Ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleoside diphosphates (dNDPs). The Escherichia coli class Ia RNR uses a mechanism of radical propagation by which a cysteine in the active site of the RNR large (α2) subunit is transiently oxidized by a stable tyrosyl radical (Y•) . These results present a structural and biochemical characterization of the active RNR complex "trapped" during turnover, and suggest that stabilization of the α2β2 state may be a regulatory mechanism for protecting the catalytic radical and ensuring the fidelity of its reactivity.conformational equilibria | radical transfer | unnatural amino acid R ibonucleotide reductase (RNR) is the sole enzyme responsible for the conversion of nucleoside diphosphates (NDPs) to 2′-deoxynucleoside diphosphates (dNDPs), providing the cell with the monomeric precursors necessary for DNA replication and repair (1, 2). The class I RNRs are composed of two subunits, α and β;the active form of the prototypical Escherichia coli class Ia enzyme is generally accepted to be α2β2 (3, 4). The α2 subunit houses the active site, where the four NDP substrates (CDP, ADP, GDP, and UDP) are reduced, and two distinct regulatory sites, where allosteric effectors (ATP, dGTP, dTTP, and dATP) bind. The specificity site dictates which of the four substrates is reduced, whereas the activity site binds ATP/dATP and regulates the overall rate of reduction (5). The β2 subunit, an obligate dimer, contains a diferric-tyrosyl radical cofactor (Y 122 •) that is essential for catalysis. Xray crystal structures of the individual E. coli β2 and α2 subunits have been solved (6, 7). However, the weak interaction between α2 and β2 (8), the conformational rearrangements induced by nucleotide binding (2, 9, 10), and complicated subunit equilibria (11) have precluded detailed structural characterization of any active RNR complexes. We now report the characterization of an active, kinetically stable α2β2 complex that forms transiently during turnover.Nearly 2 decades ago, Uhlin and Eklund (7) put forth a docking model for the active E. coli α2β2 complex based on shape complementarity between the structures of the individual subunits.Their model predicted a 35-Å distance between the diferric-Y 122 • cofactor in β2 and the active site cysteine (C 439 ) in α2, the transient oxidation of which is a prerequisite for nucleotide reduction (1). A radical transfer (RT) pathway of conserved aromatic amino acids was proposed to account for kinetically competent radical propagation over this long distance (7). The thermodynamics of Y oxidation require loss of a proton to accompany loss of an electron, and the more detailed mechanism for proton-coupled electron transfer shown in Fig. 1A has emerged from experiments conducted in our laboratories (12,13).Evidence for the utilization of an amino acid pathway in longrange RT has been derived from several types of experiments. Initial site-directed mutagenesis studies of the conserved residues (Fig. 1A) supported t...

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. Active E. coli class Ia RNR is an α2β2 complex that undergoes reversible, long-range proton-coupled electron transfer (PCET) over a pathway of redox active amino acids (β-Y122 → [β-W48] → β-Y356 → α-Y731 → α-Y730 → α-C439) that spans ∼35 Å. To unmask PCET kinetics from rate-limiting conformational changes, we prepared a photochemical RNR containing a [ReI] photooxidant site-specifically incorporated at position 355 ([Re]-β2), adjacent to PCET pathway residue Y356 in β. [Re]-β2 was further modified by replacing Y356 with 2,3,5-trifluorotyrosine to enable photochemical generation and spectroscopic observation of chemically competent tyrosyl radical(s). Using transient absorption spectroscopy, we compare the kinetics of Y· decay in the presence of substrate and wt-α2, Y731F-α2 ,or C439S-α2, as well as with 3′-[2H]-substrate and wt-α2. We find that only in the presence of wt-α2 and the unlabeled substrate do we observe an enhanced rate of radical decay indicative of forward radical propagation. This observation reveals that cleavage of the 3′-C–H bond of substrate by the transiently formed C439· thiyl radical is rate-limiting in forward PCET through α and has allowed calculation of a lower bound for the rate constant associated with this step of (1.4 ± 0.4) × 104 s–1. Prompting radical propagation with light has enabled observation of PCET events heretofore inaccessible, revealing active site chemistry at the heart of RNR catalysis.

Substrate turnover in class Ia ribonucleotide reductase (RNR) requires reversible radical transport across two subunits over 35 A, which occurs by a multi-step proton-coupled electron transfer mechanism. Using a photooxidant-labeled β2 subunit of Escherichia coli class Ia RNR, we demonstrate photoinitiated oxidation of a tyrosine in an α2:β2 complex, which results in substrate turnover. Using site-directed mutations of the redox-active tyrosines at the subunit interface—Y356F(β) and Y731F(α)—this oxidation is identified to be localized on Y356. The rate of Y356 oxidation depends on the presence of Y731 across the interface. This observation supports the proposal that unidirectional PCET across the Y356(β)–Y731(α)–Y730(α) triad is crucial to radical transport in RNR.