The mechanism of radical transport in the alpha 2 (R1) subunit of class I E. coli ribonucleotide reductase (RNR) has been investigated by the phototriggered generation of a tyrosyl radical, (center dot)Y356, on a 20-mer peptide bound to alpha 2. This peptide, Y-R2C19, is identical to the C-terminal peptide tail of the beta 2 (R2) subunit and is a known competitive inhibitor of binding of the native beta 2 protein to alpha 2. (center dot)Y356 radical initiation is prompted by excitation (lambda >= 300 nm) of a proximal anthraquinone, Anq, or benzophenone, BPA, chromophore on the peptide. Transient absorption spectroscopy has been employed to kinetically characterize the radical-producing step by time resolving the semiquinone anion (Anq(center dot-)), ketyl radical ((center dot)BPA), and Y-center dot photoproducts on (i) BPA-Y and Anq-Y dipeptides and (ii) BPA/Anq-Y-R2C19 peptides. Light-initiated, single-turnover assays have been carried out with the peptide/alpha 2 complex in the presence of [C-14]-labeled cytidine 5'-diphosphate substrate and ATP allosteric effector. We show that both the Anq- and BPA-containing peptides are competent in deoxycytidine diphosphate formation and turnover occurs via Y731 to Y730 to C439 pathway-dependent radical transport in alpha 2. Experiments with the Y730F mutant exclude a direct superexchange mechanism between C439 and Y731 and are consistent with a PCET model for radical transport in which there is a unidirectional transport of the electron and proton transport among residues of alpha 2.