogen metabolism in T. roseopersicina BBS.     

Redox properties and active center of phototrophic bacteria hydrogenases

It is shown that the activity of phototrophic bacteria hydrogenases depends on the redox potential (Eh) of the medium. Hydrogenase from the purple sulfur bacterium Thiocapsa roseopersicina strain BBS reversibly activates H2 at Eh less than -290 mV (pH 7.0). When Eh is increased from -290 to -170 mV, the enzyme is converted into an inactive form which is accompanied by one-electron oxidation of its Fe-S cluster. In contrast, the hydrogenases of the purple nonsulfur bacterium Rhodobacter capsulatus B10 and the green sulfur bacterium Chlorobium limicola forma thiosulfatophilum exhibit maximum activity at Eh greater than -300 mV, favourable only for H2 uptake. When Eh decreases the activities of these enzymes drop dramatically; this accounts for their unidirectional effect directed mainly towards H2 uptake. Such dependence on Eh of activity of hydrogenases from these bacteria correlates with their physiological function in the metabolism of phototrophic bacteria, i.e. with the catalysis of the H2 uptake reaction. Hydrogenases from purple bacteria contain nickel and a single Fe-S cluster. Metal chelators do not affect the activity of these enzymes, which indicates that iron and nickel are tightly bound to the apoprotein. Sulfhydryl compounds irreversibly inactivate T. roseopersicina hydrogenase by 30-40% in the presence of sulfide. Acetylene and carbon monoxide are reversible inhibitors of the enzyme. EPR and inhibitory analysis indicate a direct interaction of H2 with the nickel ion in the active center of the T. roseopersicina hydrogenase.