Vermeir, F. J.; Haaijer-Vroomen, S. C. M.; van der Velden, P. M. M.; Mesman, R.; Jansen, R. S.; Versantvoort, W.; van Niftrik, L.

Anaerobic ammonium-oxidizing (anammox) bacteria convert ammonium and nitrite into dinitrogen gas via the intermediates nitric oxide and hydrazine. To produce hydrazine, anammox bacteria harbor a biochemically unique enzyme: hydrazine synthase. Based on the hydrazine synthase crystal structure it was hypothesized that hydrazine is produced in a two-step mechanism. In this hypothesis, nitric oxide is first reduced to hydroxylamine (first half-reaction), followed by condensation of hydroxylamine with ammonium to hydrazine (second half-reaction). Here, we experimentally investigated the proposed molecular mechanism of hydrazine synthase and characterized and optimized the in vitro activity. First, we optimized the activity of isolated hydrazine synthase from anammox bacterium Kuenenia stuttgartiensis strain MBR1 via an anaerobic isolation method. We further compared hydrazine synthase activity measured via a coupled assay versus that of a newly established direct LC-MS assay. Next, the hypothesized second half-reaction was investigated via the direct LC-MS assay, quantifying biologically produced hydrazine from hydroxylamine and ammonium. Despite variation in hydrazine synthase activity across assays, we determined ammonium and hydroxylamine affinity and investigated product inhibition. Finally, we found that hydrazine synthesis from ammonium and hydroxylamine by isolated hydrazine synthase is oxygen-tolerant, strongly suggesting that the second half-reaction is initiated on an oxidized heme within hydrazine synthase. Taken together, the results corroborate that condensation of ammonium with hydroxylamine to form hydrazine is the second half-reaction of the proposed two-step mechanism for hydrazine synthesis by hydrazine synthase.