Effect of hydrogen and oxygen on uptake-hydrogenase activity in nitrogen-fixing and ammonium-grown Azospirillum brasilense

Hydrogen uptake by Azospirillum brasilense was studied in N2-fixing and NH4Cl-grown batch cultures. The Km for H2 of uptake hydrogenase (O2-dependent H3H uptake) in whole cells was 9 µM. In N2-flxing or NH4Cl-grown cultures hydrogenase activity reached its maximum in late log phase, followed by a rapid decline in stationary phase. In cultures sparged with H2, hydrogenase activity was markedly prolonged. Rates of O2-dependent H3H uptake and H2 uptake measured by gas chromatography were very similar. Hydrogenase derepression required microaerobic conditions, was independent of nitrogenase derepression, did not require exogenous H2, and may be enhanced by electron-donor limitation. Air caused irreversible inhibition of hydrogenase activity. In N-free cultures, theO2 optima for H2 uptake, ranging from 0.5 to 1.25% O2 depending on the phase of growth, were significantly higher than those for nitrogenase activity (C2H2 reduction), 0.15 to 0.35%, suggesting that H2 uptake may have a limited ability to aid in the protection of nitrogenase against inactivation by O2.