Reversible, Long-Range Radical Transfer in E. coli Class Ia Ribonucleotide Reductase

Abstract

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides (NDPs or NTPs where N = C, U, G, or A) to 2′-deoxynucleotides (dNDPs or dNTPs)1 and are responsible for controlling the relative ratios and absolute concentrations of cellular dNTP pools. For this reason, RNRs play a major role in ensuring the fidelity of DNA replication and repair. RNRs are found in all organisms and are classified based on the metallocofactor used to initiate catalysis,1 with the class Ia RNRs requiring a diferric-tyrosyl radical (Y•) cofactor. The prototypical class Ia RNR from E. coli, the subject of this account, is composed of two subunits, α2 and β2, and is active as an α2β2 complex, as highlighted in Figure 1. α2 houses the catalytic site for substrate (S) reduction and two allosteric effector (E = ATP, dGTP, TTP, and dATP) binding sites that govern which S is reduced (specificity site) and the overall rate of reduction (activity site). β2 contains the essential diferric-Y• cofactor. This unusually stable Y•, located at position 122, has a t1/2 of 4 days at 4 °C in contrast to the μs lifetimes observed for Y•s in solution. Nucleotide reduction occurs by a complex mechanism involving protein- and substrate-derived radicals, some details of which are summarized in Figure 2.1,3 The stable Y122• transiently oxidizes a cysteine (C439) in the catalytic site to a thiyl radical (S•), which reversibly abstracts a 3′-hydrogen atom (H•) from the NDP. The 3′-nucleotide radical rapidly loses water in the first irreversible step.1,3 The reducing equivalents are provided by two local cysteines (C225 and C462), and the resulting disulfide is re-reduced for subsequent turnovers, ultimately by thioredoxin (TR), thioredoxin reductase (TRR), and NADPH.