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Redox-linked structural changes in ribonucleotide reductase.

ABSTRACT: Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides. Class I RNRs are composed of two homodimeric proteins, alpha2 and beta2. The class Ia E. coli beta2 contains dinuclear, antiferromagnetically coupled iron centers and one tyrosyl free radical, Y122*/beta2. Y122* acts as a radical initiator in catalysis. Redox-linked conformational changes may accompany Y122 oxidation and provide local control of proton-coupled electron transfer reactions. To test for such redox-linked structural changes, FT-IR spectroscopy was employed in this work. Reaction-induced difference spectra, associated with the reduction of Y122* by hydroxyurea, were acquired from natural abundance, (2)H(4) tyrosine, and (15)N tyrosine labeled beta2 samples. Isotopic labeling led to the assignment of a 1514 cm(-1) band to the upsilon19a ring stretching vibration of Y122 and of a 1498 cm(-1) band to the upsilon7a CO stretching vibration of Y122*. The reaction-induced spectra also exhibited amide I bands, at 1661 and 1652 cm(-1). A similar set of amide I bands, with frequencies of 1675 and 1651 cm(-1), was observed when Y* was generated by photolysis in a pentapeptide, which matched the primary sequence surrounding Y122. This result suggests that reduction of Y122* is linked with structural changes at nearby amide bonds and that this perturbation is mediated by the primary sequence. To explain these data, we propose that a structural perturbation of the amide bond is driven by redox-linked electrostatic changes in the tyrosyl radical aromatic ring.

SUBMITTER: Offenbacher AR

PROVIDER: S-EPMC2917099 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Redox-linked structural changes in ribonucleotide reductase.

Offenbacher A R AR Vassiliev I R IR Seyedsayamdost M R MR Stubbe J J Barry B A BA

Journal of the American Chemical Society 20090601 22

Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides. Class I RNRs are composed of two homodimeric proteins, alpha2 and beta2. The class Ia E. coli beta2 contains dinuclear, antiferromagnetically coupled iron centers and one tyrosyl free radical, Y122*/beta2. Y122* acts as a radical initiator in catalysis. Redox-linked conformational changes may accompany Y122 oxidation and provide local control of proton-coupled electron transfer reactions. To test f ...[more]

PMID: 19489635

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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 structural changes in ribonucleotide reductase.

Publications

Redox-linked structural changes in ribonucleotide reductase.

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