Deciphering Radical Transport in the Large Subunit of Class I Ribonucleotide Reductase

Abstract

Incorporation of 2,3,6-trifluorotyrosine (F3Y) and a rhenium bipyridine ([Re]) photooxidant into a peptide corresponding to the C-terminus of the β protein (βC19) of Escherichia coli ribonucleotide reductase (RNR) allows for the temporal monitoring of radical transport into the α2 subunit of RNR. Injection of the photogenerated F3Y radical from the [Re]–F3Y–βC19 peptide into the surface accessible Y731 of the α2 subunit is only possible when the second Y730 is present. With the Y–Y established, radical transport occurs with a rate constant of 3 × 105 s−1. Point mutations that disrupt the Y–Y dyad shut down radical transport. The ability to obviate radical transport by disrupting the hydrogen bonding network of the amino acids composing the co-linear proton-coupled electron transfer pathway in α2 suggests a finely tuned evolutionary adaptation of RNR to control the transport of radicals in this enzyme.

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.…”

Generation of a stable, aminotyrosyl radical-induced α2β2 complex of Escherichia coli class Ia ribonucleotide reductase

“…The lifetime of the Y 356 • depends on the identity of the radical initiator at position 122; however, in all cases, the new radical persists on the minute time scale. Recalling that Y 356 •, located on the C-terminal tail of free β2, would likely be reduced on the microsecond time scale (19), the observation of a long-lived Y 356 • suggests a comparably long-lived α2β2 association. In retrospect, a similar stabilization of the α2β2 complex likely accounts for the observations with the mechanism-based inhibitor 2′-azido-2′-deoxynucleotide, which reacts in the presence of α2 and β2 to generate a nucleotide-based, nitrogen-centered radical (N•) in the enzyme active site.…”

Photochemical Generation of a Tryptophan Radical within the Subunit Interface of Ribonucleotide Reductase

“…9 In the modified setup, the previously used Triax 320 spectrometer has been replaced by a Horiba iHR320 spectrometer. Optical long-pass cutoff filters (λ > 375 nm) were used to filter probe light before detection to remove scattered 355 nm pump light.…”

“…8–10 Installation of a bromomethylpyridyl rhenium(I) tricarbonyl phenanthroline complex ([Re I ]) at position β 355 via cysteine ligation produces a photoβ 2 , 11 where the adjacent Y 356 has been replaced with various fluorotyrosines (F n Ys, n = 2–3) to modulate the p K a and E °′ of Y 356 within the RNR subunit interface. 12,13 This methodology has enabled spectroscopic observation of photochemically competent radical intermediates, 9,12 assignment of rate constants associated with individual PCET steps, 12 and determination of Marcus parameters within the α/β subunit interface. 13 In these studies, individual Y• species from among the β-Y 356 , α-Y 731 and α-Y 730 triad could not be spectroscopically resolved, preventing measurement of PCET rates among them.…”