Spectroscopic insights into the mechanism of anammox hydrazine synthase

Anaerobic ammonium oxidizing (anammox) bacteria oxidize ammonium with nitrite as electron acceptor. Hydrazine is a free intermediate in this metabolism, produced by the enzyme hydrazine synthase. Hydrazine synthase is a tetraheme cytochrome c, containing two proposed active site hemes (γI and αI), connected by an intra-enzymatic tunnel. These structural features resulted in an initial hypothesis of its reaction mechanism: nitric oxide is reduced to hydroxylamine which is condensed with ammonium to form hydrazine. Here, investigations by electrochemically induced optical and infrared difference spectroscopy and electron paramagnetic resonance revealed two low potential low spin hemes, αII and γII, with midpoint potentials of ∼ -330 mV (versus SHE). Heme γI showed redox transitions in the range of 0 mV, featuring both low-spin and high-spin characteristics possibly due to implication of an aspartic acid, connected to heme γI axial site by a OH^-/H₂O. Furthermore, electron paramagnetic resonance spectroscopy confirmed the ability of heme γI to bind NO in the reduced state. Heme αI exhibited a rhombic high spin signal, in line with its ligation by a proximal tyrosine observed in the crystal structure. Neither dithionite nor potentials of -610 mV reduced this heme, indicating a very low midpoint potential. In vivo chemistry at this heme αI, the candidate for the comproportionation of hydroxylamine and ammonium, is thus likely to be initiated solely on the oxidized heme, in contrast to previously reported DFT calculations.