Probing Conformational Change During Radical Propagation in the E.coli Class 1a RNR Using 3‐aminotyrosine as a Radical “Sink”

Abstract

The E. coli class Ia ribonucleotide reductase (RNR) catalyzes the de novo synthesis of deoxynucleoside 5ʹ‐diphosphates (dNDPs) using nucleoside 5ʹ‐diphosphates (NDPs) as substrates. The catalytic mechanism of RNR requires an active site thiyl radical, which is generated via reversible oxidation by a tyrosyl radical cofactor located over 35 Å away. The model for the long range oxidation involves multiple proton‐coupled electron transfer (PCET) steps through aromatic amino acid residues. Efforts to study the radical hopping mechanism have been challenging due to kinetic masking of the chemistry by a conformational gate. Lowering the midpoint potentials of pathway residues by using the amber stop codon suppression method to site‐specifically incorporate the unnatural amino acid (UAA) 3‐aminotyrosine (NH2Y) acts as a radical “sink” on pathway and changes the rate limiting step of the radical propagation mechanism from a conformational change to a PCET step. NH2Y‐incorporated RNR (NH2Y‐RNRs) have allowed the detection of radical intermediates on this PCET pathway and have established the role of three tyrosines, Y356, Y730 and Y731, in the PCET pathway. Despite the large perturbation in midpoint potential, NH2Y‐RNRs can also do multiple turnovers and produce dNDPs. The study of the rapid kinetics of NH2Y● and dNDP formation demonstrates the kinetic and chemical competence of the NH2Y● intermediates. Additionally, insight into the kinetic model of the NH2Y‐RNRs give rise to further details of multiple, more subtle conformational changes within the PCET pathway mechanism. Specifically, the NH2Y● populations exist in two distinct conformations, with one less competent in nucleotide reduction.