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Subunit Interaction Dynamics of Class Ia Ribonucleotide Reductases: In Search of a Robust Assay.

ABSTRACT: Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides (NDP) to deoxynucleotides (dNDP), in part, by controlling the ratios and quantities of dNTPs available for DNA replication and repair. The active form of Escherichia coli class Ia RNR is an asymmetric ?2?2 complex in which ?2 contains the active site and ?2 contains the stable diferric-tyrosyl radical cofactor responsible for initiating the reduction chemistry. Each dNDP is accompanied by disulfide bond formation. We now report that, under in vitro conditions, ?2 can initiate turnover in ?2 catalytically under both "one" turnover (no external reductant, though producing two dCDPs) and multiple turnover (with an external reductant) assay conditions. In the absence of reductant, rapid chemical quench analysis of a reaction of ?2, substrate, and effector with variable amounts of ?2 (1-, 10-, and 100-fold less than ?2) yields 3 dCDP/?2 at all ratios of ?2:?2 with a rate constant of 8-9 s-1, associated with a rate-limiting conformational change. Stopped-flow fluorescence spectroscopy with a fluorophore-labeled ? reveals that the rate constants for subunit association (163 ± 7 ?M-1 s-1) and dissociation (75 ± 10 s-1) are fast relative to turnover, consistent with catalytic ?2. When assaying in the presence of an external reducing system, the turnover number is dictated by the ratio of ?2:?2, their concentrations, and the concentration and nature of the reducing system; the rate-limiting step can change from the conformational gating to a step or steps involving disulfide rereduction, dissociation of the inhibited ?4?4 state, or both. The issues encountered with E. coli RNR are likely of importance in all class I RNRs and are central to understanding the development of screening assays for inhibitors of these enzymes.

SUBMITTER: Ravichandran K

PROVIDER: S-EPMC7160020 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Subunit Interaction Dynamics of Class Ia Ribonucleotide Reductases: In Search of a Robust Assay.

Ravichandran Kanchana K Olshansky Lisa L Nocera Daniel G DG Stubbe JoAnne J

Biochemistry 20200330 14

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides (NDP) to deoxynucleotides (dNDP), in part, by controlling the ratios and quantities of dNTPs available for DNA replication and repair. The active form of Escherichia coli class Ia RNR is an asymmetric α2β2 complex in which α2 contains the active site and β2 contains the stable diferric-tyrosyl radical cofactor responsible for initiating the reduction chemistry. Each dNDP is ...[more]

PMID: 32186371

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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

Subunit Interaction Dynamics of Class Ia Ribonucleotide Reductases: In Search of a Robust Assay.

Publications

Subunit Interaction Dynamics of Class Ia Ribonucleotide Reductases: In Search of a Robust Assay.

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