title: Hydrogen bond network between amino acid radical intermediates on the proton-coupled electron transfer pathway of E. coli α2 ribonucleotide reductase.
abstract: Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides in all organisms. In all Class Ia RNRs, initiation of nucleotide diphosphate (NDP) reduction requires a reversible oxidation over 35 Å by a tyrosyl radical (Y122•, Escherichia coli) in subunit β of a cysteine (C439) in the active site of subunit α. This radical transfer (RT) occurs by a specific pathway involving redox active tyrosines (Y122 ⇆ Y356 in β to Y731 ⇆ Y730 ⇆ C439 in α); each oxidation necessitates loss of a proton coupled to loss of an electron (PCET). To study these steps, 3-aminotyrosine was site-specifically incorporated in place of Y356-β, Y731- and Y730-α, and each protein was incubated with the appropriate second subunit β(α), CDP and effector ATP to trap an amino tyrosyl radical (NH2Y•) in the active α2β2 complex. High-frequency (263 GHz) pulse electron paramagnetic resonance (EPR) of the NH2Y•s reported the gx values with unprecedented resolution and revealed strong electrostatic effects caused by the protein environment. (2)H electron-nuclear double resonance (ENDOR) spectroscopy accompanied by quantum chemical calculations provided spectroscopic evidence for hydrogen bond interactions at the radical sites, i.e., two exchangeable H bonds to NH2Y730•, one to NH2Y731• and none to NH2Y356•. Similar experiments with double mutants α-NH2Y730/C439A and α-NH2Y731/Y730F allowed assignment of the H bonding partner(s) to a pathway residue(s) providing direct evidence for colinear PCET within α. The implications of these observations for the PCET process within α and at the interface are discussed.
authors.affiliation: Max Planck Institute for Biophysical Chemistry , 37077 Göttingen, Germany.
authors.forename: Thomas U
authors.initials: TU
authors.lastname: Nick
authors.forename: Wankyu
authors.initials: W
authors.lastname: Lee
authors.forename: Simone
authors.initials: S
authors.lastname: Kossmann
authors.forename: Frank
authors.initials: F
authors.lastname: Neese
authors.forename: JoAnne
authors.initials: J
authors.lastname: Stubbe
authors.forename: Marina
authors.initials: M
authors.lastname: Bennati
mesh_terms: D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism; D004579:Electron Transport; D004926:Escherichia coli / Q000201:enzymology*; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008968:Molecular Conformation; D011522:Protons; D012264:Ribonucleotide Reductases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D005609:Free Radicals; D011522:Protons; D012264:Ribonucleotide Reductases