Characterization of a nitrite-reducing octaheme hydroxylamine oxidoreductase that lacks the tyrosine cross-link

Abstract

The hydroxylamine oxidoreductase (HAO) family consists of octaheme proteins that harbor seven bis-His ligated electron-transferring hemes and one 5-coordinate catalytic heme with His axial ligation. Oxidative HAOs have a homotrimeric configuration with the monomers covalently attached to each other via a unique double cross-link between a Tyr residue and the catalytic heme moiety of an adjacent subunit. This cross-linked active site heme, termed the P460 cofactor, has been hypothesized to modulate enzyme reactivity toward oxidative catalysis. Conversely, the absence of this cross-link is predicted to favor reductive catalysis. However, this prediction has not been directly tested. In this study, an HAO homolog that lacks the heme-Tyr cross-link (HAOr) was purified to homogeneity from the nitrite-dependent anaerobic ammonium-oxidizing (anammox) bacterium Kuenenia stuttgartiensis , and its catalytic and spectroscopic properties were assessed. We show that HAOr reduced nitrite to nitric oxide and also reduced nitric oxide and hydroxylamine as nonphysiological substrates. In contrast, HAOr was not able to oxidize hydroxylamine or hydrazine supporting the notion that cross-link-deficient HAO enzymes are reductases. Compared with oxidative HAOs, we found that HAOr harbors an active site heme with a higher (at least 80 mV) midpoint potential and a much lower degree of porphyrin ruffling. Based on the physiology of anammox bacteria and our results, we propose that HAOr reduces nitrite to nitric oxide in vivo , providing anammox bacteria with NO, which they use to activate ammonium in the absence of oxygen.

Millimeter-scale vertical partitioning of nitrogen cycling in hypersaline mats reveals prominence of genes encoding multi-heme and prismane proteins

“…Turning first to hao, the top Pfam annotation for all 52 of our hao (K10535) genes was PF13447 (Table S8), a multiheme cytochrome for which the majority of sequences underlying the Pfam model were annotated as hydroxylamine dehydrogenase EC 1.7.2.6 (HAO). HAO reductases may be differentiated from oxidative HAOs by one structural characteristic, namely a tyrosine residue which cross links the active site to the catalytic heme moiety of an adjacent subunit; the presence of this cross-linked active site heme has been hypothesized to modulate enzyme reactivity toward oxidative catalysis [60]. Conversely, the absence of this cross-link is predicted to favor reductive catalysis.…”

“…52 HAO sequences recovered in the metagenome presented here (Table S8) were aligned with HAO from Nitrosomonas europea and Nitrosomonas mobilis (AOB), HAO from the anaerobic ammoniumoxidizing (anammox) bacterium Kuenenia stuttgartiensis described in the Ferousi [60], ɛHao protein (HAO subfamily) from the Campylobacterota [61] (previously known as Epsilonproteobacteria) and other microorganisms described in Hasse [62] (Fig. S6).…”

A new paradigm of multiheme cytochrome evolution by grafting and pruning protein modules

“…In this scenario, the P460 heme evolved de novo in the HAO/HDH subclade. Indeed, an HAO family protein was recently isolated from K. stuttgartiensis and showed no hydroxylamine oxidation activity, while presenting nitrite reduction to nitric oxide (Ferousi et al 2021). The second branching clade was clade 2, that latter diversified into ONR that conserved the overall heme-core arrangement of the LCA, and NrfA that further lost the three N-terminal hemes.…”

Structural and functional characterization of the intracellular filament-forming nitrite oxidoreductase multiprotein complex

“…This is particularly interesting given that the various anammox genera appear to use different types of enzymes for NO generation 10 , 17 . For instance, K. stuttgartiensis was proposed to employ a highly expressed nitrite-reducing octaheme c -type cytochrome 10 , 34 , 36 , whereas the cytochrome cd 1 -containing NirS nitrite reductase also encoded in its genome is hardly transcribed 10 . Scalindua profunda , on the other hand, contains both a highly abundant NirS and an octaheme c- type cytochrome that could perform nitrite reduction, meaning that there are two likely NO-generating enzymes in this organism 40 , and in Jettenia caeni a copper-based NirK-type nitrite reductase was proposed to be involved in nitrite reduction for NO generation 37 .…”