Hydrogen-Dependent Nitrogenase Activity and ATP Formation in Rhizobium japonicum Bacteroids

Abstract

Rhizobium japonicum 122 DES bacteroids from soybean nodules possess an active H2-oxidizing system that recycles all of the H2 lost through nitrogenasedependent H2 evolution. The addition of 72 ,uM H2 to suspensions of bacteroids increased 02 uptake 300% and the rate of C2H2 reduction 300 to 500%. The optimal partial pressure of 02 was increased, and the partial pressure of 02 range for C2H2 reduction was extended by adding H2. A supply of succinate to bacteroids resulted in effects'similar to those obtained by adding H2. Both H2 and succinate provided respiratory protection for the N2-fixing system in bacteroids. The oxidation of H2 by bacteroids increased the steady-state pool of ATP by 20 to 40%. In the presence of 50 mM iodoacetate, which caused much greater inhibition of endogenous respiration than of H2 oxidation, the addition of H2 increased the steady-state pool of ATP in bacteroids by 500%. Inhibitor evidence and an absolute requirement for 02 indicated that the H2-stimulated ATP synthesis occurred through oxidative phosphorylation. In the presence of 50 mM iodoacetate, H2-dependent ATP synthesis occurred at a rate sufficient to support nitrogenase activity. The addition of H2 to H2 uptake-negative strains of R. japonicum had no effect on ATP formation or C2H2 reduction. It is concluded that the H2-oxidizing system in H2 uptake-positive bacteroids benefits the N2-fixing process by providing respiratory protection of the 02-labile nitrogenase proteins and generating ATP to support maximal rates of C2H2 reduction by oxidation of the H2 produced from the nitrogenase system.

Effect of H2 evolution on 15N2 fixation, C2H2 reduction and relative efficiency of leguminous symbionts

“…Ihere are reports (Smith et al 1976) that hydrogenase is inhibited by acetylene (not to be confused with C2H2 inhibition of H2 production by nitrogenase). But Emerich et al (1979), Peterson and Burris (1978), and Houchins and Burris (1981) found little, if any, inhibition of hydrogenase by acetylene. When no hydrogenase or a weak hydrogenase is present, the total measurable electron tlux through the nitrogenase as measured by C2H4 + H2 produced should be equal, within experimental error, to the measurable electron flux measured by 'N2 reduction -I-H2 evolution.…”

“…Schubert et al (1978) found a positive correlation between the RE and the symbiotic effectiveness of soybeans (Hup* and Hup" strains tested), but Gibson et al, (1981) reported that soybeans inoculated with Hup* or Hup" R. japonicum strains showed no correlation between the RE and symbiotic effectiveness. When symbionts with an RE near 1,0 show a (C2H4 H-H2(c:2H2))/"''^2 of about 3, this indicates that hydrogenase action supports the nitrogenase activity and presumably increases the efficiency of the nitrogenase system (Emerich et al 1979, Nelson and Salminen 1982, Pedrosaet al 1982.…”

Hydrogen metabolism and energy costs of nitrogen fixation

“…Hydrogen enhances respiration in R. japonicum bacteroids [62]. In bacteroids of Hup + R. leguminosarum strains [63] and in bacteroids of R. japonicum [64], hydrogen increased the level of oxygen, optimal for acetylene reduction. These authors conclude that a major function of hydrogen oxidation is to provide respiratory protection of the nitrogenase proteins against oxygen.…”

“…Hydrogen-dependent ATP synthesis has been demonstrated in Azotobacter [60], Anabaena [66] and bacteroids of R. japonicum [64] and R. leguminosarum [63]. In the latter case, only 5 of the 14 Hup + R. leguminosarum phenotypes coupled hydrogen uptake to ATP synthesis.…”

Metabolism of [13C]‐labelled photosynthate in plant cytosol and bacteroids of root nodules of Glycine max

“…From the harvested plants, nodules were detached quickly and immediately fractionated into plant cytosol and bacteroids by two different methods. In experiments 1 and 3, a differential centrifugation (Emerich et al 1979) was adopted. Nodules were ground in 5 vol of 50 mM HEPES, pH 7.4, in a chUled mortar.…”