title: Glutamate 350 Plays an Essential Role in Conformational Gating of Long-Range Radical Transport in Escherichia coli Class Ia Ribonucleotide Reductase.
abstract: Escherichia coli class Ia ribonucleotide reductase (RNR) is composed of two subunits that form an active α2β2 complex. The nucleoside diphosphate substrates (NDP) are reduced in α2, 35 Å from the essential diferric-tyrosyl radical (Y122•) cofactor in β2. The Y122•-mediated oxidation of C439in α2 occurs by a pathway (Y122⇆ [W48] ⇆ Y356in β2 to Y731⇆ Y730⇆ C439in α2) across the α/β interface. The absence of an α2β2 structure precludes insight into the location of Y356and Y731at the subunit interface. The proximity in the primary sequence of the conserved E350to Y356in β2 suggested its importance in catalysis and/or conformational gating. To study its function, pH-rate profiles of wild-type β2/α2 and mutants in which 3,5-difluorotyrosine (F2Y) replaces residue 356, 731, or both are reported in the presence of E350or E350X (X = A, D, or Q) mutants. With E350, activity is maintained at the pH extremes, suggesting that protonated and deprotonated states of F2Y356and F2Y731are active and that radical transport (RT) can occur across the interface by proton-coupled electron transfer at low pH or electron transfer at high pH. With E350X mutants, all RNRs were inactive, suggesting that E350could be a proton acceptor during oxidation of the interface Ys. To determine if E350plays a role in conformational gating, the strong oxidants, NO2Y122•-β2 and 2,3,5-F3Y122•-β2, were reacted with α2, CDP, and ATP in E350and E350X backgrounds and the reactions were monitored for pathway radicals by rapid freeze-quench electron paramagnetic resonance spectroscopy. Pathway radicals are generated only when E350is present, supporting its essential role in gating the conformational change(s) that initiates RT and masking its role as a proton acceptor.
authors.forename: Kanchana
authors.identifier: 0000-0001-5050-9719
authors.initials: K
authors.lastname: Ravichandran
authors.forename: Ellen C
authors.initials: EC
authors.lastname: Minnihan
authors.affiliation: Department of Pathology and Pathophysiology, Key Laboratory of Disease Proteomics of Zhejiang Province, Research Center for Air Pollution and Health, Zhejiang University School of Medicine , Hangzhou 310058, China.
authors.forename: Qinghui
authors.initials: Q
authors.lastname: Lin
authors.forename: Kenichi
authors.initials: K
authors.lastname: Yokoyama
authors.forename: Alexander T
authors.initials: AT
authors.lastname: Taguchi
authors.affiliation: Department of Pathology and Pathophysiology, Key Laboratory of Disease Proteomics of Zhejiang Province, Research Center for Air Pollution and Health, Zhejiang University School of Medicine , Hangzhou 310058, China.
authors.forename: Jimin
authors.initials: J
authors.lastname: Shao
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University , 12 Oxford Street, Cambridge, Massachusetts 02138, United States.
authors.forename: Daniel G
authors.identifier: 0000-0001-5055-320X
authors.initials: DG
authors.lastname: Nocera
authors.forename: JoAnne
authors.identifier: 0000-0001-8076-4489
authors.initials: J
authors.lastname: Stubbe
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D001051:Apoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001667:Binding, Competitive; D055162:Biocatalysis; D003565:Cytidine Diphosphate / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005609:Free Radicals / Q000378:metabolism*; D018698:Glutamic Acid / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry
chemical_list: D001051:Apoenzymes; D029968:Escherichia coli Proteins; D005609:Free Radicals; D011994:Recombinant Proteins; C025200:3,5-difluorotyrosine; D018698:Glutamic Acid; D014443:Tyrosine; D003565:Cytidine Diphosphate; D000255:Adenosine Triphosphate; C571544:NrdB protein, E coli; D012264:Ribonucleotide Reductases