Ribonucleotide reductase (RDPR) from Escherichia coli catalyzes the conversion of nucleotides to deoxynucleotides and is composed of two homodimeric subunits: R1 and R2. (E)- and (Z)-2'-fluoromethylene-2'-deoxycytidine 5'-diphosphate (FMCDP) are time dependent inactivators of this protein, with approximately 1.5 equiv being sufficient for complete loss of catalytic activity. Inactivation results from loss of the essential tyrosyl radical on R2 and alkylation of R1. Studies using electron spin resonance spectroscopy reveal that tyrosyl radical loss is accompanied by formation of a new, substrate-based radical. Experiments using [6'-14C]-(E)-FMCDP and [5-3H]-(E)-FMCDP reveal that alkylation of R1 is accompanied by release of 0.5 equiv of cytosine and 1.4 equiv of fluoride ion. When R1 is denatured subsequent to inactivation, approximately 1 equiv of label per R1 is observed only in studies carried out with [14C]FMCDP. Under these same conditions with [3H]FMCDP, 1.5 equiv of radiolabel is detected as cytosine. Inactivation of R1 thus results from alkylation by the sugar moiety of FMCDP. While studies to isolate the alkylated amino acid on R1 were unsuccessful, studies using a variety of site-directed mutants of R1 (C462S, C225S, C754/759S, C439S, and E441Q) indicate that E441 or possibly C439 is the modified residue. Inactivation is accompanied by rapid formation of a new chromophore with a lambda max at 334 nm. Dithiothreitol does not protect the enzyme against inactivation by FMCDP, although it does prevent chromophore formation. Two possible mechanisms are proposed to accommodate these experimental observations.

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Ribonucleotide reductase (RDPR) from E. coli catalyzes the conversion of nucleotides to deoxynucleotides and contains an unusual tyrosyl radical−diferric cluster cofactor. (E)-2‘-Fluoromethylene-2‘-deoxycytidine 5‘-diphosphate [(E)-1] obtained by phosphorylation of the clinically promising antitumor agent MDL 101,731, is a potent time-dependent inactivator of this protein. Electron paramagnetic resonance (EPR) spectroscopy reveals that inactivation is accompanied by loss of the essential tyrosyl radical cofactor and formation of a new radical species. The 9.4-GHz EPR spectrum of this new radical reveals two hyperfine splittings of approximately 1.5 mT producing a triplet-like signal. Incubation of the enzyme with [6‘-2H]-(E)-1 alters this EPR spectrum, providing the first evidence for a nucleotide-based radical species generated by RDPR. The observed spectrum is a 1:1 composite of a doublet and a triplet signal, the latter being identical to that obtained with unlabeled (E)-1. Studies with (E)-1 in 2H2O also produce a 1:1 mixture of these two radical signals. The results of these isotope labeling experiments suggest wash-out or wash-in of ∼0.5 equiv of deuterium at the 6‘ position, respectively. Incubation of the enzyme with [6‘-2H]-(E)-1 in 2H2O produced only the doublet as would be expected on the basis of this hypothesis. EPR (139.5 GHz) spectra established the principal g values of the new radical species. Simulation of the 9.4- and 139.5-GHz EPR spectra yield a self-consistent set of principal hyperfine values. A structure is proposed for the radical intermediate that is consistent with the EPR data and kinetic data on release of the fluoride ion that accompanies inactivation. The proposed structure and a postulated mechanism for its formation provide further support for the hypothesis that catalysis is initiated by 3‘-hydrogen atom abstraction from the nucleotide substrate.