Investigation of the mechanism of radical propagation in E. coli ribonucleotide reductase by site-specific incorporation of unnatural amino acids

Abstract

Inside the cell, ribonucleotide reductases (RNRs) are responsible for the conversion of nucleotides to 2'-deoxynucleotides, an essential step in DNA biosynthesis and repair. The E. coli RNR is the best studied RNR to date and consists of two protein subunits, a2 and P2. a2 is the site of nucleotide reduction and 02 contains a diiron tyrosyl radical (Y122*) cofactor. Each turnover requires radical propagation from the Y122* in 32 to the active site of a2 over 35 A. The mechanism of this unprecedented, long-range radical propagation step is poorly understood. Based on structural studies, a pathway of aromatic residues has been proposed to participate in this process. Site-directed mutants of these residues have been uninformative. In an effort to understand radical propagation, we have employed expressed protein ligation and suppressor tRNA/aminoacyl-tRNA synthetase (RS) methodologies to site-specifically insert unnatural tyrosine analogues into 12 and a2, at residues believed to be involved. On the basis of results with the radical traps 3,4-dihydroxyphenylalanine (DOPA) and 3-aminotyrosine (NH2Y), which we have incorporated into 32 and a2, respectively, and a series of fluorotyrosines (FnYs, n=2, 3, 4), which we have established as probes for proton-coupled electron transfer reactions and incorporated into 12, we propose a mechanism for radical transfer in RNR. We show that binding of substrate and effector are essential for control and gating of radical propagation. We further demonstrate that three Ys, 12-Y356, a2-Y731 and a2-Y730, are redox-active and participate in hole propagation. The NH2Y. observed with NH2Y-a2s likely constitutes the first observation of a transiently oxidized intermediate during active radical propagation. In 12, Y356 participates in radical transfer by an orthogonal proton-coupled electron transfer mechanism, where long-range electron transfer is coupled to short-range, off-pathway proton transfer.

(cont) Within a2, Y731 and Y730o participate by a hydrogen atom transfer mechanism where the proton and electron originate from and arrive at the same moiety. We also establish the positions of these three Ys in the a2/32 complex and present direct evidence for the reversible nature of radical propagation.