Department of Microbiology, Institute for Water and Wetland Research, Radboud University, Nijmegen, The Netherlands.
Microbial Physiology Group, Max Planck Institute for Marine Microbiology, Bremen, Germany.
J Biol Chem. 2021 Jan-Jun;296:100476. doi: 10.1016/j.jbc.2021.100476. Epub 2021 Feb 27.
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.
羟胺氧化还原酶（HAO）家族由八血红素蛋白组成，这些蛋白含有七个双组氨酸连接的电子转移血红素和一个 5 配位的催化血红素，其轴向配位为组氨酸。氧化 HAO 具有三聚体结构，单体通过 Tyr 残基和相邻亚基的催化血红素部分之间独特的双交联共价连接在一起。这种交联的活性位点血红素，称为 P460 辅因子，据推测可以调节酶对氧化催化的反应性。相反，没有这种交联结构，有利于还原催化。然而，这一预测尚未得到直接验证。在这项研究中，从亚硝酸盐依赖的厌氧氨氧化（anammox）细菌 Kuenenia stuttgartiensis 中纯化出一种缺乏血红素-Tyr 交联的 HAO 同源物（HAOr），并评估了其催化和光谱特性。我们表明，HAOr 将亚硝酸盐还原为一氧化氮，也将非生理底物一氧化氮和羟胺还原。相比之下，HAOr 不能氧化羟胺或肼，这支持了交联缺陷的 HAO 酶是还原酶的观点。与氧化 HAO 相比，我们发现 HAOr 具有一个活性位点血红素，其中点电位更高（至少 80 mV），卟啉起皱程度更低。基于 anammox 细菌的生理学和我们的结果，我们提出 HAOr 将亚硝酸盐还原为一氧化氮，为 anammox 细菌提供了在缺氧条件下激活铵的 NO。
