Dataset Information

Hydrogen bond network between amino acid radical intermediates on the proton-coupled electron transfer pathway of E. coli ?2 ribonucleotide reductase.

ABSTRACT: Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides in all organisms. In all Class Ia RNRs, initiation of nucleotide diphosphate (NDP) reduction requires a reversible oxidation over 35 Å by a tyrosyl radical (Y122•, Escherichia coli) in subunit ? of a cysteine (C439) in the active site of subunit ?. This radical transfer (RT) occurs by a specific pathway involving redox active tyrosines (Y122 ? Y356 in ? to Y731 ? Y730 ? C439 in ?); each oxidation necessitates loss of a proton coupled to loss of an electron (PCET). To study these steps, 3-aminotyrosine was site-specifically incorporated in place of Y356-?, Y731- and Y730-?, and each protein was incubated with the appropriate second subunit ?(?), CDP and effector ATP to trap an amino tyrosyl radical (NH2Y•) in the active ?2?2 complex. High-frequency (263 GHz) pulse electron paramagnetic resonance (EPR) of the NH2Y•s reported the gx values with unprecedented resolution and revealed strong electrostatic effects caused by the protein environment. (2)H electron-nuclear double resonance (ENDOR) spectroscopy accompanied by quantum chemical calculations provided spectroscopic evidence for hydrogen bond interactions at the radical sites, i.e., two exchangeable H bonds to NH2Y730•, one to NH2Y731• and none to NH2Y356•. Similar experiments with double mutants ?-NH2Y730/C439A and ?-NH2Y731/Y730F allowed assignment of the H bonding partner(s) to a pathway residue(s) providing direct evidence for colinear PCET within ?. The implications of these observations for the PCET process within ? and at the interface are discussed.

SUBMITTER: Nick TU

PROVIDER: S-EPMC4304443 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Hydrogen bond network between amino acid radical intermediates on the proton-coupled electron transfer pathway of E. coli α2 ribonucleotide reductase.

Nick Thomas U TU Lee Wankyu W Kossmann Simone S Neese Frank F Stubbe JoAnne J Bennati Marina M

Journal of the American Chemical Society 20141229 1

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides in all organisms. In all Class Ia RNRs, initiation of nucleotide diphosphate (NDP) reduction requires a reversible oxidation over 35 Å by a tyrosyl radical (Y122•, Escherichia coli) in subunit β of a cysteine (C439) in the active site of subunit α. This radical transfer (RT) occurs by a specific pathway involving redox active tyrosines (Y122 ⇆ Y356 in β to Y731 ⇆ Y730 ⇆ C439 in α); each oxidation ...[more]

PMID: 25516424

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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

Hydrogen bond network between amino acid radical intermediates on the proton-coupled electron transfer pathway of E. coli ?2 ribonucleotide reductase.

Publications

Hydrogen bond network between amino acid radical intermediates on the proton-coupled electron transfer pathway of E. coli α2 ribonucleotide reductase.

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