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.     

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.     

Accessory proteins functioning selectively and pleiotropically in the biosynthesis of [NiFe] hydrogenases in Thiocapsa roseopersicina.

There are at least two membrane-bound (HynSL and HupSL) and one soluble (HoxEFUYH) [NiFe] hydrogenases in Thiocapsa roseopersicina BBS, a purple sulfur photosynthetic bacterium. Genes coding for accessory proteins that participate in the biosynthesis and maturation of hydrogenases seem to be scattered along the chromosome. Transposon-based mutagenesis was used to locate the hydrogenase accessory genes. Molecular analysis of strains showing mutant phenotypes led to the identification of hupK (hoxV ), hypC1, hypC2, hypD, hypE, and hynD genes. The roles of hynD, hupK and the two hypC genes were investigated in detail. The putative HynD was found to be a hydrogenase-specific endoprotease type protein, participating in the maturation of the HynSL enzyme. HupK plays an important role in the formation of the functionally active membrane-bound [NiFe] hydrogenases, but not in the biosynthesis of the soluble enzyme. In-frame deletion mutagenesis showed that HypC proteins were not specific for the maturation of either hydrogenase enzyme. The lack of either HypC protein drastically reduced the activity of every hydrogenase. Hence both HypCs might participate in the maturation of [NiFe] hydrogenases. Homologous complementation with the appropriate genes substantiated the physiological roles of the corresponding gene products in the H2 metabolism of T. roseopersicina.     

Relationships in hydrogen metabolism between hydrogenase and nitrogenase in phototrophic bacteria.

Purple bacteria Rhodospirillum rubrum and Thiocapsa roseopersicina form two enzymes, hydrogenase and nitrogenase, which participate in hydrogen metabolism. H2 photoproduction in these bacteria is associated mainly or completely with the action of nitrogenase. The soluble and membrane-bound hydrogenases of T. roseopersicina have similar physicochemical properties (mol. weight, subunit composition, N-terminal amino acids, Fe2+ and S2- content, pl. Eo'). In comparison with other hydrogenases the enzyme from R. rubrum and T. roseopersicina evolve H2 with high rate from reduced cytochrome c3, but not from ferredoxins. H2 production and N2 fixation take place in the presence of NAD(P)H. NADP-reductase, ferredoxin and cytochrome c3 participate in this reaction. Possible relationships between hydrogenase-nitrogenase in the metabolism of molecular hydrogen are discussed.     

Characterization of ammonium and methylamine transport systems in phototrophic sulfur bacteria

Abstract Transport of ammonium and methylamine into the cells of green sulfur bacterium Chlorobium limicola and purple sulfur bacterium Thiocapsa roseopersicina is carried out by a common transport system. This system has (for C. limicola and T. roseopersicina , respectively) pH optimum 7.0 and 7.5; V _max 0.6 and 4.2 nmol min⁻¹ (mg protein)⁻¹; K_m 5.9 × 10⁻⁵ M and 1.3 × 10⁻⁵ M, and is capable of forming 120- and 600-fold methylamine gradients. The methylamine transport can be energized by the artificially imposed transmembrane K⁺ diffusive potential and is inhibited by tetraphenylphosphonium or valinomycin and K⁺. The data presented indicate that methylamine transport in both studied species is exclusively driven by the membrane potential gradient (ΔΨ).     

Purification and properties of cytochrome c552 from purple sulfur bacterium Thiocapsa roseopersicina

The method of purification up to electrophoretical homogeneity of cytochrome c552 from the phototrophic bacterium Thiocapsa roseopersicina, strain BBS is described. For the cytochrome absorption spectrum the maxima at 417, 523 and 552 nm are characteristic for the reduced state and at 409 nm--for the oxidized state. The molecular weight is equal to 62000. The cytochrome contains two hemes per molecule and consists of two subunits. pI is 4.1; E0' is about 10 mV. Cytochrome c552 is a flavoprotein according to its fluorescence spectrum and subunit structure. T. roseopersicina cytochrome c552 is able to be reduced with sulphide, cysteine and ascorbate as well as with H2 in the presence of hydrogenase from the same bacterium. These data suggest that cytochrome c552 from T. roseopersicina functions in vivo at the initial stage of electron transport from hydrogen and sulphide.     

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.     

Purification and properties of hydrogenase from phototrophic bacterium Thiocapsa roseopersicina]

The isolation method and some peoperties of purple sulphur bacteria (Thiocapsa roseopersicina strain BBS) hydrogenase are described Hydrogenase molecular weight is found to be 66000; it contains 3.7 moles of S2- and 3.9 moles of Fe2+ per one mole of the enzyme;pI=4.2. The enzyme absorption spectrum has the maximum at 400-412 nm which is characteristic of proteins containing non-haem iron. Hydrogenase is suggested to consist pf 4 subunits of two types: with molar weight 27000 and 6000. Unlike other hydrogenases, this enzyme is rather resistant to O2 and is more thermostable: the inactivation of the enzyme was observed at the temperature above 80 degrees C; Hydrogenase preparation catalyses D2-H2O exchange reaction, H2 evolution from the reduced methyl viologene (MV) and H2 absorption in the presense of MV or benzylviologene but not in the presense of NAD(P), FAD, FMN, azocarmine, methylene blue and ferricyanide.     

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.     

Hydrogenase of purple bacteria: properties and regulation of synthesis

Physico-chemical properties of homogeneous preparations of soluble and membrane-bound hydrogenases from the purple sulfur bacterium Thiocapsa roseopersicina BBS and membrane-bound hydrogenase of Rhodopseudomonas capsulata, strain B10 have been studied. Compared to the enzymes from other sources, the hydrogenase of Thiocapsa roseopersicina is more stable to O₂ and products of its reduction (O ₂ ^- , H₂O₂), temperature and a number of other factors of the medium. A natural electron donor for T. roseopersicina hydrogenase is a low-potential cytochrome C₃, while the natural electron acceptors for hydrogenases of R. capsulata, T. roseopersicina, Ectothiorhodospira shaposhnikovii and Anabaena cylindrica are cytochromes of groups c and b.In different phototrophs, synthesis of hydrogenase can be inhibited by the presence of high concentrations of O₂. In some microorganisms (e.g. Rhodopseudomonas capsulata, strain B10) the repressing effect on hydrogenase formation is also exhibited by organic compounds. H₂ may not necessarily be present for hydrogenase synthesis by purple bacteria, but its presence may considerable increase the level of the enzyme.Abbreviations SDS sodium dodecylsulfate - Hipip high-potential iron — sulfur protein - R Rhodopseudomonas - T Thiocapsa - Rh Rhodospirillum - C Chromatium This paper is dedicated to Professor Dr. H.G. Schlegel in honour of his sixtieth birthday and in recognition of his great contribution in the field of physiology and biochemistry of microorganisms capable of using H₂. Professor H.G. Schlegel had a profound and most fuitful influence on the progress in the research of the laboratory headed by the author     

Low potential c-type cytochrome of Thiocapsa roseopersicina

The low potential c-type cytochrome from the phototrophic purple sulphur bacterium Thiocapsa roseopersicina, strain BBS was isolated in electrophoretically homogeneous state. The bulk of the cytochrome (approximately 90%) after disruption of the cells remained in the membrane fraction. The absorption spectrum of the cytochrome was characterized by the maxima at 420, 523 and 552 nm in the reduced state and at 408 nm in the oxidized one. The cytochrome interacted with CO in the reduced state. The molecular weight of the cytochrome is 50 000. The cytochrome contains great amounts of phenylalanine, leucine, valine, aspartic and glutamic acids and can be reduced by dithionite but not by cysteine, sulfide or ascorbate. Besides, the cytochrome can also be reduced by NAD(P)H in the presence of NAD(P)-reductases of T. roseopersicina, when ferredoxin of Spirulina platensis or benzyl viologen are added to the reaction mixture. The cytochrome can act as an electron donor (acceptor) for T. roseopersicina hydrogenase.     

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.     

Enlarging the gas access channel to the active site renders the regulatory hydrogenase HupUV of Rhodobacter capsulatus O2 sensitive without affecting its transductory activity

In the photosynthetic bacterium Rhodobacter capsulatus, the synthesis of the energy-producing hydrogenase, HupSL, is regulated by the substrate H2, which is detected by a regulatory hydrogenase, HupUV. The HupUV protein exhibits typical features of [NiFe] hydrogenases but, interestingly, is resistant to inactivation by O2. Understanding the O2 resistance of HupUV will help in the design of hydrogenases with high potential for biotechnological applications. To test whether this property results from O2 inaccessibility to the active site, we introduced two mutations in order to enlarge the gas access channel in the HupUV protein. We showed that such mutations (Ile65-->Val and Phe113-->Leu in HupV) rendered HupUV sensitive to O2 inactivation. Also, in contrast with the wild-type protein, the mutated protein exhibited an increase in hydrogenase activity after reductive activation in the presence of reduced methyl viologen (up to 30% of the activity of the wild-type). The H2-sensing HupUV protein is the first component of the H2-transduction cascade, which, together with the two-component system HupT/HupR, regulates HupSL synthesis in response to H2 availability. In vitro, the purified mutant HupUV protein was able to interact with the histidine kinase HupT. In vivo, the mutant protein exhibited the same hydrogenase activity as the wild-type enzyme and was equally able to repress HupSL synthesis in the absence of H2.     

Bacteriochlorophyll fluorescence changes related to the bacteriopheophytin photoreduction in the chromatophores of purple sulfur bacteria]

It is shown that illumination of chromatophores of sulfur bacterium Chromatium minutissimum at Eh of the medium --200 mV divided by --620 mV (when the photooxidation of pigment P890 is completely inhibited) induces a decrease in bacteriochlorophyll fluorescence yield, reversible in the dark. Under these conditions a reversible photoreduction of bacteriopheophytin is detected (bleaching of absorption bands at 543 and 760 nm and development of a band at 650 nm), which is accompanied by a blue shift of the absorption band at 8 nm. As a possible interpretation of these effects the suggestion is made on the function of bacteriopheophytin as a primary electron acceptor in reaction centers of bacteria. The bacteriopheophytin photoreduction, followed by a decrease in fluorescence yield, is also observed in other sulfur bacteria, Thiocapsa roseopersicina and Ectothiorodospira shaposhnikovii, but it is not detected in nonsulfur bacteria, Rhodospirillum rubrum and Rhodopseudomonas spheroides. This is considered as an evidence for the difference in the functional organization of the reaction centers of these two groups of bacteria,     

Modeling three-dimensional structure of two closely related Ni–Fe hydrogenases

Photosynthesis Research - The results of homology modeling of HydSL, a NiFe-hydrogenase from purple sulfur bacterium Thiocapsa roseopersicina BBS, and deep-water bacterium Alteromonas macleodii...     

Thioredoxin from Rhodospirillum rubrum: primary structure and relation to thioredoxins from other photosynthetic bacteria.

Thioredoxin was isolated from a photosynthetic purple nonsulfur bacterium, Rhodospirillum rubrum, and its primary structure was determined by high-performance tandem mass spectrometry. The sequence identity of R. rubrum thioredoxin to Escherichia coli thioredoxin was intermediate to those of the Chlorobium thiosulfatophilum and Chromatium vinosum proteins. The results indicate that R. rubrum has an NADP-thioredoxin system similar to that of other photosynthetic purple bacteria.