Hydrogen oxidation by Azospirillum brasilense Sp7 was studied in N(,2)-fixing and NH(,4)('+)-grown batch cultures. The K(,m) for H(,2) of O(,2)-dependent H('3)H oxidation in whole cells was 9 uM. The rates of H('3)H and H(,2) oxidation were very similar, indicating that the initial H(,2) activation step in the overall H(,2) oxidation reaction was not rate-limiting and that H('3)H oxidation was a valid measure of H(,2)-oxidation activity. Hydrogen-oxidation activity was inhibited irreversibly by air. In N-free cultures the O(,2) optima for O(,2)-dependent H(,2) oxidation, ranging from 0.5-1.25% O(,2) depending on the phase of growth, were significantly higher than those of C(,2)H(,2) reduction, 0.15-0.35%, suggesting that the H(,2)-oxidation system may have a limited ability to aid in the protection of nitrogenase against inactivation by O(,2). Oxygen-dependent H(,2) oxidation was inhibited by NO(,2)('-), NO, CO, and C(,2)H(,2) with apparent K(,i) values of 20, 0.4, 28, and 88 uM, respectively. These inhibitors also affected methylene blue-dependent H(,2) oxidation, presumably by acting on the hydrogenase directly. The CO inhibition was easily reversible; the NO(,2)('-) and NO inhibitions were irreversible; and the C(,2)H(,2) inhibition was not readily reversible. Hydrogen-oxidation activity was 50 to 100 times higher in denitrifying cultures when the terminal electron acceptor for growth was N(,2)O rather than NO(,3)('-), possibly due to the irreversible inhibition of hydrogenase by NO(,2)('-) and NO in NO(,3)('-)-grown cultures. THe expression of the H(,2)-oxidation system was independent of nitrogenase expression, did not require added H(,2) (and probably not endogenous H(,2)), was not affected by low concentrations of carbon substrates (less than 30 mM malate), and required low O(,2) concentrations (microaerobic or anaerobic conditions).

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