Abstract
Membranes capable of oxidizing H 2 with O 2 as terminal acceptor were obtained from free-living Rhizobium japonicum. Membranes contained highest H 2-uptake specific activities when isolated in the presence of an H 2 atmosphere, and when the oxygen radical scavenger butylated hydroxytoluene was included in the buffer used for rupturing cells. After breaking cells, all of the O 2-dependent H 2-uptake activity was associated with a particulate membrane-containing fraction, whereas approx. 75% of the methylene blue-dependent H 2-uptake activity was sedimented. The particulate and soluble fractions containing H 2-uptake activity with methylene blue were separated by sucrose gradient centrifugation. The particulate and soluble activities behaved identically with regard to artificial electron acceptor specificity and reversible inhibition by oxygen. The hydrogenase in membranes coupled H 2 uptake with the reduction of many positive potential electron acceptors, but not with negative potential acceptors. The optimal pH for H 2 uptake with O 2 as acceptor in membranes was approx. 7.2. H 2-uptake activity in membranes was associated with an inner (lighter) membrane fraction that also contained succinate oxidase activity. H 2-reduced minus O 2-oxidized difference spectroscopy of membranes indicated the involvement of b and c-type cytochromes in the H 2-oxidation pathway, with an absorption peak at 551.5 nm and a shoulder at 560 nm. The addition of sodium dithionite to H 2-reduced membranes caused additional b-type cytochrome reduction. The methylene blue-dependent H 2-uptake activity in membranes was reversibly inhibited by brief exposure to oxygen. Recovery of full activity after oxygen exposure was achieved only after several minutes of incubation under strict anaerobic conditions.
Published Version
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