Direct Interfacial Y731Oxidation in α2by a Photoβ2Subunit ofE. coliClass 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 (β2and α2) involving multiple PCET steps and the conserved tyrosine triad [Y356(β2)-Y731(α2)-Y730(α2)]. 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 Y356C-(β2) subunit and an ionizable, 2,3,5-trifluorotyrosine (2,3,5-F3Y) is incorporated in place of Y731in α2. This intersubunit PCET pathway is investigated by ns laser spectroscopy on [Re356]-β2:2,3,5-F3Y731-α2in the presence of substrate, CDP, and effector, ATP. This experiment has allowed analysis of the photoinjection of a radical into α2from β2in the absence of the interfacial Y356residue. 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 Y731(α2), which in turn highlights the importance of a well-coordinated proton exit channel involving the key residues, Y356and Y731, at the subunit interface.
Department of Chemistry and Chemical Biology, 12 Oxford Street, Cambridge, MA 02138-2902.
David Y
DY
Song
Department of Chemistry and Chemical Biology, 12 Oxford Street, Cambridge, MA 02138-2902.
Arturo A
AA
Pizano
Department of Chemistry and Chemical Biology, 12 Oxford Street, Cambridge, MA 02138-2902.
Patrick G
PG
Holder
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139-4307.
JoAnne
J
Stubbe
Department of Chemistry and Chemical Biology, 12 Oxford Street, Cambridge, MA 02138-2902.
Daniel G
DG
Nocera