Investigation of the H2 Oxidation System in Rhizobium japonicum 122 DES Nodule Bacteroids

Open Access
Publisher Website
Google Scholar
Abstract
The H2-oxidizing complex in Rhizobium japonicum 122 DES bacteroids failed to catalyze, at a measurable rate, 2H1H exchange from a mixture of 2H2 and 1H2 in presence of 2H2O and 1H2O, providing no evidence for reversibility of the hydrogenase reaction in vivo. In the H2 oxidation reaction, there was no significant discrimination between 2H2 and 1H2, indicating that the initial H2-activation step in the over-all H2 oxidation reaction is not rate-limiting. By use of improved methods, an apparent Km for H2 of 0.05 micromolar was determined. The H2 oxidation reaction in bacteroids was strongly inhibited by cyanide (88% at 0.05 millimolar), theonyltrifluoroacetone, and other metal-complexing agents. Carbonyl cyanide m-chlorophenylhydrazone at 0.005 millimolar and 2,4-dinitrophenol at 0.5 millimolar inhibited H2 oxidation and stimulated O2 uptake. This and other evidence suggest the involvement of cytochromes and nonheme iron proteins in the pathway of electron transport from H2 to O2. Partial pressures of H2 at 0.03 atmosphere and below had a pronounced inhibitory effect on endogenous respiration by bacteroid suspensions. The inhibition of CO2 evolution by low partial pressures of H2 suggests that H2 utilization may result in conservation of oxidizable substrates and benefits the symbiosis under physiological conditions. Succinate, acetate, and formate at concentrations of 50 millimolar inhibited rates of H2 uptake by 8, 29, and 25%, respectively. The inhibition by succinate was noncompetitive and that by acetate and formate was uncompetitive. A concentration of 11.6 millimolar CO2 (initial concentration) in solution inhibited H2 uptake by bacteroid suspensions by 18%. Further research is necessary to establish the significance of the inhibition of H2 uptake by succinate, acetate, formate, and CO2 in the metabolism of the H2-uptake-positive strains of Rhizobium.