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Articles

Investigation of the H₂ Oxidation System in Rhizobium japonicum 122 DES Nodule Bacteroids ¹

Laboratory for Nitrogen Fixation Research, Oregon State University, Corvallis, Oregon 97331

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 K_m 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.

³ Supported by the Program of Cultural Cooperation between the United States and Spain. Present address: Departamento de Microbiologia, Escuela Tecnica Superior de Ingenieros Agronomos, Madrid-3, Spain.

¹ This research was supported in part by Grant PCM 77-08784 from the National Science Foundation and by the Oregon Agricultural Experiment Station, from which this is Technical Paper No. 5217.