Direct Interfacial Y 731 Oxidation in α 2 by a Photoβ 2 Subunit of E. coli Class Ia Ribonucleotide Reductase.

Proton-coupled electron transfer (PCET) is a fundamental mechanism important in a wide range of biological processes including the universal reaction catalysed by ribonucleotide reductases (RNRs) in making de novo, the building blocks required for DNA replication and repair. These enzymes catalyse the conversion of nucleoside diphosphates (NDPs) to deoxynucleoside diphosphates (dNDPs). In the class Ia RNRs, NDP reduction involves a tyrosyl radical mediated oxidation occurring over 35 Å across the interface of the two required subunits (β ₂ and α ₂ ) involving multiple PCET steps and the conserved tyrosine triad [Y ₃₅₆ (β ₂ )-Y ₇₃₁ (α ₂ )-Y ₇₃₀ (α ₂ )]. We report the synthesis of an active photochemical RNR (photoRNR) complex in which a Re(I)-tricarbonyl phenanthroline ([Re]) photooxidant is attached site-specifically to the Cys in the Y ₃₅₆ C-(β ₂ ) subunit and an ionizable, 2,3,5-trifluorotyrosine (2,3,5-F ₃ Y) is incorporated in place of Y ₇₃₁ in α ₂ . This intersubunit PCET pathway is investigated by ns laser spectroscopy on [Re ₃₅₆ ]-β ₂ :2,3,5-F ₃ Y ₇₃₁ -α ₂ in the presence of substrate, CDP, and effector, ATP. This experiment has allowed analysis of the photoinjection of a radical into α ₂ from β ₂ in the absence of the interfacial Y ₃₅₆ residue. The system is competent for light-dependent substrate turnover. Time-resolved emission experiments reveal an intimate dependence of the rate of radical injection on the protonation state at position Y ₇₃₁ (α ₂ ), which in turn highlights the importance of a well-coordinated proton exit channel involving the key residues, Y ₃₅₆ and Y ₇₃₁ , at the subunit interface.