Department of Chemistry and Chemical Biology, The University of New Mexico, MSC03 2060, 1 University of New Mexico, Albuquerque, New Mexico 87131-0001, United States.
Department of Chemistry and Chemical Biology, Indiana University, 402 Blackford St., Indianapolis, Indiana 46202, United States.
Inorg Chem. 2024 Jul 22;63(29):13191-13196. doi: 10.1021/acs.inorgchem.4c01991. Epub 2024 Jul 10.
Mo K-edge X-ray absorption spectroscopy (XAS) is used to probe the structure of wild-type nitrate reductase NapA and the C176A variant. The results of extended X-ray absorption fine structure (EXAFS) experiments on NapA support an oxidized Mo(VI) hexacoordinate active site coordinated by a single terminal oxo donor, four sulfur atoms from two separate pyranopterin dithiolene ligands, and an additional S atom from a conserved cysteine amino acid residue. We found no evidence of a terminal sulfido ligand in NapA. EXAFS analysis shows the C176A active site to be a 6-coordinate structure, and this is supported by EPR studies on C176A and small molecule analogs of Mo(V) enzyme forms. The S is replaced by a hydroxide or water ligand in C176A, and we find no evidence of a coordinated sulfhydryl (SH) ligand. Kinetic studies show that this variant has completely lost its catalytic activity toward nitrate. Taken together, the results support a critical role for the conserved C176 in catalysis and an oxygen atom transfer mechanism for the catalytic reduction of nitrate to nitrite that does not employ a terminal sulfido ligand in the catalytic cycle.
钼 K 边 X 射线吸收光谱（XAS）被用于探测野生型硝酸盐还原酶 NapA 和 C176A 变体的结构。对 NapA 的扩展 X 射线吸收精细结构（EXAFS）实验结果支持氧化态 Mo(VI)六配位活性位点由一个末端氧供体、来自两个单独的吡喃并蝶啶二硫醇配体的四个硫原子以及一个保守半胱氨酸氨基酸残基的额外 S 原子配位。我们在 NapA 中没有发现末端硫代配体的证据。EXAFS 分析表明 C176A 的活性位点为 6 配位结构，这得到了 C176A 和 Mo(V)酶形式的小分子类似物的 EPR 研究的支持。C176A 中的 S 被氢氧根或水配体取代，我们没有发现配位巯基（SH）配体的证据。动力学研究表明，该变体完全失去了对硝酸盐的催化活性。总之，这些结果支持保守的 C176 在催化中的关键作用，以及一种氧原子转移机制，用于催化还原硝酸盐为亚硝酸盐，该机制在催化循环中不使用末端硫代配体。
