Incorporation of 2,3,6-trifluorotyrosine (F3Y) and a rhenium bipyridine ([Re]) photooxidant into a peptide corresponding to the C-terminus of the beta protein (beta C19) of Escherichia coli ribonucleotide reductase (RNR) allows for the temporal monitoring of radical transport into the alpha 2 subunit of RNR. Injection of the photogenerated F3Y radical from the [Re]-F3Y-beta C19 peptide into the surface accessible Y731 of the alpha 2 subunit is only possible when the second Y730 is present. With the Y-Y established, radical transport occurs with a rate constant of 3 X 10(5) s(-1). Point mutations that disrupt the Y-Y dyad shut down radical transport. The ability to obviate radical transport by disrupting the hydrogen bonding network of the amino acids composing the colinear proton-coupled electron transfer pathway in alpha 2 suggests a finely tuned evolutionary adaptation of RNR to control the transport of radicals in this enzyme.