McGarry Jennifer, Mintmier Breeanna, Metzger Mikayla C, Giri Nitai C, Britt Nicholas, Basu Partha, Wilcoxen Jarett
Department of Chemistry and Biochemistry, University of Wisconsin- Milwaukee, Milwaukee, WI, 53211, USA.
Department of Chemistry and Chemical Biology, Indiana University Indianapolis, Indianapolis, IN, 46202, USA.
J Biol Inorg Chem. 2024 Dec;29(7-8):811-819. doi: 10.1007/s00775-024-02087-5. Epub 2024 Dec 4.
Nitrate reductases play pivotal roles in nitrogen metabolism by leveraging the molybdopterin cofactor to facilitate the reduction of nitrate to nitrite. Periplasmic nitrate reductases (NapA) utilize nitrate as a terminal electron acceptor when oxygen is limiting, helping to drive anaerobic metabolism in bacteria. Despite extensive research into NapA homologs, open questions about the mechanism remain especially at the molecular level. More broadly, little is understood of how the molybdopterin cofactor is tuned for catalysis in these enzymes enabling broad substrate scope and reactivity observed in molybdenum-containing enzymes. Here, we have prepared NapA from Campylobacter jejuni under single turnover conditions to generate a singly reduced enzyme that can be further examined by electron paramagnetic resonance (EPR) spectroscopy. Our results provide new context into the known spectra and related structures of NapA and related enzymes. These insights open new avenues for understanding nitrate reductase mechanisms, molybdenum coordination dynamics, and the role of pyranopterin ligands in catalysis.
硝酸还原酶通过利用钼蝶呤辅因子促进硝酸盐还原为亚硝酸盐，在氮代谢中发挥关键作用。周质硝酸还原酶（NapA）在氧气有限时利用硝酸盐作为末端电子受体，有助于推动细菌中的厌氧代谢。尽管对NapA同源物进行了广泛研究，但关于其机制仍存在一些悬而未决的问题，尤其是在分子水平上。更广泛地说，对于钼蝶呤辅因子如何在这些酶中进行催化调节以实现含钼酶中观察到的广泛底物范围和反应性，人们了解甚少。在这里，我们在单周转条件下制备了空肠弯曲菌的NapA，以生成一种单还原酶，可通过电子顺磁共振（EPR）光谱进一步研究。我们的结果为NapA及相关酶的已知光谱和相关结构提供了新的背景信息。这些见解为理解硝酸还原酶机制、钼配位动力学以及吡喃蝶呤配体在催化中的作用开辟了新途径。
