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Redox-linked changes to the hydrogen-bonding network of ribonucleotide reductase ?2.

ABSTRACT: Ribonucleotide reductase (RNR) catalyzes conversion of nucleoside diphosphates (NDPs) to 2'-deoxynucleotides, a critical step in DNA replication and repair in all organisms. Class-Ia RNRs, found in aerobic bacteria and all eukaryotes, are a complex of two subunits: ?2 and ?2. The ?2 subunit contains an essential diferric-tyrosyl radical (Y122O(•)) cofactor that is needed to initiate reduction of NDPs in the ?2 subunit. In this work, we investigated the Y122O(•) reduction mechanism in Escherichia coli ?2 by hydroxyurea (HU), a radical scavenger and cancer therapeutic agent. We tested the hypothesis that Y122OH redox reactions cause structural changes in the diferric cluster. Reduction of Y122O(•) was studied using reaction-induced FT-IR spectroscopy and [(13)C]aspartate-labeled ?2. These Y122O(•) minus Y122OH difference spectra provide evidence that the Y122OH redox reaction is associated with a frequency change to the asymmetric vibration of D84, a unidentate ligand to the diferric cluster. The results are consistent with a redox-induced shift in H-bonding between Y122OH and D84 that may regulate proton-transfer reactions on the HU-mediated inactivation pathway in isolated ?2.

SUBMITTER: Offenbacher AR

PROVIDER: S-EPMC3694779 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Redox-linked changes to the hydrogen-bonding network of ribonucleotide reductase β2.

Offenbacher Adam R AR Minnihan Ellen C EC Stubbe JoAnne J Barry Bridgette A BA

Journal of the American Chemical Society 20130417 17

Ribonucleotide reductase (RNR) catalyzes conversion of nucleoside diphosphates (NDPs) to 2'-deoxynucleotides, a critical step in DNA replication and repair in all organisms. Class-Ia RNRs, found in aerobic bacteria and all eukaryotes, are a complex of two subunits: α2 and β2. The β2 subunit contains an essential diferric-tyrosyl radical (Y122O(•)) cofactor that is needed to initiate reduction of NDPs in the α2 subunit. In this work, we investigated the Y122O(•) reduction mechanism in Escherichia ...[more]

PMID: 23594029

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Project description:Tyrosyl radicals play essential roles in biological proton-coupled electron transfer (PCET) reactions. Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides and is vital in DNA replication in all organisms. Class Ia RNRs consist of ?2 and ?2 homodimeric subunits. In class Ia RNR, such as the E. coli enzyme, an essential tyrosyl radical (Y122O(•))-diferric cofactor is located in ?2. Although Y122O(•) is extremely stable in free ?2, Y122O(•) is highly reactive in the quaternary substrate-?2?2 complex and serves as a radical initiator in catalytic PCET between ?2 and ?2. In this report, we investigate the structural interactions that control the reactivity of Y122O(•) in a model system, isolated E. coli ?2. Y122O(•) was reduced with hydroxyurea (HU), a radical scavenger that quenches the radical in a clinically relevant reaction. In the difference FT-IR spectrum, associated with this PCET reaction, amide I (CO) and amide II (CN/NH) bands were observed. Specific (13)C-labeling of the tyrosine C1 carbon assigned a component of these bands to the Y122-T123 amide bond. Comparison to density functional calculations on a model dipeptide, tyrosine-threonine, and structural modeling demonstrated that PCET is associated with a Y122 rotation and a 7.2 Å translation of the Y122 phenolic oxygen. To test for the functional consequences of this structural change, a proton inventory defined the origin of the large solvent isotope effect (SIE = 16.7 ± 1.0 at 25 °C) on this reaction. These data suggest that the one-electron, HU-mediated reduction of Y122O(•) is associated with two, rate-limiting (full or partial) proton transfer reactions. One is attributable to HU oxidation (SIE = 11.9, net H atom transfer), and the other is attributable to coupled, hydrogen-bonding changes in the Y122O(•)-diferric cofactor (SIE = 1.4). These results illustrate the importance of redox-linked changes to backbone and ring dihedral angles in high potential PCET and provide evidence for rate-limiting, redox-linked hydrogen-bonding interactions between Y122O(•) and the iron cluster.

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Redox-linked changes to the hydrogen-bonding network of ribonucleotide reductase ?2.

Publications

Redox-linked changes to the hydrogen-bonding network of ribonucleotide reductase β2.

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