Reconstitution of H2 Oxidation Activity from H2 Uptake-negative Mutants of Rhizobium Japonicum Bacteroids

An in vitro reconstitution of both methylene blue and oxygen-dependent H2 uptake activity from extracts of Hup- (H2 uptake-negative) mutant strains of Rhizobium japonicum bacteroids is described. Cell-free extracts prepared from bacteroids formed from two different Hup- mutants were mixed, and active H2 oxidizing particles formed. Extracts from each mutant alone did not oxidize H2. The source of the components required for the complementation were soluble. Mixing of membrane particles from the two mutants did not result in reconstituted activity. The development of activity required an incubation period of several hours under anaerobic conditions, and maximal activity was obtained approximately 10 h after the mixing of the two extracts. Along with the development of H2 uptake activity with time, the soluble extract mixture became turbid. The turbidity could be correlated with an increase in the appearance of membrane structures, including closed vesicles. After reconstitution, 65% of the methylene blue-dependent H2 uptake activity was recovered in a particulate fraction.