Direct observation of a transient tyrosine radical competent for initiating turnover in a photochemical ribonucleotide reductase

Photochemical ribonucleotide reductases (photoRNRs) are developed for the generation and transport of amino acid radicals by proton-coupled electron transfer (PCET) in this enzyme. The beta(2) subunit has been replaced with the [Re]-3,5-F2Y-R2C19 peptide, which substitutes 3,5-F2Y for Y at position 356 and contains the Re(bpy)(CO)(3)CN ([Re]) photochemical radical generator. Excitation of this peptide with 355 nm light produces the [Re](0)-3,5-F2Y center dot charge-separated state within the nanosecond laser pulse, as characterized by transient absorption (TA) spectroscopy. Excitation of the bound peptide:proportional to 2 complex results in 29% turnover after 10 min of photolysis, while the corresponding [Re]-Phe-R2C19:proportional to 2 system is inactive. The 3,5-F2Y center dot radical on the peptide bound to the Y731F-proportional to 2 variant has been observed by TA spectroscopy. These data allow us to observe, for the first time, a peptide-derived, protein-bound radical that is competent for initiating RNR turnover.