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

Abstract

Rhizobium japonicum 122 DES bacteroids from soybean nodules possess an active H 2 -oxidizing system that recycles all of the H 2 lost through nitrogenase-dependent H 2 evolution. The addition of 72 μM H 2 to suspensions of bacteroids increased O 2 uptake 300% and the rate of C 2 H 2 reduction 300 to 500%. The optimal partial pressure of O 2 was increased, and the partial pressure of O 2 range for C 2 H 2 reduction was extended by adding H 2 . A supply of succinate to bacteroids resulted in effects similar to those obtained by adding H 2 . Both H 2 and succinate provided respiratory protection for the N 2 -fixing system in bacteroids. The oxidation of H 2 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 H 2 oxidation, the addition of H 2 increased the steady-state pool of ATP in bacteroids by 500%. Inhibitor evidence and an absolute requirement for O 2 indicated that the H 2 -stimulated ATP synthesis occurred through oxidative phosphorylation. In the presence of 50 mM iodoacetate, H 2 -dependent ATP synthesis occurred at a rate sufficient to support nitrogenase activity. The addition of H 2 to H 2 uptake-negative strains of R. japonicum had no effect on ATP formation or C 2 H 2 reduction. It is concluded that the H 2 -oxidizing system in H 2 uptake-positive bacteroids benefits the N 2 -fixing process by providing respiratory protection of the O 2 -labile nitrogenase proteins and generating ATP to support maximal rates of C 2 H 2 reduction by oxidation of the H 2 produced from the nitrogenase system.