Escherichia coli ribonucleotide reductase (RNR) catalyzes the reduction of nucleotides to 2′-deoxynucleotides. The active enzyme is a 1:1 complex of two homodimeric subunits, α2 and β2. The α2 is the site of nucleotide reduction, and β2 harbors a diferric tyrosyl radical (Y122•) cofactor. Turnover requires formation of a cysteinyl radical (C439•) in the active site of α2 at the expense of the Y122• in β2. A docking model for the α2β2 interaction and a pathway for radical transfer from β2 to α2 have been proposed. This pathway contains three Ys: Y356 in β2 and Y731/Y730 in α2. We have previously incorporated 3-hydroxytyrosine and 3-aminotyrosine into these residues and showed that they act as radical traps. In this study, we use these α2/β2 variants and PELDOR spectroscopy to measure the distance between the Y122• in one αβ pair and the newly formed radical in the second αβ pair. The results yield distances that are similar to those predicted by the docking model for radical transfer. Further, they support a long-range radical initiation process for C439• generation and provide a structural constraint for residue Y356, which is thermally labile in all β2 structures solved to date.
All Science Journal Classification (ASJC) codes
- Colloid and Surface Chemistry