Nitrogen gas in the atmosphere is partially replenished by microbial denitrification of ammonia. Recent study has shown Alcaligenes ammonioxydans oxidizes ammonia to dinitrogen via a process featuring the intermediate hydroxylamine, termed “Dirammox” (direct ammonia oxidation). However, the unique biochemistry of this process remains unknown. Here we report an enzyme involved in Dirammox that catalyzes the conversion of hydroxylamine to N₂. We tested previously annotated proteins involved in redox reactions, DnfA, DnfB, and DnfC, to determine their ability to catalyze the oxidation of ammonia or hydroxylamine. Our results showed that none of these proteins bound to ammonia or catalyzed its oxidation; however, we did find DnfA bound to hydroxylamine. Further experiments demonstrated, in the presence of NADH and FAD, DnfA catalyzed the conversion of ¹⁵N-labeled hydroxylamine to ¹⁵N₂. This conversion did not happen under O₂-free conditions, and the involvement of oxygen was further confirmed using radiolabeled ¹⁸O₂. Thus, we concluded DnfA encodes a hydroxylamine oxidase. We demonstrate DnfA is not homologous to any known hydroxylamine oxidoreductases and contains a diiron center, which was shown to be involved in catalysis via electron paramagnetic resonance experiments. Furthermore, enzyme kinetics of DnfA were assayed, revealing a K_m of 92.9 ± 3.0 μM for hydroxylamine and a k_cat of 0.028 ± 0.001 s^-1. Finally, we show DnfA was localized in the cytoplasm and periplasm, as well as in tubular membrane invaginations in HO-1 cells. To the best of our knowledge, we conclude DnfA is the first enzyme discovered that catalyzes oxidation of hydroxylamine to N₂.

大气中的氮气通过氨的微生物反硝化作用得到部分补充。最近的研究表明，Alcaligenes ammonioxydans通过一个以中间体羟胺为特征的过程将氨氧化成二氮，称为“Dirammox”（直接氨氧化）。然而，这个过程的独特生物化学仍然未知。在这里，我们报告了一种参与 Dirammox 的酶，该酶催化羟胺转化为 N ₂. 我们测试了先前注释的参与氧化还原反应的蛋白质 DnfA、DnfB 和 DnfC，以确定它们催化氨或羟胺氧化的能力。我们的研究结果表明，这些蛋白质都没有与氨结合或催化其氧化。然而，我们确实发现 DnfA 与羟胺结合。进一步的实验证明，在NADH和FAD存在下，DnfA催化¹⁵ N-标记的羟胺转化为¹⁵ N ₂。_{这种转化在无 O 2}条件下没有发生，并且使用放射性标记的¹⁸ O _{2进一步证实了氧气的参与}. 因此，我们得出结论 DnfA 编码羟胺氧化酶。我们证明 DnfA 与任何已知的羟胺氧化还原酶都不同源，并且包含一个二铁中心，通过电子顺磁共振实验表明该中心参与催化。此外，测定了 DnfA 的酶动力学，显示羟胺的K _m为 92.9 ± 3.0 μM，k _cat为 0.028 ± 0.001 s ^-1。最后，我们显示 DnfA 定位于细胞质和周质，以及 HO-1 细胞的管状膜内陷。据我们所知，我们得出结论，DnfA 是第一个发现的催化羟胺氧化成 N ₂的酶。