Physiology of nitrogen fixation in Azospirillum lipoferum Br 17 (ATCC 29 709)

Changes of cellular activities during batch cultures with Azospirillum lipoferum strain Br 17 (ATCC 29 709) were observed within the growth cycle, at optimal pO₂ (0.002–0.003 atm). The relative growth rate for cells growing with N₂ as sole nitrogen source during log phase was =0.13 h^-1 and the doubling time was 5.3 h. Nitrogenase activity was not accompanied by hydrogen evolution at any growth stage, and a very active uptake hydrogenase was demonstrated. The hydrogenase activity increased towards the end of the growth period when glucose became limiting and N₂ fixation reached its maximal specific activity. Oxygen consumption and oxygen tolerance at the various growth stages, increased simultaneously with the uptake hydrogenase activity indicating a possible role of this enzyme in an oxygen protection mechanism of A. lipoferum nitrogenase. The efficiency of nitrogen fixation expressed as mg total nitrogen fixed in cells and supernatant per g glucose consumed, was 20 at the early log phase and increased to 48 at the late log phase. About 25% of the total fixed nitrogen was recovered in the culture supernatant.Abbreviations DOT Dissolved oxygen tension - PHB Poly--hydroxybutyric acid - O.D. Optical density (560 nm) - A.T.C.C. American type culture collection - NTA Nitrilotriacetic acid Graduate student of the Universidade Federal Rural do Rio de Janeiro, Brazil     

Purification and characterization of membrane-bound hydrogenase from Methanosarcina barkeri MS

Hydrogenase was solubilized from the membrane of acetate-grown Methanosarcina barkeri MS and purification was carried out under aerobic conditions. The enzyme was reactivated under reducing conditions in the presence of H₂. The enzyme showed a maximal activity of 120±40 mol H₂ oxidized · min^–1 · min^–1 with methyl viologen as an electron acceptor, a maximal hydrogen production rate of 45±4 mol H₂ · min^–1 · mg^–1 with methyl viologen as electron donor, and an apparent K _m for hydrogen oxidation of 5.6±1.7 M. The molecular weight estimated by gel filtration was 98,000. SDS-PAGE showed the enzyme to consist of two polypeptides of 57,000 and 35,000 present in a 1:1 ratio. The native protein contained 8±2 mol Fe, 8±2 mol S^2–, and 0.5 mol Ni/mol enzyme. Cytochrome b was reduced by hydrogen in a solubilized membrane preparation. The hydrogenase did not couple with autologous F₄₂₀ or ferredoxin, nor with FAD, FMN, or NAD(P)⁺. The physiological function of the membrane-bound hydrogenase in hydrogen consumption is discussed.Abbreviation CoM-S-S-HTP the heterodisulfide of 7-mercaptoheptanoylthrconine phosphate and coenzyme M (mercaptoethanesulfonic acid)     

Mass-spectrometric kinetic studies of the nitrogenase and hydrogenase activities in in-vivo cultures of Azospirillum brasilense Sp. 7

Acetylene reduction, deuterium uptake and hydrogen evolution were followed in in-vivo cultures of Azospirillum brasilense, strain Sp 7, by a direct mass-spectrometric kinetic method. Although oxygen was needed for nitrogenase functioning, the enzyme was inactivated by a fairly low oxygen concentration in the culture and an equilibrium had to be found between the rate of oxygen diffusion and bacterial respiration. A nitrogenase-mediated hydrogen evolution was observed only in the presence of carbon monoxide inhibiting the uptake hydrogenase activity which normally recycles all the hydrogen produced. However, under anaerobic conditions and in the presence of deuterium, a bidirectional hydrogenase activity was observed, consisting in D₂ uptake and in H₂ and HD evolution. In contrast to the nitrogenase-mediated H₂ production, this anaerobic H₂ and HD evolution was insensitive to the presence of acetylene and was partly inhibited by carbon monoxide. It was moreover relatively unaffected by the deuterium partial pressure. These results suggest that the anaerobic H₂ and HD evolution can be ascribed to a reverse hydrogenase activity under conditions where D₂ is saturating the uptake process and scavenging the electron acceptors. Although the activities of both nitrogenase and hydrogenase were thus clearly differentiated, a close relationship was found between their respective functioning conditions.     

Regulation and function of ferredoxin-linked versus cytochrome b-linked hydrogenase in electron transfer and energy metabolism of Methanosarcina barkeri MS

Acetate-grown cells of Methanosarcina barkeri MS were found to form methane from H₂:CO₂ at the same rate as hydrogen-grown cells. Cells grown on acetate had similar levels of soluble F₄₂₀-reactive hydrogenase I, and higher levels of cytochrome-linked hydrogenase II compared to hydrogen-grown cells. The hydrogenase I and II activities in the crude extract of acetate-grown cells were separated by differential binding properties to an immobilized Cu²⁺ column. Hydrogenase II did not react with ferredoxin or F₄₂₀, whereas hydrogenase I coupled to both ferredoxin and F₄₂₀. A reconstituted soluble protein system composed of purified CO dehydrogenase, F₄₂₀-reactive hydrogenase I fraction, and ferredoxin produced H₂ from CO oxidation at a rate of 2.5 nmol/min · mg protein. Membrane-bound hydrogenase II coupled H₂ consumption to the reduction of CoM-S-S-HTP and the synthesis of ATP. The differential function of hydrogenase I and II is ascribed to ferredoxin-linked hydrogen production from CO and cytochrome b-linked H₂ consumption coupled to methanogenesis and ATP synthesis, respectively.     

Mutants of Alcaligenes eutrophus defective in autotrophic metabolism

Forty-four mutants of Alcaligenes eutrophus H 16 were isolated which grew poorly or not at all under autotrophic conditions. Four types were characterized with respect to their defects and their physiological properties. One mutant lacked both enzymes specific for autotrophic CO₂ fixation, another one lacked both hydrogenases, and two mutants lacked either the membrane-bound or the soluble hydrogenase. Comparing the results of studies on these mutant types, the following conclusions were drawn: the lack of each hydrogenase enzyme could be partially compensated by the other one; the lack of membrane-bound hydrogenase did not affect autotrophic growth, whereas the lack of the soluble hydrogenase resulted in a decreased autotrophic growth rate. When pyruvate as well as hydrogen were supplied to the wild-type, the cell yield was higher than in the presence of pyruvate alone. Mutant experiments under these conditions indicated that either of both hydrogenases was able to add to the energy supply of the cell. Only the soluble hydrogenase was involved in the control of the rate of hydrogen oxidation by carbon dioxide; the mutant lacking this enzyme did not respond to the presence or absence of CO₂. The suppression of growth on fructose by hydrogen could be mediated by either of both hydrogenases alone.     

A study on electron transport-driven proton translocation in Desulfovibrio desulfuricans

Proton translocation by washed cells of the sulfate-reducing bacterium Desulfovibrio desulfuricans strain Essex 6 was studied by means of pH and sulfide electrodes. Reversible extrusion of protons could be induced either by addition of electron acceptors to cells incubated under hydrogen, or by addition of hydrogen to cells incubated in the presence of an appropriate electron acceptor. Proton translocation was increased in the presence of ionophores that dissipate the membrane potential (thiocyanate, methyl triphenylphosphonium cation, but not valinomycin) and was sensitive to the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). Upon micromolar additions of H₂, usually sulfide was formed in stoichiometric amounts, and extrapolated H⁺/H₂ ratios were 1.8±0.5 with sulfate, 2.3±0.3 with sulfite and 0.5±0.1 with thiosulfate. In several experiments hydrogen pulses caused increased proton extrusion not associated with sulfide production. This was a hint that sulfite might be reduced via intermediates. In the absence of H₂S formation, extrapolated H⁺/H₂ ratios were 3.1±0.8 with sulfate, 3.4±1.1 with sulfite, 4.4±0.8 with thiosulfate and 6.3±1.2 with oxygen. Micromolar pulses of electron acceptors to cells incubated under H₂ caused less proton translocation than H₂ pulses in presence of excess of electron acceptor; extrapolated H⁺/H₂ ratios were 1.3±0.4 with sulfite, 3.3±0.9 with nitrite and 4.2±0.5 with oxygen. No proton translocation was observed after micromolar pulses of sulfate, thiosulfate or nitrate to cells incubated under hydrogen in the presence of thiocyanate. Inhibition experiments with CO and CuCl₂ revealed that the hydrogenase activity was localized in the intracellular space, and that no periplasmic hydrogenase was present. The results indicate that D. desulfuricans can generate a proton gradient by pumping protons across the cytoplasmic membrane.Abbreviations APS adenosine 5-phosphosulfate - CCCP carbonyl cyanide m-chlorophenylhydrazone - MTTP⁺ methyl triphenylphosphonium cation     

Molybdate and sulfide inhibit H2 and increase formate production from glucose by Ruminococcus albus

H₂ production from glucose by Ruminococcus albus was almost completely inhibited by 10^–5 M molybdate only when sulfide was present in the growth medium. Inhibition was accompanied by a significant increase in the production of formate. Extracts of molybdate-sulfide-grown cells did not contain hydrogenase activity. Active enzyme in extracts of uninhibited cells was not inhibited by the molybdate-sulfide-containing growth medium. The results indicate that a complex formed from molybdate and sulfide prevents the formation of active hydrogenase and electrons otherwise used to form H₂ are used to reduce CO₂ to formate. Growth was significantly inhibited when molybdate was increased to 10^–4 M. Reversal of growth inhibition but not inhibition of H₂ production occurred between 10^–4 and 10^–3 M molybdate. H₂ production by R. bromei but not by R. flavefaciens, Butyrivibrio fibrisolvens, Veillonella alcalescens, Klebsiella pneumoniae and Escherichia coli was inhibited by molybdate and sulfide.     

<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.     

Studies on a gram-positive hydrogen bacterium,Nocardia opaca strain 1b

Summary A new hydrogen bacterium has been isolated by enrichment culture on propane. It is a strictly aerobic, Gram-positive, non acid-fast bacterium, characterized by filamentous growth, and has been tentatively assigned to Nocardia opaca (strain 1 b).It grows heterotrophically, on many organic compounds (71 out of 138 tested substrates including organic acids and sugars), on hydrocarbons (C₁₁–C₁₈) as well as under autotrophic conditions (under an atmosphere of hydrogen, oxygen, and carbon dioxide=8:1:1) In the absence of a nitrogen source storage materials, mainly carbohydrates, are accumulated.Hydrogenase is an inducible enzyme. Under appropriate growth conditions the specific hydrogenase activity reaches high values: 2700 enzyme units/g cell protein. The formation of hydrogenase is repressed by fructose. With increasing oxygen concentrations during growth the specific hydrogenase activity decreases. In resting cell oxygen progressively inhibits the oxyhydrogen reaction.Cell-free extracts of autotrophically grown cells are able to reduce oxygen benzyl-and methyl viologen, dichlorphenolindophenol, methylene blue and nicotinamide adeninedinucleotide with hydrogen.     

Reversible inactivation of nitrate reductase in Chlorella vulgaris in vivo

Summary The NADH-nitrate oxidoreductase of Chlorella vulgaris has an inactive form which has previously been shown to be a cyanide complex of the reduced enzyme. This inactive enzyme can be reactivated by treatment with ferricyanide in vitro. In the present study, the activation state of the enzyme was determined after different prior in vivo programs involving environmental variations. Oxygen, nitrate, light and CO₂ all affect the in vivo inactivation of the enzyme in an interdependent manner. In general, the inactivation is stimulated by O₂ and inhibited by nitrate and CO₂. Light may stimulate or inhibit, depending on conditions. Thus, the effects of CO₂ and nitrate (inhibition of reversible inactivation) are clearly manifested only in the light. In contrast, light stimulates the inactivation in the presence of oxygen and the absence of CO₂ and nitrate. Since the inactivation of the enzyme requires HCN and NADH, and it is improbable that O₂ stimulates NADH formation, it is reasonable to conclude that HCN is formed as the result of an oxidation reaction (which is stimulated by light). The formation of HCN is probably stimulated by Mn²⁺, since the formation of reversibly-inactivated enzyme is impaired in Mn²⁺-deficient cells. The prevention of enzyme inactivation by nitrate in vivo is in keeping with previous in vitro results showing that nitrate prevents inactivation by maintaining the enzyme in the oxidized form. A stimulation of nitrate uptake by CO₂ and light could account for the effect of CO₂ (prevention of inactivation) which is seen mainly in the presence of nitrate and light. Ammonia added in the presence of nitrate has the same effect on the enzyme as removing nitrate (promotion of reversible inactivation). Ammonia added in the absence of nitrate has little extra effect. It is therefore likely that ammonia acts by preventing nitrate uptake. The uncoupler, carbonylcyanide-m-chloro-phenylhydrazone, causes enzyme inactivation because it acts as a good HCN precursor, particularly in the light. Nitrite, arsenate and dinitrophenol cause an enzyme inactivation which can not be reversed by ferricyanide in crude extracts. This suggests that there are at least two different ways in which the enzyme can be inactivated rather rapidly in vivo.     

Localization of hydrogenase and nitrate reductase in Campylobacter sputorum subsp. bubulus

Campylobacter sputorum subsp. bubulus contained hydrogenase activity after growth with lactate and nitrate and after growth with hydrogen and nitrate. After growth with hydrogen and nitrate a molar growth yield (g dry cells/mol hydrogen) of 5.6 was measured. Hydrogenase and nitrate reductase were membrane-bound enzymes. In cells with high hydrogenase activity the H⁺/O, H⁺/NO _inf2 ^sup- and H⁺/NO _inf3 ^sup- values with hydrogen as the electron donor were 3.74, 2.61 and 4.36 respectively. In cells with low hydrogenase activity these values were 2.33,-0.86 and 1.31 respectively. These values and the stoichiometry of respiration-driven proton translocation (H⁺/2e=2) led to the conclusion that hydrogenase is located at the periplasmic side of the cytoplasmic membrane. In cells with low lactate dehydrogenase activity or low hydrogenase activity the reduction of nitrate to nitrite could be separated from the reduction of nitrite to ammonia. Positive H⁺/NO _inf3 ^sup- values (between 0.9 and 1.7) with lactate or hydrogen as the electron donor were measured in these cells whereas H⁺/NO _inf2 ^sup- values were negative. From this result it was concluded that nitrate reductase is located at the cytoplasmic face of the cytoplasmic membrane. The results explain the previous observation that molar growth yields with nitrate were somewhat higher than those with nitrite.     

Hydrogenase activity in nitrate-grown cells of the unicellular cyanobacterium Cyanothece PCC 7822

The activity of hydrogenase in intact cells of the unicellular cyanobacterium Cyanothece PCC 7822 was investigated using a mass spectrometer with a permeable membrane inlet. A small hydrogenase-catalyzed hydrogen production was observed with nitrate-grown cells under anoxic conditions in the dark. The same cells were also capable of a much greater rate of hydrogen uptake, induced by oxygen as well as light. Light-induced hydrogen uptake was inhibited by uncoupler. In contrast, addition of uncoupler caused a four-fold stimulation of anoxic hydrogen production in the dark. It is suggested that anoxic hydrogen production is the result of fermentative metabolism.Cyanobacteria are generally considered to have at least two distinct hydrogenases (Houchins 1984). One is a membrane-bound uptake hydrogenase which appears to be associated with nitrogen fixation, removing the hydrogen produced by nitrogenase with the concomitant production of reductant or ATP (Eisbrenner et al. 1978). The second is a reversible hydrogenase located in the cytoplasm and not closely linked to nitrogen metabolism. The reversible character of this enzyme can be demonstrated in the presence of suitable electron donors or acceptors; hydrogen consumption and evolution occur at similar rates (Lambert and Smith 1980).A reversible hydrogenase capable of reducing protons with the artificial electron donor couple dithionite and methyl viologen is widely distributed amongst cyanobacteria. However its physiological role remains unclear. The enzyme appears to be sensitive to oxygen, and consequently in vivo activity can only be demonstrated under anoxic conditions (Houchins 1984).On the basis of in vivo measurements with tritium and the observed low K _m for hydrogen, the function of the reversible hydrogenase of the heterocystous cyanobacterium Anabaena has been proposed to be the uptake of hydrogen as a means of collecting additional reducing power during growth in light-limited anoxic environments (Spiller et al. 1983; Houchins 1984). However, Hallenbeck et al. (1981) reported a modest production of hydrogen by intact filaments of Anabaena.An example of a function of the reversible hydrogenase in the production of hydrogen is provided by the nonheterocystous filamentous cyanobacterium Oscillatoria limnetica. This organism is capable of shifting between oxygenic and anoxygenic photosynthesis (Oren and Padan 1978). In the latter case sulfide is the electron donor supporting photoreduction of CO₂ via photosystem I only. However when CO₂ is limiting, excess reducing equivalents are removed by a reversible hydrogenase (Belkin and Padan 1978). This hydrogen production probably enables the organism to continue photophosphorylation under these conditions.We recently reported that the unicellular cyanobacterium Cyanothece 7822 is capable of hydrogenase-catalyzed hydrogen production in vivo, without the addition of artificial reductants (Van der Oost et al. 1987). In this paper we have investigated the in vivo activity of the hydrogenase in Cyanothece by monitoring the concentrations of dissolved H₂ and O₂ in the cell suspension using a mass spectrometer with a permeable membrane inlet.Abbreviations DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU N-(3,4-dichlorophenyl) N,N-dimethylurea - FCCP carbonylcyanide-p-trifluoromethoxy phenylhydrazone - PBQ phenyl benzoquinone     

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     

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     

The occurrence of the hydrogenase in some blue-green algae

Several blue-green algae were surveyed for the occurrence of the hydrogenase which was assayed by the oxyhydrogen or Knallgas reaction in the intact organisms. In aerobically grown cultures, the reaction was detectable in Anabaena cylindrica, Nostoc muscorum and in two Anabaena variabilis species, whereas virtually no activity was observed in Anacystis nidulans and Cyanophora paradoxa. In these latter two algae, the reaction was, however, found after growth under molecular hydrogen for several days, which drastically increased the activity levels with all the algae tested. In the nitrogen fixing species, the activity of the Knallgas reaction was enhanced when all combined nitrogen was omitted from the media. H₂ and hydrogenase could not significantly support the CO₂-fixation in photoreduction experiments with all blue-green algae investigated here. Hydrogenase was assayed by the dithionite and methyl viologen dependent evolution of hydrogen and was found to be present with essentially the same specific activity levels in preparations of both heterocysts and vegetative cells from Anabaena cylindrica. Na₂S₂O₄ as well as H₂ supported the C₂H₂-reduction of the isolated heterocysts. The H₂-dependent C₂H₂-reduction did not require the presence of oxygen but was strictly light-dependent where H₂ served as an electron donor to photosystem I of these cells. It is concluded that hydrogen can be utilized by two different pathways in blue-green algae.Abbreviations Chl chlrophyll - CP creatine phosphate - CP kinase creatine phosphokinase - DCMU N-(3,4-dichlorophenyl)N,N-dimethylurea     

Nitrogenase — hydrogenase relationships in Rhizobium japonicum

The conditions necessary for coordinate derepression of nitrogenase and O₂-dependent hydrogenase activities in free-living cultures of Rhizobium japonicum were studied. Carbon sources were screened for their ability to support nitrogenase, and then hydrogenase activities. There was a positive correlation between the level of nitrogenase and corresponding hydrogenase activities among the various carbon substrates. The carbon substrate -ketoglutarate was able to support the highest levels of both nitrogenase and hydrogenase activities. When cells were incubated in -ketoglutarate-containing medium, without added H₂ but in the presence of acetylene (to block H₂ evolution from nitrogenase) significant hydrogenase activity was still observed. Complete inhibition of nitrogenase-dependent H₂ evolution by acetylene was verified by the use of a Hup^- mutant. Hydrogen is therefore not required to induce hydrogenase. The presence of 10% acetylene inhibited derepression of hydrogenase. Constitutive (Hup^c) mutants were isolated which contained up to 9 times the level of hydrogenase acitivity than the wild type in nitrogenase induction medium. These mutants did not have greater nitrogenase activities than the wild type.This is contribution number 1254 from the Department of Biology and the McCollum-Pratt Institute Abbreviations: -Ketoglutarate-containing medium (LOKG) and pre-adaptation medium (SRM) as described in Materials and methods     

Chemical Modification of Catalytically Essential Functional Groups of NAD-Dependent Hydrogenase from Ralstonia eutropha H16

Amino acid residues His and Cys of the NAD-dependent hydrogenase from the hydrogen-oxidizing bacterium Ralstonia eutropha H16 were chemically modified with specific reagents. The modification of His residues of the nonactivated hydrogenase resulted in decrease in both hydrogenase and diaphorase activities of the enzyme. Activation of NADH hydrogenase under anaerobic conditions additionally modified a His residue (or residues) significant only for the hydrogenase activity. The rate of decrease in the diaphorase activity was unchanged. The modification of thiol groups of the nonactivated enzyme did not affect the hydrogenase activity. The effect of thiol-modifying agents on the activated hydrogenase was accompanied by inactivation of both diaphorase and hydrogenase activities. The modification degree and changes in the corresponding catalytic activities depended on conditions of the enzyme activation. Data on the modification of cysteine and histidine residues of the hydrogenase suggested that the enzyme activation should be associated with significant conformational changes in the protein globule.     

Enzymes of the autotrophic pathway in mating partners and transconjugants of Nocardia opaca 1 b and Rhodococcus erythropolis

Enzyme activities have been measured in the partners of a bacterial mating system consisting of the hydrogen autotroph Nocardia opaca (donor and Aut^- recipient), the heterotroph Rhodococcus erythropolis (recipient) and intra- and interspecies transconjugants after growth on fructose, pyruvate and under autotrophic conditions. Specific activities of each of the enzymes hydrogenase, phosphoribulokinase and ribulosebisphosphate carboxylase were high in autotrophically grown cells of the donor and the transconjugants: they amounted to only 10% after growth on pyruvate. The recipient cells did not grow autotrophically and the enzymes mentioned were not detectable even after growth on pyruvate. Other enzymes of the Calvin cycle were constitutively formed in all strains examined.The properties of hydrogenase (K _m for NAD, R_f in gel electrophoresis) and of ribulosebisphosphate carboxylase (K _m for RuBP and R_f) were the same in the donor and transconjugant cells. The properties of glucose-6-phosphate dehydrogenase (K _m for G-6-P and mode of inhibition by ATP and phosphoenolpyruvate) were the same in the recipient and the interspecies transconjugant cells and differed from those of the donor cells. The curves of growth under autotrophic conditions in batch culture of the donor and interspecies transconjugant were almost congruent. The specific activities of hydrogenase, phosphoribulokinase and ribulosebisphosphate carboxylase increased from 40% at the beginning to 100% at the end of the exponential growth phase; these enzymes were under coordinate control.The results are in accordance with genetic studies: the genetic information for autotrophic growth is localized on a so far unidentified genetic element and is transferred en bloc from N. opaca to Aut^- mutants of the same strain or to recipient bacteria such as R. erythropolis; expression in the wild type and transconjugant cells is the same.Abbreviations G-6-P glucose-6-phosphate - 6-PG 6-phosphogluconate - FBP fructose-1,6-bisphosphate - SBP sedoheptulose-1,7-bisphosphate - RuBP ribulose-1,5-bisphosphate     

FTIR spectroelectrochemical study of the activation and inactivation processes of [NiFe] hydrogenases: effects of solvent isotope replacement and site-directed mutagenesis

The kinetics of the activation and anaerobic inactivation processes of Desulfovibrio gigas hydrogenase have been measured in D₂O by FTIR spectroelectrochemistry. A primary kinetic solvent isotope effect was observed for the inactivation process but not for the activation step. The kinetics of these processes have been also measured after replacement of a glutamic residue placed near the active site of an analogous [NiFe] hydrogenase from Desulfovibrio fructosovorans. Its replacement by a glutamine affected greatly the kinetics of the inactivation process but only slightly the activation process. The interpretation of the experimental results is that the rate-limiting step for anaerobic inactivation is the formation from water of a -OH^– bridge at the hydrogenase active site, and that Glu25 has a role in this step.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0559-7     

Nitrogenase activity in Rhodopseudomonas sulfidophila

The marine purple nonsulfur bacterium, Rhodopseudomonas sulfidophila, strain W4, was capable of photosynthetic growth on dinitrogen and malate. Higher growth rates were observed when either glutamate or ammonia replaced dinitrogen as nitrogen source and when bicarbonate was omitted from the culture medium. Although ammonia was released from cells growing on malate and N₂, no nitrogenase activity could be detected unless -ketoglutarate was added to the culture medium. No nitrogenase activity was found in cultures grown in the presence of NH ₄ ⁺ . In cultures grown on glutamate as nitrogen source, nitrogenase and hydrogenase activities were found to be 5.4 nmol C₂H₂ reduced · min^-1 · mg^-1 dry weight and 50 nmol methylene blue reduced · min^-1 · mg^-1 dry weight respectively. Such activities are significantly lower than those observed for other members of the Rhodospirillaceae e.g. Rhodopseudomonas capsulata. However, the hydrogenase activity would be sufficient to recycle all H₂ produced by nitrogenase. It was indeed observed that growing cells did not evolve molecular hydrogen during photoheterotrophic growth and that H₂ stimulated nitrogenase activity in resting cells of R. sulfidophila. The nitrogenase from this bacterium proved to be extremely sensitive to low concentrations of oxygen, half-inhibition occurring at between 1–1.5% O₂ in the gas phase, depending on the bacterial concentration. Light was essential for nitrogenase activity. No activity was found during growth in the dark under extremely low oxygen concentrations (1–2% O₂), which are still sufficient to support good growth. Resting cell suspensions prepared from such cultures were unable to reduce acetylene upon illumination. Optimum nitrogenase activities were broadly defined over the temperature range, 30–38°C, and between pH 6.9 and 8.0. The results are discussed in comparison with the non-marine purple nonsulfur bacterium, R. capsulata, which somewhat resembles R. sulfidophila.