Electron Transfer Properties of the R2 Protein of Ribonucleotide Reductase from Escherichia coli

Kathleen E. Silva, Timothy E. Elgren, Lawrence Que, Marian T. Stankovich

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47 Scopus citations

Abstract

The enzyme ribonucleotide reductase from Escherichia coli consists of two proteins, R1 and R2. The active R2 protein contains two dinuclear iron centers and the catalytically essential tyrosyl radical. We have explored the redox properties of the tyrosyl radical and estimate an apparent redox potential of + 1000 ±100 mV (vs SHE) on the basis of the behavior of numerous mediators. The inability of most of these mediators to equilibrate with the tyrosyl radical supports the notion that the radical exists in an extremely protected hydrophobic pocket that prevents most radical scavengers from interacting with the radical, resulting in its unusual stability. The formal midpoint potential of the diiron clusters of the R2 protein was determined to be - 115 ± 2 mV at pH 7.6 and 4 °C. This reduction is a two-electron transfer process, making the R2 protein the first of the nonheme diiron proteins not to stabilize a mixed valence intermediate at ambient temperature. The formal midpoint potential of the dinuclear iron centers is pH dependent, exhibiting a 30 mV/pH unit variance, which indicates that one proton is accepted from the solvent per two electrons transferred to the dinuclear iron center upon reduction. The midpoint potential of the site-directed mutant Y122F R2 protein was also investigated under the same conditions, and this midpoint potential was determined to be -178 mV, providing the first direct evidence that the presence of the Y122 residue modulates the redox properties of the diiron clusters. The redox potentials of both the wild type and Y122F proteins experience cathodic shifts when measured in the presence of azide or the R1 protein. For the latter, the midpoint potentials were determined to be -226 mV for the wild type protein and -281 mV for the Y122F mutant protein, representing a negative shift of over 100 mV for both proteins. These results indicate that the presence of the Y122 residue does not influence the effect of R1 binding, that the R1 protein preferentially binds the oxidized form of R2, and that the binding of R1 acts as a regulatory control mechanism to prevent unnecessary turnover of the dinuclear iron centers.

Original languageEnglish (US)
Pages (from-to)14093-14103
Number of pages11
JournalBiochemistry
Volume34
Issue number43
DOIs
StatePublished - Oct 1995

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