The activities of hydrogenase and enoate reductase in two Clostridium species,their interrelationship and dependence on growth conditions

The enzyme activities of Clostridium La 1 and Clostridium kluyveri involved in the stereospecific hydrogenation of ,-unsaturated carbonyl compounds with hydrogen gas were measured. In C. La 1 the specific activities of hydrogenase and enoate reductase depended heavily on the growth phase and the composition of the medium. During growth in batch cultures on 70 mM crotonate the specific activity of hydrogenase increased and then dropped to about 10% of its maximum value, whereas the activity of enoate reductase reached its maximum in cells of the stationary phase. Under certain conditions during growth the activity ratio hydrogenase: enoate reductase changed from 120 to 1. Thus, the rate limiting enzyme for the hydrogenation can be either the hydrogenase or the enoate reductase, depending on the growth conditions of the cells.The specific activities of ferredoxin-NAD reductase and butyryl-CoA dehydrogenase increased 3-4-fold during growth on crotonate. By turbidostatic experiments it was shown that at constant input of high crotonate concentrations (200 mM) the enoate reductase activity was almost completely suppressed; it increased steadily with decreasing crotonate down to an input concentration of 35 mM.Glucose as carbon source led to high hydrogenase and negligible enoate reductase activities. The latter could be induced by changing the carbon source of the medium from glucose to crotonate. Tetracycline inhibited the formation of enoate reductase.A series of other carbon sources was tested. They can be divided into ones which result in high hydrogenase and rather low enoate reductase activities and others which cause the reverse effect.When the Fe²⁺ concentration in crotonate medium was growth limiting, cells with relatively high hydrogenase activity and very low enoate reductase activity in the stationary phase were obtained. At Fe²⁺ concentrations above 3·10^-7 M enoate reductase increased and hydrogenase activity reached its minimum. The ratio of activities changes by a factor of about 200. In a similar way the dependence of enzyme activities on the concentration of sulfate was studied.In batch cultures of Clostridium kluyveri a similar opposite time course of enoate reductase and hydrogenase was found.The possible physiological significance of this behavior is discussed.Non Standard Abbreviations O.D.₅₇₈ Optical density at 578 nm Dedicated to Professor Dr. O. Kandler on the occasion of his 60th birthday     

A simplified method for assay of hydrogenase activities of H<Subscript>2</Subscript> evolution and uptake in <Emphasis Type="Italic">Enterobacter aerogenes</Emphasis>

Assay of hydrogenase activity pertaining to H₂ production needs anaerobic conditions. To establish a simplified method for assay of hydrogenase activities by using intact cells of Enterobater aerogenes, different chemicals capable of enhancing the cell-wall permeability to electron mediators were examined. As a result, Triton X-100 and CTAB were found to be appropriate for H₂ uptake and evolution activities of the intact cells, respectively. This method enabled H₂ uptake and evolution activities of the intact cells to be easily detected. This is also the first report of the presence of H₂ uptake hydrogenase activity in E. aerogenes.Revisions requested 2 March 2005 and 21 April 2005; Revisions received 12 April 2005 and 17 May 2005     

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.     

Connection between the membrane electron transport system and Hyn hydrogenase in the purple sulfur bacterium, Thiocapsa roseopersicina BBS

Thiocapsa. roseopersicina BBS has four active [NiFe] hydrogenases, providing an excellent opportunity to examine their metabolic linkages to the cellular redox processes. Hyn is a periplasmic membrane-associated hydrogenase harboring two additional electron transfer subunits: Isp1 is a transmembrane protein, while Isp2 is located on the cytoplasmic side of the membrane. In this work, the connection of HynSL to various electron transport pathways is studied. During photoautotrophic growth, electrons, generated from the oxidation of thiosulfate and sulfur, are donated to the photosynthetic electron transport chain via cytochromes. Electrons formed from thiosulfate and sulfur oxidation might also be also used for Hyn-dependent hydrogen evolution which was shown to be light and proton motive force driven. Hyn-linked hydrogen uptake can be promoted by both sulfur and nitrate. The electron flow from/to HynSL requires the presence of Isp2 in both directions. Hydrogenase-linked sulfur reduction could be inhibited by a Q_B site competitive inhibitor, terbutryne, suggesting a redox coupling between the Hyn hydrogenase and the photosynthetic electron transport chain. Based on these findings, redox linkages of Hyn hydrogenase are modeled.     

Effect of carbon source on growth,nitrogenase and uptake hydrogenase activities of Frankia isolates from Casuarina sp.

The effect of different carbon sources on the growth of Frankia isolates for Casuarina sp. was studied. In addition, regulation of nitrogenase and uptake hydrogenase activity by carbon sources was investigated. For each of the three isolates, JCT287, KB5 and HFPCcI3, growth was greatest on the carbon sources pyruvate and propionate. In general the carbon sources which gave the greatest growth gave the highest levels of nitrogenase activity, but repressed the activity of uptake hydrogenase. The regulation of growth, uptake hydrogenase activity and nitrogenase activity is discussed.     

Temperature dependence of intracellular pH in higher plant cells

The recent introduction of ³¹P nuclear magnetic resonance spectroscopy offers a new approach to the problem of obtaining a simultaneous and direct evaluation of both the cytoplasmic and vacuolar pH in higher plant cells (J. K. M. Roberts, P.M. Ray, N. Waderlardetzky and O. Sardetzky, 1980, Nature 283, 870–872; 1981, Planta 152, 74–78). Using this method we have been able to detect a selective pH decrease of about 0.5 units at the level of the cytoplasmic compartment of maize root tips when the temperature was increased from 4 to 28°C. This effect was completely reversible with temperature. No pH variation could be detected at the level of the vacuolar compartment.     

Nickel and iron incorporation into soluble hydrogenase of Alcaligenes eutrophus

Archives of Microbiology - Qualitative and quantitative determination of proteins of the soluble hydrogenase (hydrogen: NAD+ oxidoreductase, EC 1.12.1.2) from Alcaligenes eutrophus H16 was done by...     

Polysulfide utilization by Thiocapsa roseopersicina

The purple sulfur bacterium Thiocapsa roseopersicina, being the dominant anoxygenic phototroph in microbial mats, was tested for growth on polysulfide as the electron donor for carbon dioxide fixation. Data collected in continuous cultures revealed _max to be 0.065 h^-1 and the saturation affinity constant K _s to be 6.7 M. The value of the inhibition constant K _i was estimated in batch cultures and was found to be approximately 1100 M. When grown on monosulfide, the organism was capable of trisulfide utilization without lag. Monosulfide-limited growth was established to have a _max of 0.091 h^-1 and K _s of 8.0 M. Field observations revealed polysulfide, present at supra-optimal concentrations, as a major pool of reduced sulfur in a laminated marine sediment ecosystem.Non-standard abbreviations DLP Direct Linear Plot - TS Total Sugar - SS Structural Sugar - P Protein - R _R concentration of growth limiting nutrient in reservoir vessel - S _nutrient residual concentration of growth-limiting nutrient in the culture vessel - S _{sulfur compound} concentration of sulfur in the corresponding compound - D dilution rate - _max maximum specific growth rate - K _s saturation constant - K _i inhibition constant Dedicated to Prof. Dr. Norbert Pfennig on the occasion of his 65th birthday     

<Emphasis Type="Italic">Escherichia coli</Emphasis> hydrogenase 3 is a reversible enzyme possessing hydrogen uptake and synthesis activities

In the past, it has been difficult to discriminate between hydrogen synthesis and uptake for the three active hydrogenases in Escherichia coli (hydrogenase 1, 2, and 3); however, by combining isogenic deletion mutations from the Keio collection, we were able to see the role of hydrogenase 3. In a cell that lacks hydrogen uptake via hydrogenase 1 (hyaB) and via hydrogenase 2 (hybC), inactivation of hydrogenase 3 (hycE) decreased hydrogen uptake. Similarly, inactivation of the formate hydrogen lyase complex, which produces hydrogen from formate (fhlA) in the hyaB hybC background, also decreased hydrogen uptake; hence, hydrogenase 3 has significant hydrogen uptake activity. Moreover, hydrogen uptake could be restored in the hyaB hybC hycE and hyaB hybC fhlA mutants by expressing hycE and fhlA, respectively, from a plasmid. The hydrogen uptake results were corroborated using two independent methods (both filter plate assays and a gas-chromatography-based hydrogen uptake assay). A 30-fold increase in the forward reaction, hydrogen formation by hydrogenase 3, was also detected for the strain containing active hydrogenase 3 activity but no hydrogenase 1 or 2 activity relative to the strain lacking all three hydrogenases. These results indicate clearly that hydrogenase 3 is a reversible hydrogenase.     

Rhizobium strains expressing uptake hydrogenase in different host species of cowpea miscellany

Uptake hydrogenase activity in nodules of green gram (Vigna radiata (L.) (Wilczek)), black gram (Vigna mungo (L.) (Hepper)), cowpea (Vigna unguiculata (L.) and cluster bean (Cyamopsis tetragonoloba (L.) (Taub.)), formed with two Hup⁺ (S24 and CT2014) and one Hup⁻ (M11)Rhizobium strains, was determined at different levels of external H2 in air atmosphere. Nodules of all the 4 host species formed by inoculation with strains S24 and CT2014, showed H2 uptake but not those formed with strain M11. H₂ uptake rates were higher in 1 and 2% H₂ in air atmosphere (v/v) than at 5 or 10% levels in all the host species. Variations in the relative rates of H₂ uptake were observed both, due to host species as well as due toRhizobium strains. However, no host dependent complete repression of the expression of H₂ uptake activity was observed in nodules of any of the host species formed with Hup⁺ strains.     

Reversible hydrogenase in Anabaena variabilis ATCC 29413

The presence and localization of a reversible hydrogenase in non-N₂-fixing cells of the filamentous cyanobacterium Anabaena variabilis were investigated by in vitro activity measurements, native-PAGE/activity stain, SDS-PAGE/Western immunoblots, and immunogold localization. Reversible hydrogenase activity was induced approximately 100-fold by sparging the cell suspensions with a mixture of 99% argon and 1% CO₂ for 20–26 h. Native-PAGE/activity stain demonstrated the presence of an in vitro functional enzyme with an apparent molecular mass of 118 kDa. Native-PAGE/Western immunoblots, using polyclonal antisera directed against purified hydrogenase from the purple sulphur bacterium Thiocapsa roseopersicina, detected two native proteins with molecular masses of 118 and 133 kDa, respectively. SDS-PAGE/Western immunoblots confirmed the presence of a single polypeptide with a molecular mass of approximately 40 kDa in both induced and non-induced cells. Immunocytolocalization experiments using ultrathin sections again demonstrated the presence of hydrogenase in both induced and non-induced cells. A higher specific labeling was associated with the thylakoid regions, which, using an image analyzer, was calculated to be approximately 4 x higher per cell area compared to in the centroplasm. It is suggested that anaerobic incubation induces higher reversible hydrogenase activity, regulated mainly at the level of activating (pre)existing form(s) of inactive enzyme(s)/protein(s), maybe in combination with synthesis of additional subunit(s).     

Bidirectional measurement of hydrogenase activity by the pH-stat method

The protons produced by the catalytic activity of hydrogenase in H2 evolution from dithionite-reduced methyl viologen or through benzyl viologen reduction by H2 gas are automatically titrated by a pH-stat device. This approach allows the measurement of hydrogenase activity and ensures the constancy of pH during the reaction in absence of buffers. Kinetic assays and pH and temperature-dependence experiments with Desulfovibrio gigas hydrogenase performed by this method basically confirm the results obtained with customary manometric assay.     

Effect of redox mediators on nitrogenase and hydrogenase activities in Azotobacter vinelandii

In bioelectrochemical studies, redox mediators such as methylene blue, natural red, and thionine are used to studying the redox characteristics of enzymes in the living cell. Here we show that nitrogenase activity in Azotobacter vinelandii is completely inhibited by oxidized methylene blue (MB_o) when the concentration of this mediator in the medium is increased up to 72 M. This activity in A. vinelandii is somewhat inhibited by a coenzyme, ascorbic acid (AA). However, the nitrogenase activity within the A. vinelandii cell is unchanged even for a high concentration of oxidized natural red (NR_o) alone. Interestingly, these mediators and AA do not have the capacity to inhibit the H₂ uptake activity of the hydrogenase in A. vinelandii. Average active rates of 66 nM H₂ evolved/mg cell protein/min from the nitrogenase and 160 nM H₂-uptake/mg cell protein/min from the hydrogenase in A. vinelandii are found in aid of the activities of the enzymes for H₂ evolution and for H₂ uptake are compared. The activities of both enzymes in A. vinelandii are strongly inhibited by thionine having high oxidative potential. Mechanisms of various mediators acting in vivo for both enzymes in A. vinelandii are discussed.     

In vivo inactivation of soluble hydrogenase of Alcaligenes eutrophus

The soluble, NAD⁺-reducing hydrogenase in intact cells of Alcaligenes eutrophus was inactivated by oxygen when electron donors such as hydrogen or pyruvate were available. The sole presence of either oxygen or oxidizable substrates did not lead to inactivation of the enzyme. Inactivation occurred similarly under autotrophic growth conditions with hydrogen, oxygen and carbon dioxide. The inactivation followed first order reaction kinetics, and the half-life of the enzyme in cells exposed to a gas atmosphere of hydrogen and oxygen (8:2, v/v) at 30° C was 1.5 h. The process of inactivation did not require ATP-synthesis. There was no experimental evidence that the inactivation is a reversible process catalyzed by a regulatory protein. The possibility is discussed that the inactivation is due to superoxide radical anions (O ₂ ^- ) produced by the hydrogenase itself.     

Energetics of sulfate transport in Desulfomicrobium baculatum

The freshwater sulfate reducer Desulfomicrobium baculatum accumulated ³⁵S-sulfate up to 120-fold by an energy-dependent transport system, as was concluded from inhibition of transport by tetrachlorosalicylanilide (TCS). Sulfate accumulation was completely reversible and depended on the presence of sodium ions. The sodium ion gradient ([Na⁺]_out/[Na⁺]_in) was eightfold and was built up by electrogenic Na⁺/H⁺ antiport. Together with a membrane potential of-145 m V, the sodium ion motive force was-199 m V, from which a symport stoichiometry of two sodium ions per sulfate was calculated. This is the first report of a freshwater sulfate reducer taking up sulfate electroneutrally in symport with sodium ions and not with protons.Abbreviations ETH 2120 N,N'-Dibenzyl-N,N'-diphenyl-1,2-phenylendioxydiacetamide - TCS Tetrachlorosalicylanilide     

Hydrogenase 3 but not hydrogenase 4 is major in hydrogen gas production by Escherichia coli formate hydrogenlyase at acidic pH and in the presence of external formate

Fermenting Escherichia coli is able to produce formate and molecular hydrogen (H₂) when grown on glucose. H₂ formation is possessed by two hydrogenases, 3 (Hyd-3) and 4 (Hyd-4), those, in conjunction with formate dehydrogenase H (Fdh-H), constitute distinct membrane-associated formate hydrogenylases. At slightly alkaline pH (pH 7.5), the production of H₂ was found to be dependent on Hyd-4 and the F₀F₁-adenosine triphosphate (ATPase), whereas external formate increased the activity of Hyd-3. In this study with cells grown without and with external formate H₂ production dependent on pH was investigated. In both types of cells, H₂ production was increased after lowering of pH. At acidic pH (pH 5.5), this production became insensitive either to N,N′-dicyclohexylcarbodiimide or to osmotic shock and it became largely dependent on Fdh-H and Hyd-3 but not Hyd-4 and the F₀F₁-ATPase. The results indicate that Hyd-3 has a major role in H₂ production at acidic pH independently on the F₀F₁-ATPase.     

Occurrence and localization of an uptake hydrogenase in the filamentous heterocystous cyanobacteriumNostoc PCC 73102

Summary Free-living nitrogen-fixingNostoc PCC 73102, a filamentous heterocystous cyanobacterium originally isolated from coralloid roots of the cycadMacrozamia sp., were examined for the presence of an uptake hydrogenase (H₂ase) enzyme. In vivo and in vitro hydrogen uptake measurements were used to study activities and SDS-PAGE and Western immunoblots to reveal occurrence of the hydrogenase protein. Also, transmission electron microscopy and immunocytological labeling were used to study the cellular and subcellular distribution of H₂ase in theNostoc cells. In vivo measurements demonstrated an active uptake of hydrogen in both light and darkness. Light stimulated in vivo hydrogen uptake with approximately 100%, and this was further doubled by increasing the pH₂, from 56 to 208 M H₂. An in vitro hydrogen uptake of 1.1 mol H₂/ mg (protein)/h was observed when using phenazinemethosulphate as e^–-acceptor. Western immunoblots revealed that a polypeptide with a molecular weight of about 55 kDa was immunologically related to uptake H₂ase holoenzyme purified fromAlcaligenes latus. Immunolocalization demonstrated that the H₂ase protein was located both in heterocysts and vegetative cells. A higher specific labeling was associated with the cytoplasmic membranes where the vegetative cells are in contact with each other and where they actually are dividing into two vegetative cells. Using the particle analysis of an image processor, approximately equal H₂ase-gold labeling per cell area was observed in the nitrogen-fixing heterocysts compared to the photosynthetic vegetative cells. This study also shows that there was no correlation between presence of phycoerythrin and uptake H₂ase activity.Abbreviations H₂ase hydrogenase - IgG immunoglobulin G     

Ammonia inhibition of nitrogenase activity in purple and green bacteria

Ammonia reversibly inhibits the nitrogenase activity not only in purple nonsulfur bacteria but in purple (Thiocapsa roseopersicina) and green (Chlorobium limicola forma thiosulfatophilum) sulfur bacteria as well.The complete inhibition of nitrogenase activity (acetylene reduction) is observed about 30 s after addition of NH ₄ ⁺ (2.5×10^-6 M) to cell suspensions. The pattern of ammonia inhibition of acetylene reduction in T. roseopersicina does not differ from the action of tetrabutylammonium and tetraphenylphosphonium (3 · 10^-6-5·10^-5 M) on nitrogenase activity of this bacterium.Simultaneously with the switch-off effect of NH ₄ ⁺ a considerable increase of ATP in cells of Rhodobacter sphaeroides and C. limicola f. thiosulphatophilum was observed.