Photo-ribonucleotide reductase β2 by selective cysteine labeling with a radical phototrigger

Photochemical radical initiation is a powerful tool for studying radical initiation and transport in biology. Ribonucleotide reductases (RNRs), which catalyze the conversion of nucleotides to deoxynucleotides in all organisms, are an exemplar of radical mediated transformations in biology. Class Ia RNRs are composed of two subunits: α2 and β2. As a method to initiate radical formation photochemically within β2, a single surface-exposed cysteine of the β2 subunit of Escherichia coli Class Ia RNR has been labeled (98%) with a photooxidant ([Re ] = tricarbonyl(1,10-phenanthroline)(methylpyridyl)rhenium(I)). The labeling was achieved by incubation of S355C-β2 with the 4-(bromomethyl)pyridyl derivative of [Re] to yield the labeled species, [Re]-S355C-β2. Steady-state and time-resolved emission experiments reveal that the metal-to-ligand charge transfer (MLCT) excited-state (3)[Re ](∗) is not significantly perturbed after bioconjugation and is available as a phototrigger of tyrosine radical at position 356 in the β2 subunit; transient absorption spectroscopy reveals that the radical lives for microseconds. The work described herein provides a platform for photochemical radical initiation and study of proton-coupled electron transfer (PCET) in the β2 subunit of RNR, from which radical initiation and transport for this enzyme originates.