Dataset Information

Use of 2,3,5-F(3)Y-?2 and 3-NH(2)Y-?2 to study proton-coupled electron transfer in Escherichia coli ribonucleotide reductase.

ABSTRACT: Escherichia coli ribonucleotide reductase is an ?2?2 complex that catalyzes the conversion of nucleoside 5'-diphosphates (NDPs) to deoxynucleotides (dNDPs). The active site for NDP reduction resides in ?2, and the essential diferric-tyrosyl radical (Y(122)(•)) cofactor that initiates transfer of the radical to the active site cysteine in ?2 (C(439)), 35 Å removed, is in ?2. The oxidation is proposed to involve a hopping mechanism through aromatic amino acids (Y(122) ? W(48) ? Y(356) in ?2 to Y(731) ? Y(730) ? C(439) in ?2) and reversible proton-coupled electron transfer (PCET). Recently, 2,3,5-F(3)Y (F(3)Y) was site-specifically incorporated in place of Y(356) in ?2 and 3-NH(2)Y (NH(2)Y) in place of Y(731) and Y(730) in ?2. A pH-rate profile with F(3)Y(356)-?2 suggested that as the pH is elevated, the rate-determining step of RNR can be altered from a conformational change to PCET and that the altered driving force for F(3)Y oxidation, by residues adjacent to it in the pathway, is responsible for this change. Studies with NH(2)Y(731(730))-?2, ?2, CDP, and ATP resulted in detection of NH(2)Y radical (NH(2)Y(•)) intermediates capable of dNDP formation. In this study, the reaction of F(3)Y(356)-?2, ?2, CDP, and ATP has been examined by stopped-flow (SF) absorption and rapid freeze quench electron paramagnetic resonance spectroscopy and has failed to reveal any radical intermediates. The reaction of F(3)Y(356)-?2, CDP, and ATP has also been examined with NH(2)Y(731)-?2 (or NH(2)Y(730)-?2) by SF kinetics from pH 6.5 to 9.2 and exhibited rate constants for NH(2)Y(•) formation that support a change in the rate-limiting step at elevated pH. The results together with kinetic simulations provide a guide for future studies to detect radical intermediates in the pathway.

SUBMITTER: Seyedsayamdost MR

PROVIDER: S-EPMC3076197 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Publications

Use of 2,3,5-F(3)Y-β2 and 3-NH(2)Y-α2 to study proton-coupled electron transfer in Escherichia coli ribonucleotide reductase.

Seyedsayamdost Mohammad R MR Yee Cyril S CS Stubbe JoAnne J

Biochemistry 20110208 8

Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleoside 5'-diphosphates (NDPs) to deoxynucleotides (dNDPs). The active site for NDP reduction resides in α2, and the essential diferric-tyrosyl radical (Y(122)(•)) cofactor that initiates transfer of the radical to the active site cysteine in α2 (C(439)), 35 Å removed, is in β2. The oxidation is proposed to involve a hopping mechanism through aromatic amino acids (Y(122) → W(48) → Y(356) in β2 to Y(7 ...[more]

PMID: 21182280

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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.

Dataset Information

Use of 2,3,5-F(3)Y-?2 and 3-NH(2)Y-?2 to study proton-coupled electron transfer in Escherichia coli ribonucleotide reductase.

Publications

Use of 2,3,5-F(3)Y-β2 and 3-NH(2)Y-α2 to study proton-coupled electron transfer in Escherichia coli ribonucleotide reductase.

Similar Datasets

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