Dataset Information

Glutamate 350 Plays an Essential Role in Conformational Gating of Long-Range Radical Transport in Escherichia coli Class Ia Ribonucleotide Reductase.

ABSTRACT: Escherichia coli class Ia ribonucleotide reductase (RNR) is composed of two subunits that form an active ?2?2 complex. The nucleoside diphosphate substrates (NDP) are reduced in ?2, 35 Å from the essential diferric-tyrosyl radical (Y122•) cofactor in ?2. The Y122•-mediated oxidation of C439 in ?2 occurs by a pathway (Y122 ? [W48] ? Y356 in ?2 to Y731 ? Y730 ? C439 in ?2) across the ?/? interface. The absence of an ?2?2 structure precludes insight into the location of Y356 and Y731 at the subunit interface. The proximity in the primary sequence of the conserved E350 to Y356 in ?2 suggested its importance in catalysis and/or conformational gating. To study its function, pH-rate profiles of wild-type ?2/?2 and mutants in which 3,5-difluorotyrosine (F2Y) replaces residue 356, 731, or both are reported in the presence of E350 or E350X (X = A, D, or Q) mutants. With E350, activity is maintained at the pH extremes, suggesting that protonated and deprotonated states of F2Y356 and F2Y731 are active and that radical transport (RT) can occur across the interface by proton-coupled electron transfer at low pH or electron transfer at high pH. With E350X mutants, all RNRs were inactive, suggesting that E350 could be a proton acceptor during oxidation of the interface Ys. To determine if E350 plays a role in conformational gating, the strong oxidants, NO2Y122•-?2 and 2,3,5-F3Y122•-?2, were reacted with ?2, CDP, and ATP in E350 and E350X backgrounds and the reactions were monitored for pathway radicals by rapid freeze-quench electron paramagnetic resonance spectroscopy. Pathway radicals are generated only when E350 is present, supporting its essential role in gating the conformational change(s) that initiates RT and masking its role as a proton acceptor.

SUBMITTER: Ravichandran K

PROVIDER: S-EPMC5689411 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Publications

Glutamate 350 Plays an Essential Role in Conformational Gating of Long-Range Radical Transport in Escherichia coli Class Ia Ribonucleotide Reductase.

Ravichandran Kanchana K Minnihan Ellen C EC Lin Qinghui Q Yokoyama Kenichi K Taguchi Alexander T AT Shao Jimin J Nocera Daniel G DG Stubbe JoAnne J

Biochemistry 20170202 6

Escherichia coli class Ia ribonucleotide reductase (RNR) is composed of two subunits that form an active α2β2 complex. The nucleoside diphosphate substrates (NDP) are reduced in α2, 35 Å from the essential diferric-tyrosyl radical (Y122•) cofactor in β2. The Y122•-mediated oxidation of C439 in α2 occurs by a pathway (Y122 ⇆ [W48] ⇆ Y356 in β2 to Y731 ⇆ Y730 ⇆ C439 in α2) acros ...[more]

PMID: 28103007

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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

Glutamate 350 Plays an Essential Role in Conformational Gating of Long-Range Radical Transport in Escherichia coli Class Ia Ribonucleotide Reductase.

Publications

Glutamate 350 Plays an Essential Role in Conformational Gating of Long-Range Radical Transport in Escherichia coli Class Ia Ribonucleotide Reductase.

Similar Datasets

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