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Active Site Characterization of a Campylobacter jejuni Nitrate Reductase Variant Provides Insight into the Enzyme Mechanism.

التفاصيل البيبلوغرافية
العنوان: Active Site Characterization of a Campylobacter jejuni Nitrate Reductase Variant Provides Insight into the Enzyme Mechanism.
المؤلفون: Yang J; 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., Mintmier B; Department of Chemistry and Chemical Biology, Indiana University, 402 Blackford St., Indianapolis, Indiana 46202, United States., Kc K; 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., Metzger MC; Department of Chemistry and Chemical Biology, Indiana University, 402 Blackford St., Indianapolis, Indiana 46202, United States., Radhakrishnan M; Department of Chemistry and Chemical Biology, Indiana University, 402 Blackford St., Indianapolis, Indiana 46202, United States., McGarry J; Department of Chemistry and Chemical Biology, Indiana University, 402 Blackford St., Indianapolis, Indiana 46202, United States.; Department of Chemistry and Biochemistry, University of Wisconsin, 3210 N. Cramer St., Milwaukee, Wisconsin 53211, United States., Wilcoxen J; Department of Chemistry and Biochemistry, University of Wisconsin, 3210 N. Cramer St., Milwaukee, Wisconsin 53211, United States., Basu P; Department of Chemistry and Chemical Biology, Indiana University, 402 Blackford St., Indianapolis, Indiana 46202, United States., Kirk ML; 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.
المصدر: Inorganic chemistry [Inorg Chem] 2024 Jul 22; Vol. 63 (29), pp. 13191-13196. Date of Electronic Publication: 2024 Jul 10.
نوع المنشور: Journal Article
اللغة: English
بيانات الدورية: Publisher: American Chemical Society Country of Publication: United States NLM ID: 0366543 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1520-510X (Electronic) Linking ISSN: 00201669 NLM ISO Abbreviation: Inorg Chem Subsets: MEDLINE
أسماء مطبوعة: Original Publication: [Easton, Pa.] American Chemical Society.
مواضيع طبية MeSH: Campylobacter jejuni*/enzymology , Nitrate Reductase*/chemistry , Nitrate Reductase*/metabolism , Catalytic Domain*, Models, Molecular ; X-Ray Absorption Spectroscopy
مستخلص: Mo K-edge X-ray absorption spectroscopy (XAS) is used to probe the structure of wild-type Campylobacter jejuni nitrate reductase NapA and the C176A variant. The results of extended X-ray absorption fine structure (EXAFS) experiments on wt 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 wt 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 Cys 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.
المشرفين على المادة: EC 1.7.99.4 (Nitrate Reductase)
تواريخ الأحداث: Date Created: 20240710 Date Completed: 20240722 Latest Revision: 20240722
رمز التحديث: 20240722
DOI: 10.1021/acs.inorgchem.4c01991
PMID: 38984973
قاعدة البيانات: MEDLINE
الوصف
تدمد:1520-510X
DOI:10.1021/acs.inorgchem.4c01991