Dataset Information

Radical transfer in E. coli ribonucleotide reductase: a NH2Y731/R411A-? mutant unmasks a new conformation of the pathway residue 731.

ABSTRACT: Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides in all living organisms. The catalytic cycle of E. coli RNR involves a long-range proton-coupled electron transfer (PCET) from a tyrosyl radical (Y122?) in subunit ?2 to a cysteine (C439) in the active site of subunit ?2, which subsequently initiates nucleotide reduction. This oxidation occurs over 35 Å and involves a specific pathway of redox active amino acids (Y122 ? [W48?] ? Y356 in ?2 to Y731 ? Y730 ? C439 in ?2). The mechanisms of the PCET steps at the interface of the ?2?2 complex remain puzzling due to a lack of structural information for this region. Recently, DFT calculations on the 3-aminotyrosyl radical (NH2Y731?)-?2 trapped by incubation of NH2Y731-?2/?2/CDP(substrate)/ATP(allosteric effector) suggested that R411-?2, a residue close to the ?2?2 interface, interacts with NH2Y731? and accounts in part for its perturbed EPR parameters. To examine its role, we further modified NH2Y731-?2 with a R411A substitution. NH2Y731?/R411A generated upon incubation of NH2Y731/R411A-?2/?2/CDP/ATP was investigated using multi-frequency (34, 94 and 263 GHz) EPR, 34 GHz pulsed electron-electron double resonance (PELDOR) and electron-nuclear double resonance (ENDOR) spectroscopies. The data indicate a large conformational change in NH2Y731?/R411A relative to the NH2Y731? single mutant. Particularly, the inter-spin distance from NH2Y731?/R411A in one ?? pair to Y122? in a second ?? pair decreases by 3 Å in the presence of the R411A mutation. This is the first experimental evidence for the flexibility of pathway residue Y731-?2 in an ?2?2 complex and suggests a role for R411 in the stacked Y731/Y730 conformation involved in collinear PCET. Furthermore, NH2Y731?/R411A serves as a probe of the PCET process across the subunit interface.

SUBMITTER: Kasanmascheff M

PROVIDER: S-EPMC5968753 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Publications

Radical transfer in E. coli ribonucleotide reductase: a NH2Y731/R411A-α mutant unmasks a new conformation of the pathway residue 731.

Kasanmascheff Müge M Lee Wankyu W Nick Thomas U TU Stubbe JoAnne J Bennati Marina M

Chemical science 20151209 3

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides in all living organisms. The catalytic cycle of E. coli RNR involves a long-range proton-coupled electron transfer (PCET) from a tyrosyl radical (Y122˙) in subunit β2 to a cysteine (C439) in the active site of subunit α2, which subsequently initiates nucleotide reduction. This oxidation occurs over 35 Å and involves a specific pathway of redox active amino acids (Y<sub ...[more]

PMID: 29899944

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Project description:Ribonucleotide reductases (RNRs) catalyze the conversionof nucleotides to 2'-deoxynucleotides and are classified on the basis of the metallo-cofactor used to conduct this chemistry. The class Ia RNRs initiate nucleotide reduction when a stable diferric-tyrosyl radical (Y•, t1/2 of 4 days at 4 °C) cofactor in the ?2 subunit transiently oxidizes a cysteine to a thiyl radical (S•) in the active site of the ?2 subunit. In the active ?2?2 complex of the class Ia RNR from E. coli , researchers have proposed that radical hopping occurs reversibly over 35 Å along a specific pathway comprised of redox-active aromatic amino acids: Y122• ? [W48?] ? Y356 in ?2 to Y731 ? Y730 ? C439 in ?2. Each step necessitates a proton-coupled electron transfer (PCET). Protein conformational changes constitute the rate-limiting step in the overall catalytic scheme and kinetically mask the detailed chemistry of the PCET steps. Technology has evolved to allow the site-selective replacement of the four pathway tyrosines with unnatural tyrosine analogues. Rapid kinetic techniques combined with multifrequency electron paramagnetic resonance, pulsed electron-electron double resonance, and electron nuclear double resonance spectroscopies have facilitated the analysis of stable and transient radical intermediates in these mutants. These studies are beginning to reveal the mechanistic underpinnings of the radical transfer (RT) process. This Account summarizes recent mechanistic studies on mutant E. coli RNRs containing the following tyrosine analogues: 3,4-dihydroxyphenylalanine (DOPA) or 3-aminotyrosine (NH2Y), both thermodynamic radical traps; 3-nitrotyrosine (NO2Y), a thermodynamic barrier and probe of local environmental perturbations to the phenolic pKa; and fluorotyrosines (FnYs, n = 2 or 3), dual reporters on local pKas and reduction potentials. These studies have established the existence of a specific pathway spanning 35 Å within a globular ?2?2 complex that involves one stable (position 122) and three transient (positions 356, 730, and 731) Y•s. Our results also support that RT occurs by an orthogonal PCET mechanism within ?2, with Y122• reduction accompanied by proton transfer from an Fe1-bound water in the diferric cluster and Y356 oxidation coupled to an off-pathway proton transfer likely involving E350. In ?2, RT likely occurs by a co-linear PCET mechanism, based on studies of light-initiated radical propagation from photopeptides that mimic the ?2 subunit to the intact ?2 subunit and on [(2)H]-ENDOR spectroscopic analysis of the hydrogen-bonding environment surrounding a stabilized NH2Y• formed at position 730. Additionally, studies on the thermodynamics of the RT pathway reveal that the relative reduction potentials decrease according to Y122 < Y356 < Y731 ? Y730 ? C439, and that the pathway in the forward direction is thermodynamically unfavorable. C439 oxidation is likely driven by rapid, irreversible loss of water during the nucleotide reduction process. Kinetic studies of radical intermediates reveal that RT is gated by conformational changes that occur on the order of >100 s(-1) in addition to the changes that are rate-limiting in the wild-type enzyme (?10 s(-1)). The rate constant of one of the PCET steps is ?10(5) s(-1), as measured in photoinitiated experiments.

Dataset Information

Radical transfer in E. coli ribonucleotide reductase: a NH2Y731/R411A-? mutant unmasks a new conformation of the pathway residue 731.

Publications

Radical transfer in <i>E. coli</i> ribonucleotide reductase: a NH<sub>2</sub>Y<sub>731</sub>/R<sub>411</sub>A-α mutant unmasks a new conformation of the pathway residue 731.

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