Expression of uptake hydrogenase and hydrogen oxidation during heterotrophic growth of Bradyrhizobium japonicum.

Strains I-110 ARS, SR, USDA 136, USDA 137, and AK13 1c of Bradyrhizobium japonicum induced Hup activity when growing heterotrophically in medium with carbon substrate and NH4Cl in the presence of 2% H2 and 2% O2. Hup activity was induced during heterotrophic growth in the presence of carbon substrates, which were assimilated during the time of H2 oxidation. Strains I-110 ARS and SR grown heterotrophically or chemoautotrophically for 3 days had similar rates of H2 oxidation. Similar rates of Hup activity were also observed when cell suspensions were induced for 24 h in heterotrophic or chemoautotrophic growth medium with 1% O2, 10% H2, and 5% CO2 in N2. These results are contrary to the reported repression of Hup activity by carbon substrates in B. japonicum. Bradyrhizobial Hup activity during heterotrophic growth was limited by H2 and O2 and repressed by aerobic conditions, and CO2 addition had no effect. Nitrogenase and ribulosebisphosphate carboxylase activities were not detected in H2-oxidizing cultures of B. japonicum during heterotrophic growth. Immunoblot analysis of cell extracts with antibodies prepared against the 65-kilodalton subunit of uptake hydrogenase indicated that Hup protein synthesis was induced by H2 and repressed under aerobic conditions.     

Nickel is a component of hydrogenase in Rhizobium japonicum

The derepression of H2-oxidizing activity in free-living Rhizobium japonicum does not require the addition of exogenous metal to the derepression media. However, the addition of EDTA (6 microM) inhibited derepression of H2 uptake activity by 80%. The addition of 5 microM nickel to the derepression medium overcame the EDTA inhibition. The addition of 5 microM Cu or Zn also relieved EDTA inhibition, but to a much lesser extent; 5 microM Fe, Co, Mg, or Mn did not. The kinetics of induction and magnitude of H2 uptake activity in the presence of EDTA plus Ni were similar to those of normally derepressed cells. Nickel also relieved EDTA inhibition of methylene blue-dependent Hup activity, suggesting that nickel is involved directly with the H2-activating hydrogenase enzyme. Adding nickel or EDTA to either whole cells or crude extracts after derepression did not affect the hydrogenase activity. Cells were grown in 63Ni and the hydrogenase was subsequently purified by gel electrophoresis. 63Ni comigrated with the H2-dependent methylene blue reducing activity on native polyacrylamide gels and native isoelectric focusing gels. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the nickel-containing hydrogenase band revealed a single polypeptide with a molecular weight of ca. 67,000. We conclude that the hydrogenase enzyme in R. japonicum is a nickel-containing metalloprotein.     

Hydrogen metabolism of Azospirillum brasilense in nitrogen-free medium

Production of H2 by Azospirillum brasilense under N2-fixing conditions was studied in continuous and batch cultures. Net H2 production was consistently observed only when the gas phase contained CO. Nitrogenase activity (C2H2 reduction) and H2 evolution (in the presence of 5% CO) showed a similar response to O2 and were highest at 0.75% dissolved O2. Uptake hydrogenase activity, ranging from 0.3 to 2.5 mumol H2/mg protein per hour was observed in batch cultures under N2. Such rates were more than sufficient to recycle nitrogenase-produced H2. Tritium-exchange assay showed that H2 uptake was higher under Ar than under N2. Uptake hydrogenase was strongly inhibited by CO and C2H2. Cyclic GMP inhibited both nitrogenase and uptake hydrogenase activities.     

Selenium increases hydrogenase expression in autotrophically cultured Bradyrhizobium japonicum and is a constituent of the purified enzyme.

We have investigated the effect of added selenite on autotrophic growth and the time course of hydrogen oxidation derepression in Bradyrhizobium japonicum 122DES cultured in a medium purified to remove selenium compounds. In addition, hydrogenase was purified to near homogeneity and examined for the specific incorporation of Se into the enzyme. The addition of Se at 0.1 microM significantly increased total cell protein and hydrogenase specific activity of harvested cells. Also, the addition of SeO3(2-) enhanced the time course of hydrogenase derepression by 133%, whereas VO3, AsO2(2-), SO2(2-), and TeO3(2-) failed to substantially affect hydrogenase derepression. During the final chromatographic purification of hydrogenase, a striking coincidence in peaks of protein content, Se radioactivity, and hydrogenase activity of fractions was obtained. The total Se content expressed per milligram of protein increased manyfold during the purification procedure. The mean Se content of the purified hydrogenase was 0.56 +/- 0.13 mol of Se per mol of enzyme. These results indicate that Se is an important element in the H2 metabolism of B. japonicum and that hydrogenase from B. japonicum is a seleno protein.     

Rhizobium japonicum mutants that are hypersensitive to repression of H2 uptake by oxygen.

The synthesis of an H2 oxidation system in free-living Rhizobium japonicum wild-type strain SR is repressed by oxygen. Maximal H2 uptake rates were obtained in strain SR after derepression in 11 microM or less dissolved oxygen. Oxygen levels above 45 microM completely repressed H2 uptake in strain SR. Five R. japonicum mutant strains that are hypersensitive to repression or H2 oxidation by oxygen were derived from strain SR. The mutants were obtained by screening H2 uptake-negative mutants that retained the ability to oxidize H2 as bacteroids from soybean nodules. As bacteroids, the five mutant strains were capable of H2 oxidation rates comparable to that of the wild type. The mutants did not take up H2 when derepressed in 22 microM dissolved oxygen, whereas strain SR had substantial activity at this oxygen concentration. The O2 repression of H2 uptake in both the wild-type and two mutant strains, SR174 and SR200, was rapid and was similar to the effect of inhibiting synthesis of H2 uptake system components with rifampin. None of the mutant strains was able to oxidize H2 when the artificial electron acceptors methylene blue or phenazine methosulfate were provided. The mutant strains were not sensitive to killing by oxygen, they took up O2 at rates similar to strain SR, and they did not produce an H2 uptake system that was oxygen labile. Cyclic AMP levels were comparable in strain SR and the five mutant strains after subjection of the cultures to the derepression conditions.     

Interaction between hydrogenase, nitrogenase, and respiratory activities in a Frankia isolate from Alnus rubra

H2 uptake and H2-supported O2 uptake were measured in N2-fixing cultures of Frankia strain ArI3 isolated from root nodules of Alnus rubra. H2 uptake by intact cells was O2 dependent and maximum rates were observed at ambient O2 concentrations. No hydrogenase activity could be detected in NH4+-grown, undifferentiated filaments cultured aerobically indicating that uptake hydrogenase activity was associated with the vesicles, the cellular site of nitrogen fixation in Frankia. Hydrogenase activity was inhibited by acetylene but inhibition could be alleviated by pretreatment with H2. H2 stimulated acetylene reduction at supraoptimal but not suboptimal O2 concentrations. These results suggest that uptake hydrogenase activity in ArI3 may play a role in O2 protection of nitrogenase, especially under conditions of carbon limitation.     

Occurrence and localization of two distinct hydrogenases in the heterocystous cyanobacterium Anabaena sp. strain 7120

Two distinct types of hydrogenase occur in Anabaena 7120 and are distinguishable in whole filaments by the application of selective assay methods. A reversible hydrogenase occurs both in heterocysts and vegetative cells and can be selectively assayed by measuring H2 evolution from reduced methyl viologen. Activities in aerobically grown filaments were low but could be increased by 2 to 3 orders of magnitude by growing cells microaerobically. The presence of the reversible hydrogenase was independent of the N2-fixing properties of the organism, and activity did not respond to added H2 in the culture. Illumination was necessary during derepression of the reversible hydrogenase, and addition of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea increased the amount of enzyme that was synthesized. An uptake hydrogenase occurred only in heterocysts of aerobically grown filaments, but a small amount of activity also was present in the vegetative cells of filaments grown microaerobically with 20% H2. It was assayed selectively by measuring an oxyhydrogen reaction at atmospheric levels of O2. Additional uptake hydrogenase could be elicited by including H2 or by removing O2 from the sparging gas of a culture.     

Regulation of hydrogen utilisation in Rhizobium japonicum by cyclic AMP.

Utilisation (uptake) of hydrogen gas by whole cells of Rhizobium japonicum was found to be influenced by the carbon source(s) present in the growth medium, with activity being highest in a medium containing sugars. Tricarboxylic acid cycle intermediates, such as malate, significantly reduced H2 utilisation. No reduction in the hydrogenase activity is observed when the enzyme is assayed directly by the tritium exchange method, indicating that the decrease in hydrogen uptake activity is not due to repression of hydrogenase biosynthesis. Cyclic AMP was found to alleviate the inhibition of H2 uptake by malate, and this requires new protein synthesis. Addition of chloramphenicol or rifampicin simultaneously with cyclic AMP eliminated the stimulation of H2 uptake in the malate medium. These results show that in R. japonicum cyclic AMP plays a major role in the regulation of H2 metabolism.     

Regulation of two nickel-requiring (inducible and constitutive) hydrogenases and their coupling to nitrogenase in Methylosinus trichosporium OB3b.

Two uptake hydrogenases were found in the obligate methanotroph Methylosinus trichosporium OB3b; one was constitutive, and a second was induced by H2. Both hydrogenases could be assayed by measuring methylene blue reduction anaerobically or by coupling their activity to nitrogenase acetylene reduction activity in vivo in an O2-dependent reaction. The H2 concentration for half-maximal activity of the inducible and constitutive hydrogenases in both assays was 0.01 and 0.5 bar (1 and 50 kPa), respectively, making it easy to distinguish these enzymes from one another both in vivo and in vitro. Hydrogen uptake was shown to be coupled to ATP synthesis in methane-starved cells. Methane, methanol, formate, succinate, and glucose all repressed the H2-mediated synthesis of the inducible hydrogenase. Furthermore, this enzyme was only expressed in N-starved cultures and was repressed by NH4+ and NO3-; synthesis of the constitutive hydrogenase was not affected by excess N in the growth medium. In nickel-free, EDTA-containing medium, the activities of these two enzymes were negligible; however, both enzyme activities appeared rapidly following the addition of nickel to the culture. Chloramphenicol, when added along with nickel, had no effect on the rapid appearance of either the constitutive or inducible activity, indicating that nickel is not required for synthesis of the hydrogenase apoproteins. These observations all suggest that these hydrogenases are nickel-containing enzymes. Finally, both hydrogenases were soluble and could be fractionated by 20% ammonium sulfate; the constitutive enzyme remained in the supernatant solution, while the inducible enzyme was precipitated under these conditions.     

Effects of cultivation gas phase on hydrogenase of the acetogen Clostridium thermoaceticum.

The effect of cultivation gas phase on the expression and activity of hydrogenase in heterotrophic cultures of Clostridium thermoaceticum was examined. Of the five gas phases tested, hydrogenase was maximal from cells cultivated under CO. Correlations were observed between the level of hydrogenase and the evolution of H2 by growing cultures. Activity stains of polyacrylamide gels revealed a single hydrogenase band in CO2 cells and multiple hydrogenase bands in CO cells.     

浑球红假单胞菌及其谷氨酸合成酶突变株氢酶活性和合成

浑球红假单胞菌野生型菌株的氢酶表达被有机碳、氮底物所抑制。在光照和黑暗时,氧浓度变化对氢酶的作用不同,但高氧浓度都阻遏氢酶的表达。微量Ni~(2+)能专一性地促进氢酶活性,固氮酶的产氢也可以调节氢酶的表达水平。该野生菌株的GOGAT突变株缺乏固氮酶和氢酶活性,在加入谷氨酰胺合成酶抑制剂MSX后,固氮酶和氢酶以相关联的方式合成出来,固氮酶产生的氢看来诱导了氢酶的合成。然而在固氮酶不表达的情况下,外源氢也可诱导氢酶的合成。     

Uptake hydrogenase activity and ATP formation in Rhizobium leguminosarum bacteroids.

The role of uptake hydrogenase was studied in Rhizobium leguminosarum bacteroids from the nodules of Pisum sativum L. cv. Homesteader. Uptake hydrogenase activity, measured by the 3H2 uptake method, was dependent on O-consumption and was similar to H2 uptake measured by gas chromatography. Km for O2 of 0.0007 atm (0.0709 kPa) and a Km for H2 of 0.0074 atm (0.7498, kPa) were determined. H2 increased the rate of endogenous respiration by isolates with uptake hydrogenase (Hup+) but had no effect on an isolate lacking uptake hydrogenase (Hup-). A survey of 14 Hup+ isolates indicated a wide range of H2 uptake activities. Four of the isolates tested had activities similar to or higher than those found in two Hup+ Rhizobium japonicum strains. H2 uptake was strongly coupled to ATP formation in only 5 of the 14 isolates. H2 increased the optimal O2 level of C2H2 reduction by 0.01 atm and permitted enhanced C2H2 reduction at O2 levels above the optimum in both a coupled and an uncoupled isolate. At suboptimal O2 concentrations a small enhancement of C2H2 reduction by H2 was seen in two out of three isolates in which H2 oxidation was coupled to ATP formation. Thus, the main function of uptake hydrogenase in R. leguminosarum appears to be in the protection of nitrogenase from O2 damage.     

Aerobic purification of hydrogenase from Rhizobium japonicum by affinity chromatography.

We purified active hydrogenase from free-living Rhizobium japonicum by affinity chromatography. The uptake hydrogenase of R. japonicum has been treated previously as an oxygen-sensitive protein. In this purification, however, reducing agents were not added nor was there any attempt to exclude oxygen. In fact, the addition of sodium dithionite to aerobically purified protein resulted in the rapid loss of activity. Purified hydrogenase was more stable when stored under O2 than when stored under Ar. Sodium-chloride-washed hydrogen-oxidizing membranes were solubilized in Triton X-100 and deoxycholate and loaded onto a reactive red 120-agarose column. Purified hydrogenase elutes at 0.36 M NaCl, contains a nickel, and has a pH optimum of 6.0. There was 452-fold purification resulting in a specific activity of 76.9 mumol of H2 oxidized per min per mg of protein and a yield of 17%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed subunits with estimated molecular weights of 65,000 and 33,000. Hydrogenase prepared in this manner was used to raise and affinity purify antibodies against both subunits.     

Continuous monitoring, by mass spectrometry, of H2 production and recycling in Rhodopseudomonas capsulata.

Hydrogen evolution and consumption by cell and chromatophore suspensions of the photosynthetic bacterium Rhodopseudomonas capsulata was measured with a sensitive and specific mass spectrometric technique which directly monitors dissolved gases. H2 production by nitrogenase was inhibited by acetylene and restored by carbon monoxide. An H2 evolution activity coupled with HD formation and D2 uptake (H-D exchange) was unaffected by C2H2 and CO. Cultures lacking nitrogenase activity also exhibited H-D exchange activity, which was catalyzed by a membrane-bound hydrogenase present in the chromatophores of R. capsulata. A net hydrogen uptake, mediated by hydrogenase, was observed when electron acceptors such as CO2, O2, or ferricyanide were present in the medium.     

Transport of Ca2+ and Na+ across the chromaffin-granule membrane.

The soluble hydrogenase (hydrogen-NAD+ oxidoreductase, EC 1.12.1.2) of Alcaligenes eutrophus H16 was shown to be stabilized by oxidation with oxygen and ferricyanide as long as electron donors and reducing compounds were absent. The simultaneous presence of H2, NADH and O2 in the enzyme solution, however, caused an irreversible inactivation of hydrogenase that was dependent on the O2 concentration. The half-life periods of 4 degrees C under partial pressures of 0.1, 5, 20 and 50% O2 were 11, 5, 2.5 and 1.5 h respectively. Evidence has been obtained that hydrogenase produces superoxide free radical anions (O2-.), which were detected by their ability to oxidize hydroxylamine to nitrite. The correlation between O2 concentration, nitrite formation and inactivation rates and the stabilization of hydrogenase by addition of superoxide dismutase indicated that superoxide radicals are responsible for enzyme inactivation. During short-term activity measurements (NAD+ reduction, H2 evolution from NADH), hydrogenase activity was inhibited by O2 only very slightly. In the presence of 0.7 mM-O2 an inhibition of about 20% was observed.     

Rhizobitoxine inhibition of hydrogenase synthesis in free-living Bradyrhizobium japonicum.

Rhizobitoxine produced by Bradyrhizobium species strongly prevented derepression of hydrogenase expression in free-living Bradyrhizobium japonicum, although the toxin had no effect on the activity of cells which had already synthesized hydrogenase protein. Dihydrorhizobitoxine, a structural analog of rhizobitoxine, proved to be a less potent inhibitor of hydrogenase derepression. Rhizobitoxine did not cause cell death at a concentration sufficient to eliminate hydrogenase expression. The large subunit of hydrogenase was not detectable with antibody after derepression in the presence of rhizobitoxine. The general pattern of proteins synthesized from 14C-labeled amino acids during derepression was not significantly different in the presence or absence of rhizobitoxine. These results indicated that rhizobitoxine inhibited hydrogenase synthesis in free-living B. japonicum. Cystathionine and methionine strongly prevented the inhibition of hydrogenase derepression by rhizobitoxine, suggesting that the inhibition involves the level of sulfur-containing amino acids in the cell.     

Nickel affects expression of the nickel-containing hydrogenase of Alcaligenes latus.

The effects of nickel on the expression of hydrogenase in the hydrogen-oxidizing bacterium Alcaligenes latus were studied. In the absence of added nickel, both hydrogenase activity, measured as O2-dependent H2 uptake, and hydrogenase protein, measured in a Western immunoblot, were very low compared with the levels in cells induced for hydrogenase in the presence of nickel. Hydrogenase activity and protein levels were dependent on the added nickel concentration and were saturated at 30 nM added Ni2+. The amount of hydrogenase protein in a culture at a given nickel concentration was calculated from the H2 uptake activity of the culture at that Ni2+ concentration. Between 0 and 30 nM added Ni2+, the amount of hydrogenase protein (in nanomoles) was stoichiometric with the amount of added Ni2+. Thus, all of the added Ni2+ could be accounted for in hydrogenase. Between 0 and 50 nM added Ni2+, all the Ni present in the cultures was associated with the cells after 12 h; above 50 nM added Ni2+, some Ni remained in the medium. No other divalent metal cations tested were able to substitute for Ni2+ in the formation of active hydrogenase. We suggest two possible mechanisms for the regulation of hydrogenase activity and protein levels by nickel.