Function of the Diiron Cluster ofEscherichia coliClass Ia Ribonucleotide Reductase in Proton-Coupled Electron Transfer

Abstract

The class Ia ribonucleotide reductase (RNR) from Escherichia coli (Ec) employs a free-radical mechanism, which involves bidirectional translocation of a radical equivalent or “hole” over a distance of ∼35 Å from the stable diferric/tyrosyl-radical (Y122•) cofactor in the β subunit to cysteine 439 (C439) in the active site of the α subunit. This long-range, inter-subunit electron transfer occurs by a multi-step “hopping” mechanism via formation of transient amino acid radicals along a specific pathway and is thought to be conformationally gated and coupled to local proton transfers. Whereas constituent amino acids of the hopping pathway have been identified, details of the proton-transfer steps and conformational gating within the β sununit have remained obscure; specific proton couples have been proposed, but no direct evidence has been provided. In the key first step, the reduction of Y122• by the first residue in the hopping pathway, a water ligand to Fe1 of the diferric cluster was suggested to donate a proton to yield the neutral Y122. Here we show that forward radical translocation is associated with perturbation of the Mössbauer spectrum of the diferric cluster, especially the quadrupole doublet associated with Fe1. Density functional theory (DFT) calculations verify the consistency of the experimentally observed perturbation with that expected for deprotonation of the Fe1-coordinated water ligand. The results thus provide the first evidence that the diiron cluster of this prototypical class Ia RNR functions not only in its well-known role as generator of the enzyme's essential Y122•, but also directly in catalysis.

A >200 meV Uphill Thermodynamic Landscape for Radical Transport in Escherichia coli Ribonucleotide Reductase Determined Using Fluorotyrosine-Substituted Enzymes

“…This is primarily due to the unique nature of residue 122’s environment compared to that of the other pathway Y’s. The Y 122 site is not in equilibrium with solvent 48 over the time course of our experiments (<20 s); its reduction potential is pH-independent and is directly determined by the dielectric constant of the protein environment. Due to these reasons, we turned our attention to an alternate way to monitor equilibration of Y 122 • and Y 356 • where the native Y 122 • remains intact but Y 356 is replaced with F 2 Y 356 .…”

“…We have previously proposed that the conformational change that triggers RT targets the initial PT step from Fe1–H 2 O to Y 122 • (Scheme 2). 48 Uncoupled PT and ET in NO 2 Y 122 •-β2, and potentially F 3 Y 122 •-β2, suggest that we may have overcome this conformational gating and obtained direct insight into the thermodynamic effect of replacing Y 122 with these unnatural analogs. Further support for this model is obtained when the forward RT rate constants in NO 2 Y 122 •-β2 and F 3 Y 122 •-β2 are predicted using the Moser–Dutton equation 52 (eq 3) for dependence of k ET on distance ( R ) and driving force (Δ G ).…”

Electron flow through biological molecules: does hole hopping protect proteins from oxidative damage?

“…Functional radical transfer pathways have been identified in several enzymes, including ribonucleotide reductase (Argirevic et al, 2012; Holder et al, 2012; Offenbacher et al, 2013a; Offenbacher et al, 2013b; Sjöberg, 1997; Stubbe et al, 2003; Stubbe & van der Donk, 1998; Worsdorfer et al, 2013; Yokoyama et al, 2011), photosystem II (Boussac et al, 2013; Keough et al, 2013; Sjoholm et al, 2012), DNA photolyase (Aubert et al, 1999; Aubert et al, 2000; Byrdin et al, 2003; Kodali, Siddiqui & Stanley, 2009; Li, Heelis & Sancar, 1991; Lukacs et al, 2006; Sancar, 2003; Taylor, 1994; Woiczikowski et al, 2011), and MauG (Davidson & Liu, 2012; Davidson & Wilmot, 2013; Geng et al, 2013; Yukl et al, 2013). If radical transfer pathways do indeed provide protection mechanisms for enzymes operating at high electrochemical potentials, then it is likely that they will be found in many more redox-active enzymes.…”

Mono- and binuclear non-heme iron chemistry from a theoretical perspective

“…Prediction of Mössbauer spectral changes allowed to conclude the proton source in the radical transfer of a class Ia RNR to be the iron-bound water molecule. [28] On the other hand, correlation with spectroscopic data can be used to validate or actually choose ("calibrate") the DFT methodology for the description of electronic structure or reactivity. This approach was chosen in a comparative study of the high-spin/low-spin Fe…”