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Selenocysteine Substitution in a Class I Ribonucleotide Reductase.

ABSTRACT: Ribonucleotide reductases (RNRs) employ a complex radical-based mechanism during nucleotide reduction involving multiple active site cysteines that both activate the substrate and reduce it. Using an engineered allo-tRNA, we substituted two active site cysteines with distinct function in the class Ia RNR of Escherichia coli for selenocysteine (U) via amber codon suppression, with efficiency and selectivity enabling biochemical and biophysical studies. Examination of the interactions of the C439U ?2 mutant protein with nucleotide substrates and the cognate ?2 subunit demonstrates that the endogenous Y122• of ?2 is reduced under turnover conditions, presumably through radical transfer to form a transient U439• species. This putative U439• species is formed in a kinetically competent fashion but is incapable of initiating nucleotide reduction via 3'-H abstraction. An analogous C225U ?2 protein is also capable of radical transfer from Y122•, but the radical-based substrate chemistry partitions between turnover and stalled reduction akin to the reactivity of mechanism-based inhibitors of RNR. The results collectively demonstrate the essential role of cysteine redox chemistry in the class I RNRs and establish a new tool for investigating thiyl radical reactivity in biology.

SUBMITTER: Greene BL

PROVIDER: S-EPMC6994234 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Selenocysteine Substitution in a Class I Ribonucleotide Reductase.

Greene Brandon L BL Stubbe JoAnne J Nocera Daniel G DG

Biochemistry 20191206 50

Ribonucleotide reductases (RNRs) employ a complex radical-based mechanism during nucleotide reduction involving multiple active site cysteines that both activate the substrate and reduce it. Using an engineered allo-tRNA, we substituted two active site cysteines with distinct function in the class Ia RNR of <i>Escherichia coli</i> for selenocysteine (U) via amber codon suppression, with efficiency and selectivity enabling biochemical and biophysical studies. Examination of the interactions of th ...[more]

PMID: 31774661

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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

Selenocysteine Substitution in a Class I Ribonucleotide Reductase.

Publications

Selenocysteine Substitution in a Class I Ribonucleotide Reductase.

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