Dataset Information

Structural examination of the transient 3-aminotyrosyl radical on the PCET pathway of E. coli ribonucleotide reductase by multifrequency EPR spectroscopy.

ABSTRACT: E. coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides, a process that requires long-range radical transfer over 35 A from a tyrosyl radical (Y(122)*) within the beta2 subunit to a cysteine residue (C(439)) within the alpha2 subunit. The radical transfer step is proposed to occur by proton-coupled electron transfer via a specific pathway consisting of Y(122) --> W(48) --> Y(356) in beta2, across the subunit interface to Y(731) --> Y(730) --> C(439) in alpha2. Using the suppressor tRNA/aminoacyl-tRNA synthetase (RS) methodology, 3-aminotyrosine has been incorporated into position 730 in alpha2. Incubation of this mutant with beta2, substrate, and allosteric effector resulted in loss of the Y(122)* and formation of a new radical, previously proposed to be a 3-aminotyrosyl radical (NH(2)Y*). In the current study [(15)N]- and [(14)N]-NH(2)Y(730)* have been generated in H(2)O and D(2)O and characterized by continuous wave 9 GHz EPR and pulsed EPR spectroscopies at 9, 94, and 180 GHz. The data give insight into the electronic and molecular structure of NH(2)Y(730)*. The g tensor (g(x) = 2.0052, g(y) = 2.0042, g(z) = 2.0022), the orientation of the beta-protons, the hybridization of the amine nitrogen, and the orientation of the amino protons relative to the plane of the aromatic ring were determined. The hyperfine coupling constants and geometry of the NH(2) moiety are consistent with an intramolecular hydrogen bond within NH(2)Y(730)*. This analysis is an essential first step in using the detailed structure of NH(2)Y(730)* to formulate a model for a PCET mechanism within alpha2 and for use of NH(2)Y in other systems where transient Y*s participate in catalysis.

SUBMITTER: Seyedsayamdost MR

PROVIDER: S-EPMC4703294 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Publications

Structural examination of the transient 3-aminotyrosyl radical on the PCET pathway of E. coli ribonucleotide reductase by multifrequency EPR spectroscopy.

Seyedsayamdost Mohammad R MR Argirević Tomislav T Minnihan Ellen C EC Stubbe JoAnne J Bennati Marina M

Journal of the American Chemical Society 20091101 43

E. coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides, a process that requires long-range radical transfer over 35 A from a tyrosyl radical (Y(122)*) within the beta2 subunit to a cysteine residue (C(439)) within the alpha2 subunit. The radical transfer step is proposed to occur by proton-coupled electron transfer via a specific pathway consisting of Y(122) --> W(48) --> Y(356) in beta2, across the subunit interface to Y(731) --> Y(730) --> C(439) in ...[more]

PMID: 19821570

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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

Structural examination of the transient 3-aminotyrosyl radical on the PCET pathway of E. coli ribonucleotide reductase by multifrequency EPR spectroscopy.

Publications

Structural examination of the transient 3-aminotyrosyl radical on the PCET pathway of E. coli ribonucleotide reductase by multifrequency EPR spectroscopy.

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