Hydrogen Bond Network between Amino Acid Radical Intermediates on the Proton-Coupled Electron Transfer Pathway of E. coli α2 Ribonucleotide Reductase

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 angstrom by a tyrosyl radical (Y-122, Escherichia coli) in subunit beta of a cysteine (C-439) in the active site of subunit a. This radical transfer (RT) occurs by a specific pathway involving redox active tyrosines (Y-122 reversible arrow Y-356 in beta to Y-731 reversible arrow Y-730 reversible arrow C-439 in alpha); 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-beta, Y731- and Y730-alpha, and each protein was incubated with the appropriate second subunit beta(alpha), CDP and effector ATP to trap an amino tyrosyl radical (NH2Y center dot) in the active alpha 2 beta 2 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. H-2 electronnuclear 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 a-NH2Y730/C(439)A and alpha-NH2Y731/Y730F allowed assignment of the H bonding partner(s) to a pathway residue(s) providing direct evidence for colinear PCET within a. The implications of these observations for the PCET process within a and at the interface are discussed.