Nitrogenase of Klebsiella pneumoniae. Hydrazine is a product of azide reduction

Abstract

K. pneumoniae nitrogenase reduced azide, at 30.degree. C and pH 6.8-8.2, to yield NH3, dinitrogen (N2) and hydrazine (N2H4). Reduction of (15N.dbd.14N.dbd.14N)- followed by mass spectrometric analysis showed that no new N.sbd.N bonds were formed. During azide reduction, added 15N2H4 did not contribute 15N to NH3, indicating lack of equilibration between enzyme-bound intermediates giving rise to N2H4 and N2H4 in solution. When azide reduction to N2H4 was partially inhibited by 15N2, label appeared in NH3 but not in N2H4. Product balances combined with the labeling data indicate that azide is reduced according to the following equations (e is electron): N3- + 3H+ + 2e .fwdarw. N2 + NH3; N3- + 7H+ + 6e .fwdarw. N2H4 + NH3; and N3+ + 9H+ + 8e .fwdarw. 3NH3. N2 was a competitive inhibitor and CO a non-competitive inhibitor of azide reduction to N2H4. The percentage of total electron flux used for H2 evolution concomitant with azide reaction fell from 26% at pH 6.8 to 0% at pH 8.2. Pre-steady-state kinetic data suggest that N2H4 is formed by the cleavage of the .alpha.-.beta. N.sbd.N bond of bound azide to leave a nitride (.tbd.N) intermediate that subsequently yields NH3.