Photochemical ribonucleotide reductases (photoRNRs) have been developed to study the proton-coupled electron transfer (PCET) mechanism of radical transport in Escherichia coli class I ribonucleotide reductase (RNR). The transport of the effective radical occurs along several conserved aromatic residues across two subunits: beta2((*)Y122 --> W48 --> Y356) --> alpha2(Y731 --> Y730 --> C439). The current model for RNR activity suggests that radical transport is strongly controlled by conformational gating. The C-terminal tail peptide (Y-betaC19) of beta2 is the binding determinant of beta2 to alpha2 and contains the redox active Y356 residue. A photoRNR has been generated synthetically by appending a Re(bpy)(CO)(3)CN ([Re]) photo-oxidant next to Y356 of the 20-mer peptide. Emission from the [Re] center dramatically increases upon peptide binding, serving as a probe for conformational dynamics and the protonation state of Y356. The diffusion coefficient of [Re]-Y-betaC19 has been measured (k(d1) = 6.1 x 10(-7) cm(-1) s(-1)), along with the dissociation rate constant for the [Re]-Y-betaC19-alpha2 complex (7000 s(-1) > k(off) > 400 s(-1)). Results from detailed time-resolved emission and absorption spectroscopy reveal biexponential kinetics, suggesting a large degree of conformational flexibility in the [Re]-Y-betaC19-alpha2 complex that engenders partitioning of the N-terminus of the peptide into both bound and solvent-exposed fractions.