Hydrogenase in pea root nodule bacteriods

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 Hi and was not inhibited by the presence of 0.10 atmosphere CiH2, which prevented H2 evolution from nitrogenase. Total Hi production from nitrogenase was estimated as net H2 evolution in air plus H2 exchange in 0.10 atmosphere CGH2. 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. Ieguminosarum 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.Reduction of C2H2 to C2H4 by nitrogenase is measured easily by gas chromatography (12). As a consequence, this assay has become a major research technique for estimating biological N2 fixation. In the process of interacting with nitrogenase, however, C2H2 inhibits ATP-dependent H2 evolution by this enzyme complex (6). The first reproducible demonstration of H2 evolution from soybean root nodules (13) apparent that a better understanding of uptake hydrogenase activity and H2 production by nitrogenase is needed to comprehend factors which affect the efficiency of N2 fixation.Whole-plant studies revealed that plant ontogeny (5) and irradiance (4) during 4 weeks of growth markedly altered the relative efficiency of N2 fixation calculated according to Schubert and Evans (18). Although those experiments demonstrated that the host legume could affect the efficiency of bacteroid functioning, there were no data to indicate whether the altered levels of H2 evolution resulted from changes in uptake hydrogenase activity or from differences in H2 production by nitrogenase. The present experiments were performed to separate the effects of ontogeny and growth irradiance on nitrogenase and hydrogenase activity in Rhizobium leguminosarum 128C53. In addition, other strains of this bacterial species were examined for hydrogenase and nitrogenase activity in vivo to determine the mechanisms underlying different N2 reduction rates which alter photosynthetic efficiency (3). MATERIALS AND METHODS

supporting

confidence: 70%

Variation in Nitrogenase and Hydrogenase Activity of Alaska Pea Root Nodules

Bethlenfalvay¹,

Phillips²

1979

“…However, RE of strain 128C53 was significantly (P c (25). That work was confirmed by Dixon (11), who localized uptake hydrogenase activity in pea root nodule bacteroids (12) and found it to be similar to Azotobacter hydrogenase in all respects examined (13 Many data suggest that at least 25% of the reductant used by nitrogenase is allocated to protons for H2 formation, while the remaining fraction of reductant is used to convert N2 to NH3 (6). The EAC4 is a convenient expression that reflects the partitioning of reductant among protons and alternative substrates such as N2 or C2H2 (6): EAC = (exogenous substrate reduced/H30 reduced + exogenous substrate reduced).…”

mentioning

confidence: 70%

“…Hydrogen uptake by leguminous root nodules was first reported in the pea system in 1941 (25). That work was confirmed by Dixon (11), who localized uptake hydrogenase activity in pea root nodule bacteroids (12) and found it to be similar to Azotobacter hydrogenase in all respects examined (13). ' Supported by a Fulbright Fellowship, a postgraduate fellowship from the Natural Sciences and Engineering Research Council of Canada, and the United States National Science Foundation Grant PCM 8217187.…”

mentioning

confidence: 85%

Host Plant Cultivar Effects on Hydrogen Evolution by Rhizobium leguminosarum

Bedmar¹,

Edie²,

Phillips³

1983

The effect of host plant cultivar on H2 evolution by root nodules was examined in symbioses between Pisum sativum L. and selected strains of Rhizobium leguminosarum. Hydrogen evolution from root nodules containing Rhizobium represents the sum of H2 produced by the nitrogenase enzyme complex and H2 oxidized by any uptake hydrogenase present in those bacterial cells. Relative efficiency (RE) calculated as RE -1 -(H2 evolved in air/C2H2 reduced) did not vary significantly among 'Feltham First,"Alaska,' and 'JI1205' peas inoculated with R. kguminosarum strain 300, which lacks uptake hydrogenase activity (Hup-). That observation suggests that the three host cultivars had no effect on H2 production by nitrogenase. However, RE of strain 128C53 was significantly (P c (25). That work was confirmed by Dixon (11), who localized uptake hydrogenase activity in pea root nodule bacteroids (12) and found it to be similar to Azotobacter hydrogenase in all respects examined (13 Many data suggest that at least 25% of the reductant used by nitrogenase is allocated to protons for H2 formation, while the remaining fraction of reductant is used to convert N2 to NH3 (6). The EAC4 is a convenient expression that reflects the partitioning of reductant among protons and alternative substrates such as N2 or C2H2 (6): EAC = (exogenous substrate reduced/H30 reduced + exogenous substrate reduced). In the presence of saturating levels of C2H2, all reductant is used to produce C2H4 and no H2 formation is detected. Schubert and Evans (28) defined the relative efficiency of N2 fixation as RE = 1 -(H2 evolved in air/C2H2 reduced) and showed that RE varied greatly among different Rhizobium-legume symbioses. Such RE values depend on the EAC of nitrogenase and on the capacity of the rhizobial cells to recover H2 by a separate uptake hydrogenase. Thus, in the absence ofuptake hydrogenase activity (Hup-phenotype), RE presumably is a measure of apparent EAC.Previous reports indicate that both plant and bacterial factors can affect H2 evolution from leguminous root nodules. Dixon (13) showed that R leguminosarum strain ONA 311 expressed strongly Hup+, slightly Hup+, or Hup-phenotypes on Pisum sativum, Vicia bengalensis, and V. faba, respectively. Similar reversals in uptake hydrogenase phenotype have been observed for two R. japonicum strains associated with Vigna unguiculata and Glycine max (20). Both strains were Hup+ on three cowpea cultivars and Hup-on three soybean cultivars. Host plant effects on apparent rhizobial EAC have been indicated by the observation that lengthening the normal dark period for P. sativum and Trifolium subterraneum increased RE of Hup-Rhizobium symbionts (15). Host plant age and long-term environmental factors such as availability of combined nitrogen, irradiance, and CO2 level also can affect apparent EAC (16). These latter findings are consistent with a previous study which concluded that both EAC and uptake hydrogenase activity of a Hup+ rhizobial strain were affected by irradiance treatment of the host pea pl...

“…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 hydrogenases have been found in several aerobic N2-fixing organisms (1,6,12), and these enzymes share several common properties. They are membrane-bound (7,12,28), couple to 02 consumption via a Cyt-containing electron transport system, and through this reaction provide a source of ATP (7, 9, 21).…”

mentioning

confidence: 99%

“…Uptake hydrogenases have been found in several aerobic N2-fixing organisms (1,6,12), and these enzymes share several common properties. They are membrane-bound (7,12,28), couple to 02 consumption via a Cyt-containing electron transport system, and through this reaction provide a source of ATP (7,9,21).…”

mentioning

confidence: 99%

Light and Dark Reactions of the Uptake Hydrogenase in Anabaena 7120

Houchins¹,

Burris²

1981

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 ifiaments 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 N03-or N02-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 cyanidesensitive oxyhydrogen reaction that was inactivated by 02 at 02 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 02, light-dependent H2 uptake occurred sunultaneously 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 hydrogenases have been found in several aerobic N2-fixing organisms (1,6,12), and these enzymes share several common properties. They are membrane-bound (7,12,28), couple to 02 consumption via a Cyt-containing electron transport system, and through this reaction provide a source of ATP (7, 9, 21). Several authors have suggested that these uptake hydrogenases function to recycle the H2 evolved by nitrogenase (4,7,9,21).In blue-green algae, membrane-bound hydrogenases can couple to two different electron transport pathways. Peschek has demonstrated respiratory H2 uptake and photosynthetic H2 uptake in Anacystis nidulans (19,20) in which the hydrogenase was induced by growth under a gas phase containing H2. In the dark, both H2 uptake systems could react only with acceptors having a positive midpoint potential; however, upon illumination acceptors with a negative potential could also be reduced.