Abstract
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 of 10 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 HAO-related hydroxylamine-oxidizing enzyme kustc1061 from K. stuttgartiensis Interestingly, the HDH trimers formed octamers in solution, each octamer harboring an amazing 192 c-type heme moieties. Whereas HAO and kustc1061 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 kustc1061. 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.
Highlights
Anaerobic ammonium-oxidizing bacteria derive their energy for growth from the oxidation of ammonium with nitrite as the electron acceptor
Whereas hydroxylamine (NH2OH) oxidoreductase (HAO) and kustc1061 are capable of hydrazine oxidation as well, KsHDH was highly specific for this activity
hydrazine dehydrogenase (HDH) Is a Homotrimeric Protein with Covalently Bound Subunits Forming Octamers in Solution—Hydrazine dehydrogenase from K. stuttgartiensis was purified as a bright red protein that, when resolved by native PAGE, displayed one prominent band with an estimated molecular mass of ϳ200 –220 kDa (Fig. 2A)
Summary
The four electrons that are released during hydrazine oxidation drive the reduction reactions (Reactions 1 and 2). A hydrazine-oxidizing enzyme highly related to kustc0694 was isolated before from the anammox enrichment culture KSU-1, but the exact reaction catalyzed by it has so far remained unknown (8). In the genome of K. stuttgartiensis, kustc0694 is one of the 10 paralogs of hydroxylamine oxidoreductase (HAO)-like proteins (3, 6). HAO-like proteins have two structurally well characterized representatives, NeHAO from Nitrosomonas europaea (9 –11) and hydroxylamine oxidase from K. stuttgartiensis (kustc1061; here denoted as KsHOX) (12). NeHAO is a key enzyme in aerobic ammonium-oxidizing bacteria, which catalyzes the four-electron oxidation of hydroxylamine to nitrite (Reaction 4), whereas KsHOX is a dominant protein in the anammox bacterium catalyzing the three-electron oxidation of hydroxylamine to NO (Reaction 5)
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