Characterization of Anammox Hydrazine Dehydrogenase, a Key N2-producing Enzyme in the Global Nitrogen Cycle

Abstract

Anaerobic ammonium-oxidizing (anammox) bacteria derive their energy for growth from the oxidation of ammonium with nitrite as the electron acceptor. N 2 , the end product of this metabolism, is produced from the oxidation of the intermediate, hydrazine (N 2 H 4 ). Previously, we identified N 2 -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 (NH 2 OH) 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 (P 460 ) and of the electron-wiring circuit comprising seven His/His-ligated c-type hemes in each subunit. These nuances make HDH a globally prominent N 2 -producing enzyme, next to nitrous oxide (N 2 O) reductase from denitrifying microorganisms.An estimated 30 -70% of all N 2 that is released into the atmosphere is produced by anaerobic ammonium-oxidizing (anammox) 4 bacteria (1, 2), which represent one of the latest scientific discoveries in the biogeochemical nitrogen cycle. These organisms gain their energy for growth from the oxidation of ammonium, with nitrite as the electron acceptor, to produce N 2 . With the employment of advanced molecular tools, these bacteria have been detected in nearly every anoxic environment where fixed nitrogen compounds are degraded (3). Besides its biogeochemical and ecological relevance, the anammox process has found worldwide application in ammonium removal from wastewater as an environment-friendly and cost-effective alternative to conventional systems (4).In our current understanding, anammox catabolism is composed of three consecutive, coupled reactions with two intermediates, nitric oxide (NO) and hydrazine (N 2 H 4 ): 1) the oneelectron reduction of the substrate nitrite to NO (Reaction 1); 2) the activation of the second substrate ammonium with NO and the concomitant input of three electrons to synthesize N 2 H 4 (Reaction 2); and 3) the oxidation of hydrazine, the most powerful reductant in nature, to N 2 (Reaction 3) (5-7). NO

The pH dependency of N‐converting enzymatic processes, pathways and microbes: effect on net N2O production

“…The iron ions in the active sites are often (e.g., HAO, cd-NIR, HZS, HDH, cNOR), but not always (e.g., cNOR; cNOR contains one heme and one non-heme Fe) part of heme groups. The Fe-ions in heme groups commonly interact with proximal histidine (his) residues (pI 5 7.6) (Igarashi et al, 1997;Maalcke et al, 2016). Protonated his-residues move the Fe-ion out of the heme plane and cause the Fe-ion to change its spin state (Perutz et al, 1998).…”

Geomicrobiology of the carbon, nitrogen and sulphur cycles in Powell Lake: a permanently stratified water column containing ancient seawater

“…For this, we used prodigal (Hyatt et al, 2010) to call genes from the metagenomes and subsequently scanned hidden Markov models (HMMs), which were either retrieved from the FunGene repository (Fish et al, 2013) or from the custom-made based on Uniprot entries, using HMMer (Eddy, 2011). We further note that the marker gene hao (hydroxylamine oxidoreductase; Supporting Information Table S7) is not process-specific based on KEGG orthology (K10535), because the gene for hydroxylamine oxidoreductase used by AOBs (Kozlowski et al, 2016) and the hydrazine dehydrogenase gene (hdh) found in anammox bacteria are both paralogues of octaheme hydroxylamine oxidoreductase (Maalcke et al, 2016). Hao was thus interpreted to represents the sum of aerobic and anaerobic bacterial ammonium oxidation.…”

Evidence of differential adaptation to decreased temperature by anammox bacteria

“…The K. stuttgartiensis genome encodes 10 different hao-like paralogs (Kartal et al, 2010;de Almeida et al, 2011) and these HAO-like proteins dominated the anammox bacterium proteome. Potentially these catalyse the three-electron oxidation of hydroxylamine to nitric oxide and nitrite (Maalcke et al, 2014(Maalcke et al, , 2016. At 20 C, the reduced anammox reaction rates (when compared with that 35 C) may result in nitrite accumulation in both the cytoplasm and the anammoxsome.…”