Mechanistic studies of proton-coupled electron transfer in aminotyrosine- and fluorotyrosine- substituted class Ia Ribonucleotide reductase

Abstract

Ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to 2'- deoxynucleotides in all organisms. The class Ia RNR from Escherichia coli is active as an a2p2 complex and utilizes an unprecedented mechanism of reversible proton-coupled electron transfer (PCET) to propagate a stable tyrosyl radical (Yi22-) in P2 over a distance of >35 A to an active site cysteine (C4 3 9) in a2 on each turnover. Generation of the cysteinyl radical (C4 3 9-) initiates active site nucleotide reduction. Radical propagation over 35 A by a pure tunneling mechanism would be too slow to support the observed turnover number. Instead, long-range, reversible PCET is proposed to occur by radical hopping along a specific pathway of redox-active amino acids: ... The details of this mechanism are kinetically masked in wild-type RNR, and mutation of any of these residues to another native amino acid inactivates the enzyme. Recent development of technology for the in vivo, site-specific incorporation of unnatural amino acids into proteins has provided the opportunity to systematically perturb the native PCET pathway by introduction of tyrosine analogues with modified redox potentials and/or pKas. This thesis focuses on 3-aminotyrosine (NH2Y) and fluorotyrosines (FnYs). NH2Y has a lower reduction potential than Y and, when incorporated at the three sites of transient Ye formation, generates a thermodynamic minimum and reduces kcat sufficiently to allow characterization of NH2Y. intermediates. A kinetic model for catalysis by NH2Y-RNRs has been proposed from the mechanistic studies described herein. Furthermore, the ability to generate NH2Y* on the pathway has afforded the first characterization of a kinetically stable c2p2 complex. FnYs span a wide range of solution pKas and reduction potentials and thus may be used to investigate both PT and ET events. The evolution of an orthogonal, polyspecific tRNA/tRNA synthetase pair for FnYs is reported. FnYs at positions 356, 730, and 731 have been used to measure the pH dependence of RNR activity, whereas FnY-s at position 122 of $2 have been used as radical initiators to begin mapping the relative thermodynamic landscape of the PCET pathway.