The acetylene reduction assay inactivates root nodule uptake hydrogenase in some actinorhizal plants

Actinorhizal nodules do not usually evolve H₂ due to the action of an uptake hydrogenase. We have found that nodules of several Frankia symbioses evolved large amounts of H₂ gas when returned to air following exposure to 10 kPa C₂HT₂ during an acetylene reduction assay. Increased H₂ evolution in air persisted for several days when intact root systems of Alnus incana (L.) Moench (inoculated with Frankia UGL 011101) were treated with 10 kPa C.H₂ for 1 h. Full recovery of uptake hydrogenase activity required 4 to 8 days. Studies with crude homogenates of nodules of the same plants showed that hydrogenase (measured amperometrically with phenazine metho-sulfate as electron acceptor) was directly affected, since activity in treated nodules was only 10% of that in untreated nodules. A survey of actinorhizal symbioses revealed variation in the effect of an acetylene reduction assay on hydrogen metabolism. Nodules of three species, including Alnus rubra Bong, inoculated with Frankia HFPArD. showed complete inactivation of hydrogenase. H₂ evolution in air was 25% of the C₂H₂ reduction rate and H, evolution in Ar/O₂ was equal to the QH₂ reduction rate. Two symbioses, Ceanothus americanus L. (soil inoculant) and Batista glomerata Baill. (soil inoculant) showed no change following an acetylene reduction assay. A third group of symbioses showed an intermediate response.     

Uptake and evolution of H2 and reduction of C2H2 by root nodules and nodule homogenates ofAlnus glutinosa

Summary In the growing season no net H₂ evolution is detected when root nodules ofAlnus glutinosa are incubated in air or in argon containing 20% O₂. Due to the hydrogenase activity, N₂-fixing root nodules consume added H₂ at a rate of about 1.4 moles H₂.g fresh nodule^–1.h^–1. The uptake of H₂ is only found in summer. At the end of the season, in autumn, nodules evolve significant quantities of H₂ although the nodules still continue to fix nitrogen. In-vitro studies with fractionated homogenates of summer-harvested nodules show that the recovery of the hydrogenase is high when using methylene-blue or phenazine metasulfate as electron acceptors. No hydrogenase activity is detected in homogenates of autumn-harvested nodules.The hydrogenase is localised in the microsymbiont.     

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

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

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

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

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.     

Purification and properties of the particulate hydrogenase from the bacteroids of soybean root nodules.

The uptake hydrogenase (hydrogen:ferricytochrome c3 oxidoreductase, EC 1.12.2.1) from the bacteroids of soybean root nodules infected with Rhizobium japonicum 110 has been purified and characterized. Bacteroids were prepared, then broken by sonication. The particulate enzyme was solubilized by treatment with Triton X-100 and further purified by polyethylene glycol fractionation, DEAE-cellulose and Sephadex G-100 chromatography. The specific activity has been increased 196-fold to 19.6 units/mg protein. The molecular weight is 63 300 as determined by gel filtration and 65 300 as determined by SDS-polyacrylamide gel electrophoresis, indicating that the enzyme is a monomer. The enzyme is O2 sensitive, with a half-life of 70 min when exposed to air. The pH optimum of the solubilized enzyme is near 5.5; the Km for H2 is 1.4 microM. Suitable electron acceptors are methylene blue, ferricyanide, 2,6-dichlorophenolindophenol, and cytochrome c. Benzyl viologen is reduced slowly; methyl viologen, NAD(P)+, FAD, FMN, and O2 are not reduced. The optimum temperature for activity is 65-70 degrees C with an activation energy of 9.2 kcal. H2 evolution by the enzyme has been demonstrated. The hydrogenase is well-suited to function in an environment where all the available H2 is generated in situ.     

Aerobic hydrogenase activity in Anacystis nidulans. The oxyhydrogen reaction.

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

Purification and properties of membrane-bound hydrogenase from Azotobacter vinelandii

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

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.     

Role of HoxE subunit in Synechocystis PCC6803 hydrogenase

Cyanobacterial NAD(P)(+)-reducing reversible hydrogenases comprise five subunits. Four of them (HoxF, HoxU, HoxY, and HoxH) are also found in the well-described related enzyme from Ralstonia eutropha. The fifth one (HoxE) is not encoded in the R. eutropha genome, but shares homology with the N-terminal part of R. eutropha HoxF. However, in cyanobacteria, HoxE contains a 2Fe-2S cluster-binding motif that is not found in the related R. eutropha sequence. In order to obtain some insights into the role of HoxE in cyanobacteria, we deleted this subunit in Synechocystis PCC6803. Three types of interaction of the cyanobacterial hydrogenase with pyridine nucleotides were tested: (a) reductive activation of the NiFe site, for which NADPH was found to be more efficient than NADH; (b) H(2) production, for which NADH appeared to be a more efficient electron donor than NADPH; and (c) H(2) oxidation, for which NAD(+) was a much better electron acceptor than NADP(+). Upon hoxE deletion, the Synechocystis hydrogenase active site remained functional with artificial electron donors or acceptors, but the enzyme became unable to catalyze H(2) production or uptake with NADH/NAD(+). However, activation of the electron transfer-independent H/D exchange reaction by NADPH was still observed in the absence of HoxE, whereas activation of this reaction by NADH was lost. These data suggest different mechanisms for diaphorase-mediated electron donation and catalytic site activation in cyanobacterial hydrogenase.     

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

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

Effect of redox potential on activity of hydrogenase 1 and hydrogenase 2 in Escherichia coli

This report elucidates the distinctions of redox properties between two uptake hydrogenases in Escherichia coli. Hydrogen uptake in the presence of mediators with different redox potential was studied in cell-free extracts of E. coli mutants HDK103 and HDK203 synthesizing hydrogenase 2 or hydrogenase 1, respectively. Both hydrogenases mediated H(2) uptake in the presence of high-potential acceptors (ferricyanide and phenazine methosulfate). H(2) uptake in the presence of low-potential acceptors (methyl and benzyl viologen) was mediated mainly by hydrogenase 2. To explore the dependence of hydrogen consumption on redox potential of media in cell-free extracts, a chamber with hydrogen and redox ( E(h)) electrodes was used. The mutants HDK103 and HDK203 exhibited significant distinctions in their redox behavior. During the redox titration, maximal hydrogenase 2 activity was observed at the E(h) below -80 mV. Hydrogenase 1 had maximum activity in the E(h) range from +30 mV to +110 mV. Unlike hydrogenase 2, the activated hydrogenase 1 retained activity after a fast shift of redox potential up to +500 mV by ferricyanide titration and was more tolerant to O(2). Thus, two hydrogenases in E. coli are complementary in their redox properties, hydrogenase 1 functioning at higher redox potentials and/or at higher O(2) concentrations than hydrogenase 2.     

Light and dark reactions of the uptake hydrogenase in anabaena 7120

Reactions of the uptake hydrogenase from Anabaena 7120 (A.T.C.C. 27893, Nostoc muscorum) were examined in whole filaments, isolated heterocysts, and membrane particles. Whole filaments or isolated heterocysts that contained nitrogenase consumed H₂ in the presence of C₂H₂ or N₂ in a light-dependent reaction. If nitrogenase was inactivated by O₂ shock, filaments catalyzed H₂ uptake to an unidentified endogenous acceptor in the light. Addition of NO₃⁻ or NO₂⁻ enhanced these rates. Isolated heterocysts consumed H₂ in the dark in the presence of electron acceptors with positive midpoint potentials, and these reactions were not enhanced by light. With acceptors of negative midpoint potential, significant light enhancement of H₂ uptake occurred. Maximum rates of light-dependent uptake were approximately 25% of the maximum dark rates observed. Membrane particles prepared from isolated heterocysts showed similar specificity for electron acceptors. These particles catalyzed a cyanide-sensitive oxyhydrogen reaction that was inactivated by O₂ at O₂ concentrations above 2%. Light-dependent H₂ uptake to low potential acceptors by these particles was inhibited by dibromothymoquinone but was insensitive to cyanide. In the presence of O₂, light-dependent H₂ uptake occurred simultaneously with the oxyhydrogen reaction. The pH optima for both types of H₂ uptake were near 7.0. These results further clarify the role of uptake hydrogenase in donating electrons to both the photosynthetic and respiratory electron transport chains of Anabaena.     

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.     

Hydrogenases of phototrophic microorganisms

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

Relative efficiency of nitrogen fixation and the occurrence of hydrogenase in pea root nodules

Summary The apparent K_m(hydrogen) for uptake of hydrogen by pea root nodules was determined. This enabled the concentration gradient necessary for the evolution of hydrogen to be calculated for nodules with no hydrogenase activity. This indicated that hydrogen inhibition of nitrogenase is not likely to be the cause of the low relative efficiency of legume root nodules. The factors that affect electron allocation between protons and nitrogen in nitrogenase are reviewed and it is concluded that there must be some as yet unknown factor that affects electron distribution inRhizobium nitrogenase. One possibility is put forward and considered. A strain ofRhizobium was used that was found to possess hydrogenase activity in combination with pea variety Feltham First but not with variety Meteor. The control of this enzyme is briefly discussed.     

花生根瘤菌类菌体氢酶的分离提纯及特性

花生根瘤菌类菌体经超声波破碎,ＴｒｉｔｏｎＸ－１００溶解,正已烷－硫酸铵处理后,再经ＤＥＡＥ－纤维素和Ｓｅｐｈａｃｒｙｌ凝胶柱层析等纯化步骤,获得凝胶电泳纯的膜结合态氢酶,比活为７１．４μｍｏｌＨ２ｍｇ－１Ｐｒｏｔｍｉｎ－１,为类菌体吸Ｈ２活性的２１１倍。纯化的氢酶分子量为１１０ｋＤ。经ＳＤＳ－ＰＡＧＥ后,呈现两个蛋白带,分子量分利为６５ｋＤ和３５ｋＤ。纯酶的Ｎｉ含量为０．６２ｍｏｌＮｉ／ｍｏｌ氢酶。在磷酸缓冲液中其活性的最适ｐＨ为６．５。ＤＣＩＰ、亚甲蓝、铁氰化钾、细胞色素Ｃ均可作为氢酶的电子受体,其中以ＤＣＩＰ为最适。     

Variation in nitrogenase and hydrogenase activity of alaska pea root nodules

Hydrogenase activity of root nodules in the symbiotic association between Pisum sativum L. and Rhizobium leguminosarum was determined by incubating unexcised nodules with tritiated H₂ and measuring tissue HTO. Hydrogenase activity saturated at 0.50 millimolar H₂ and was not inhibited by the presence of 0.10 atmosphere C₂H₂, which prevented H₂ evolution from nitrogenase. Total H₂ production from nitogenase was estimated as net H₂ evolution in air plus H₂ exchange in 0.10 atmosphere C₂H₂. Although such an estimate of nitrogenase function may not be quantitatively exact, due to uncertain relationships between H₂ exchange and H₂ uptake activity of hydrogenase, differences observed in H₂ exchange under various conditions represent an indication of changes in hydrogenase activity. Hydrogenase activity was lower in associations grown under higher photosynthetic photon flux densities and decreased relative to total H₂ production by nitrogenase. Total H₂ production and hydrogenase activity were maximum 28 days after planting. Thereafter, hydrogenase activity and H₂ production declined, but the potential proportion of nitrogenase-produced H₂ recovered by the uptake hydrogenase system increased. Of five R. leguminosarum strains tested two possessed hydrogenase activity. Strains which had the potential to reassimilate H₂ had significantly higher rates of N₂ reduction than those which did not exhibit hydrogenase activity.     

Hydrogenase activities in cyanobacteria

In the unicellular Anacystis nidulans, the expression of both the H2-uptake (with phenazine methosulfate or methylene blue as the electron acceptor) and H2-evolution (with methyl viologen reduced by Na2S2O4) was dependent on Ni in the culture medium. In extracts from Anacystis and Anabaena 7119, H2-evolution and uptake activities were strongly inhibited by Cu2+, p-chloromercuribenzoate and HgCl2 suggesting that at least one functional SH-group is involved in catalysis by hydrogenase. Extracts from the N2-fixing Anabaena 7119 contained two different hydrogenase fractions which could be separated by chromatography on DE-52 cellulose using a linear NaCl concentration gradient. The fraction eluting with 0.13 M NaCl from the column catalyzed only the uptake of H2 with methylene blue as the electron acceptor but virtually not the evolution of H2 ("uptake" hydrogenase fraction). The fraction eluting at a NaCl strength of 0.195 M catalyzed both H2-uptake with methylene blue and H2-evolution with reduced methyl viologen ("reversible" hydrogenase fraction). Growth under anaerobic conditions drastically enhanced the activity levels of the "reversible" but not of the "uptake" hydrogenase fraction. The "uptake" hydrogenase but not the "reversible" protein was activated by reduced thioredoxin. It is suggested that thioredoxin activates the H2-uptake by the membrane-bound "uptake" hydrogenase also in intact cells. The occurrence of the number of hydrogenases in cyanobacteria will be reevaluated.     

Non-leguminous root nodules in Japan

Summary Among plants native to Japan, nodule formation is confirmed in 14 species and varieties of Alnus, 10 of Elacagnus, 2 of Myrica and 1 of Coriaria, in a number of instances for the first time. Plants of 20 foreign species, in 8 genera, which bear nodules in their native countries, were raised in the nursery in Tokyo; only species of Alnus, Myrica and Ceanothus formed nodules. No nodules were found on native plants ofDryas octopetala var.asiatica. In a trial extending over 12 years evidence was obtained that the growth ofPinus thunbergii was benefited by underplanting withMyrica rubra, a result attributed to nitrogen fixation in the root nodules of the latter species. In the attempted isolation of the endophytes from the nodules of Alnus and other non-legume Angiosperms, although actinomycetes peculiar to the host species were usually obtained from the nodules, none of the isolates induced nodules in re-inoculation tests. Also from Podocarpus nodules actinomycetal and bacterial strains were commonly isolated; re-inoculation tests with these are in progress.