Characterization of anammox hydrazine dehydrogenase, a key N₂-producing enzyme in the global nitrogen cycle

Anaerobic ammonium-oxidizing (anammox) bacteria derive their energy for growth from the oxidation of ammonium with nitrite as the electron acceptor. N₂, the end product of this metabolism, is produced from the oxidation of the intermediate, hydrazine (N₂H₄). Previously, we identified N₂-producing hydrazine dehydrogenase (KsHDH) from the anammox organism Kuenenia stuttgartiensis as the gene product of kustc0694 and determined some of its catalytic properties. In the genome of K. stuttgartiensis, kustc0694 is one out of ten paralogs related to octaheme hydroxylamine (NH₂OH) oxidoreductase (HAO). Here, we characterized KsHDH as a covalently cross-linked homotrimeric octaheme protein as found for HAO and HAOrelated hydroxylamine-oxidizing enzyme kustc1061 (KsHOX) from K. stuttgartiensis. Interestingly, the HDH trimers formed octamers in solution, each octamer harbouring an amazing 192 c-type heme moieties. While HAO and KsHOX are capable of hydrazine oxidation as well, KsHDH was highly specific for this activity. To understand this specificity, we performed detailed amino acid sequence analyses and investigated the catalytic and spectroscopic (electronic absorbance, EPR) properties of KsHDH in comparison with the well-defined HAO and HOX. We conclude that HDH specificity is most likely derived from structural changes around the catalytic heme 4 (“P460”) and of the electron-wiring circuit comprising seven His/His-ligated _c-type hemes in each subunit. These nuances make HDH a globally prominent N₂-producing enzyme, next to nitrous oxide (N₂O) reductase from denitrifying microorganisms.