Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. Active E. coli class la RNR is an alpha(2)beta(2) complex that undergoes reversible, long-range proton-coupled electron transfer (PCET) over a pathway of redox active amino acids (beta-Y-122 -> [beta-W-48] -> beta-Y-356 -> alpha-Y-731 -> alpha-Y-730 -> alpha-C-439) that spans similar to 35 angstrom. To unmask PCET kinetics from rate-limiting conformational changes, we prepared a photochemical RNR containing a [Re-1] photooxidant site-specifically incorporated at position 355 [Re]-beta(2)), adjacent to PCET pathway residue Y-356 in beta. [Re]-beta(2) was further modified by replacing Y-356 with 2,3,5-trifluorotyrosine to enable photochemical generation and spectroscopic observation of chemically competent tyrosyl radical(s). Using transient absorption spectroscopy, we compare the kinetics of Y. decay in the presence of substrate and wt-alpha(2), Y731F-alpha(2),or C439S-alpha(2), as well as with 3'-[H-2]-substrate and wt-alpha(2). We find that only in the presence of wt-alpha(2) and the unlabeled substrate do we observe an enhanced rate of radical decay indicative of forward radical propagation. This observation reveals that cleavage of the 3'-C-H bond of substrate by the transiently formed C-439; thiyl radical is rate-limiting in forward PCET through alpha and has allowed calculation of a lower bound for the rate constant associated with this step of (1.4 +/- 0.4) x 10(4) s(-1). Prompting radical propagation with light has enabled observation of PCET events heretofore inaccessible, revealing active site chemistry at the heart of RNR catalysis.