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Escherichia coli and herpes-simplex-virus ribonucleotide reductase R2 subunit. Compared reactivities of the redox centres.

ABSTRACT: Protein R2, the small subunit of ribonucleotide reductase, contains a diferric centre and a tyrosyl radical absolutely required for enzyme activity. The reduction of the tyrosyl radical and the mobilization of the iron centre result in the inhibition of the enzyme and thus of DNA synthesis. The chemical reactivity of the iron-radical centre of Escherichia coli and herpes simplex virus has been studied by u.v.-visible and e.p.r. spectroscopies. The tyrosyl radical is efficiently scavenged by hydroxamic acids and phenols during reactions controlled by steric hindrance and hydrophobic interactions. The reaction with o-disubstituted phenols yields the corresponding diphenoquinones. The reactivity of the bacterial radical greatly contrasts with that of the viral radical, and the iron centre in herpes-simplex-virus R2 is much more labile than that in E. coli R2, as shown from the facile mobilization of iron by chelators such as catechol. These results suggest that the active sites of the two enzymes are significantly different and might be useful for designing new antiviral agents.

SUBMITTER: Atta M

PROVIDER: S-EPMC1132353 | biostudies-other | 1993 Mar

REPOSITORIES: biostudies-other

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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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Escherichia coli and herpes-simplex-virus ribonucleotide reductase R2 subunit. Compared reactivities of the redox centres.

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