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: β2(•Y122 → W48 → Y356) → α2(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-βC19) of β2 is the binding determinant of β2 to α2 and contains the redox active Y356 residue. A photoRNR has been generated synthetically by appending a Re(bpy)(CO)3CN ([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-βC19 has been measured (kd1=6.1 ± 10-7 cm-1 s-1), along with the dissociation rate constant for the [Re]-Y-βC19-α2 complex (7000 s-1 koff > 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-βC19-α2 complex that engenders partitioning of the N-terminus of the peptide into both bound and solvent-exposed fractions.
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