A Hot Oxidant, 3-NO<sub>2</sub>Y<sub>122</sub> Radical, Unmasks Conformational Gating in Ribonucleotide Reductase

Yokoyama, Kenichi; Uhlin, Ulla; Stubbe, JoAnne

doi:10.1021/ja1069344

Cited by 61 publications

(220 citation statements)

References 57 publications

(179 reference statements)

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“…For example, we have recently reported the replacement of Y 122 • with several high-energy radical initiators (NO 2 Y 122 • or F n Y 122 •s). Incubation of these mutant β2s with wt-α2, substrate, and effector converts up to 50% of the initial radical at position 122 to a Y 356 • intermediate (28,29). The lifetime of the Y 356 • depends on the identity of the radical initiator at position 122; however, in all cases, the new radical persists on the minute time scale.…”

Section: Discussionmentioning

confidence: 99%

Generation of a stable, aminotyrosyl radical-induced α2β2 complex of Escherichia coli class Ia ribonucleotide reductase

Minnihan¹,

Ando

Brignole

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleoside diphosphates (dNDPs). The Escherichia coli class Ia RNR uses a mechanism of radical propagation by which a cysteine in the active site of the RNR large (α2) subunit is transiently oxidized by a stable tyrosyl radical (Y•) . These results present a structural and biochemical characterization of the active RNR complex "trapped" during turnover, and suggest that stabilization of the α2β2 state may be a regulatory mechanism for protecting the catalytic radical and ensuring the fidelity of its reactivity.conformational equilibria | radical transfer | unnatural amino acid R ibonucleotide reductase (RNR) is the sole enzyme responsible for the conversion of nucleoside diphosphates (NDPs) to 2′-deoxynucleoside diphosphates (dNDPs), providing the cell with the monomeric precursors necessary for DNA replication and repair (1, 2). The class I RNRs are composed of two subunits, α and β;the active form of the prototypical Escherichia coli class Ia enzyme is generally accepted to be α2β2 (3, 4). The α2 subunit houses the active site, where the four NDP substrates (CDP, ADP, GDP, and UDP) are reduced, and two distinct regulatory sites, where allosteric effectors (ATP, dGTP, dTTP, and dATP) bind. The specificity site dictates which of the four substrates is reduced, whereas the activity site binds ATP/dATP and regulates the overall rate of reduction (5). The β2 subunit, an obligate dimer, contains a diferric-tyrosyl radical cofactor (Y 122 •) that is essential for catalysis. Xray crystal structures of the individual E. coli β2 and α2 subunits have been solved (6, 7). However, the weak interaction between α2 and β2 (8), the conformational rearrangements induced by nucleotide binding (2, 9, 10), and complicated subunit equilibria (11) have precluded detailed structural characterization of any active RNR complexes. We now report the characterization of an active, kinetically stable α2β2 complex that forms transiently during turnover.Nearly 2 decades ago, Uhlin and Eklund (7) put forth a docking model for the active E. coli α2β2 complex based on shape complementarity between the structures of the individual subunits.Their model predicted a 35-Å distance between the diferric-Y 122 • cofactor in β2 and the active site cysteine (C 439 ) in α2, the transient oxidation of which is a prerequisite for nucleotide reduction (1). A radical transfer (RT) pathway of conserved aromatic amino acids was proposed to account for kinetically competent radical propagation over this long distance (7). The thermodynamics of Y oxidation require loss of a proton to accompany loss of an electron, and the more detailed mechanism for proton-coupled electron transfer shown in Fig. 1A has emerged from experiments conducted in our laboratories (12,13).Evidence for the utilization of an amino acid pathway in longrange RT has been derived from several types of experiments. Initial site-directed mutagenesis studies of the conserved residues (Fig. 1A) supported t...

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Section: Discussionmentioning

confidence: 99%

Generation of a stable, aminotyrosyl radical-induced α2β2 complex of Escherichia coli class Ia ribonucleotide reductase

Minnihan¹,

Ando

Brignole

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…The reversible long range radical-transfer process, triggered by substrate and effector binding to α2, is proposed to involve multiple PCET steps via a conserved pathway (Y 122 ⇔ [W 48 ] ⇔ Y 356 in β2 to Y 731 ⇔ Y 730 ⇔ C 439 in α2; 31). We have recently described the site-specific insertion of 3-nitrotyrosine (NO 2 Y) in place of Y 122 inβ2 (48). The diferric-NO 2 Y 122 • cofactor was generated and studies with α2, substrate (CDP) and effector (ATP) allowed the first observation of a transient, kinetically competent Y• on pathway by electron paramagnetic resonance (EPR) spectroscopy (48).…”

Section: Discussionmentioning

confidence: 99%

“…We have recently described the site-specific insertion of 3-nitrotyrosine (NO 2 Y) in place of Y 122 inβ2 (48). The diferric-NO 2 Y 122 • cofactor was generated and studies with α2, substrate (CDP) and effector (ATP) allowed the first observation of a transient, kinetically competent Y• on pathway by electron paramagnetic resonance (EPR) spectroscopy (48). Pulsed electron-electron double resonance (PELDOR) spectroscopy was used to establish the primary location of the new radical as Y 356 •-β2 and suggested the formation of a small percentage of radical at either Y 731 or Y 730 in α2.…”

Section: Discussionmentioning

confidence: 99%

Formal Reduction Potential of 3,5-Difluorotyrosine in a Structured Protein: Insight into Multistep Radical Transfer

et al. 2013

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The reversible Y-O•/Y-OH redox properties of the α3Y model protein enable access to the electrochemical and thermodynamic properties of 3,5-difluorotyrosine. The unnatural amino acid has been incorporated at position 32, the dedicated radical site in α3Y, by in vivo nonsense codon suppression. Incorporation of 3,5-difluorotyrosine gives rise to very minor structural changes in the protein scaffold at pH below the apparent pK (8.0 ± 0.1) of the unnatural residue. Square-wave voltammetry on α3(3,5)F2Y provides an E°′(Y-O•/Y-OH) of 1026 ± 4 mV versus the NHE (pH 5.70 ± 0.02) and shows that the fluoro-substitutions lower E°′ by –30 ± 3 mV. These results illustrate the utility of combining the optimized α3Y tyrosine radical system with in vivo nonsense codon suppression to obtain the formal reduction potential of an unnatural aromatic residue residing within a well-structured protein. It is further observed that the protein E°′ values differ significantly from peak potentials derived from irreversible voltammograms of the corresponding aqueous species. This is significant since solution potentials have been the main thermodynamic data available for amino-acid radicals. These findings are discussed relative to recent mechanistic studies on the multistep radical-transfer process in E. coli ribonucleotide reductase site-specifically labeled with unnatural tyrosine residues.

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“…11 Each turnover of the catalytic cycle involves net transfer of e – about 35 Å from the substrate-binding site (on the α subunit) to a di-iron-tyrosyl site in the β subunit (Figure 2b); substrate- and effector-triggered conformational changes play key roles in the redox events. 12 For these, and any PCET reaction, the p K a (describing the PT component) and E ° (describing the ET component) values provide valuable insights. To that end, workers have incorporated modified tyrosine into the PCET chain of RNR (Figure 2b) to test for p K a perturbations 13 and relative redox levels of the redox-active amino acids.…”

Section: A a View From The Top: Examples Of Diverse Pcet In Enzymesmentioning

confidence: 99%

Moving Protons and Electrons in Biomimetic Systems

Warren

Mayer

2015

Biochemistry

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An enormous variety of biological redox reactions are accompanied by changes in proton content at enzyme active sites, in their associated cofactors, in substrates/products, and between protein interfaces. Understanding this breadth of reactivity is an ongoing chemical challenge. A great many workers have developed and investigated biomimetic model complexes to build new ways of thinking about the mechanistic underpinnings of such complex biological proton-coupled electron transfer (PCET) reactions. Of particular importance are those model reactions that involve transfer of one proton (H+) and one electron (e–), equivalently transfer of a hydrogen atom (H•). In this Current Topic Article we review key concepts in PCET reactivity, and describe important advances in biomimetic PCET chemistry, with special emphasis on research that has enhanced efforts to understand biological PCET reactions.

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A Hot Oxidant, 3-NO₂Y₁₂₂ Radical, Unmasks Conformational Gating in Ribonucleotide Reductase

Cited by 61 publications

References 57 publications

Generation of a stable, aminotyrosyl radical-induced α2β2 complex of Escherichia coli class Ia ribonucleotide reductase

Generation of a stable, aminotyrosyl radical-induced α2β2 complex of Escherichia coli class Ia ribonucleotide reductase

Formal Reduction Potential of 3,5-Difluorotyrosine in a Structured Protein: Insight into Multistep Radical Transfer

Moving Protons and Electrons in Biomimetic Systems

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A Hot Oxidant, 3-NO2Y122 Radical, Unmasks Conformational Gating in Ribonucleotide Reductase

Cited by 61 publications

References 57 publications

Generation of a stable, aminotyrosyl radical-induced α2β2 complex of Escherichia coli class Ia ribonucleotide reductase

Generation of a stable, aminotyrosyl radical-induced α2β2 complex of Escherichia coli class Ia ribonucleotide reductase

Formal Reduction Potential of 3,5-Difluorotyrosine in a Structured Protein: Insight into Multistep Radical Transfer

Moving Protons and Electrons in Biomimetic Systems

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A Hot Oxidant, 3-NO₂Y₁₂₂ Radical, Unmasks Conformational Gating in Ribonucleotide Reductase