Electron injection through a specific pathway determines the outcome of oxygen activation at the diiron cluster in the F208Y mutant of Escherichia coli ribonucleotide reductase protein R2

Protein R2 of ribonucleotide reductase from Escherichia coli contains a dinuclear iron cluster, which reductively activates O-2 to produce the enzyme's functionally essential tyrosyl radical by one-electron oxidation of residue Y122. A I;ey step in this reaction is the rapid injection of a single electron from an exogenous reductant (Fe2+ or ascorbate) during formation of the radical-generating intermediate, cluster X, from the diiron(II) cluster and O-2. As this step leaves only one of the two oxidizing equivalents of ?he initial diiron(II)-O-2 adduct, it commits the reaction to a one-electron oxidation outcome and precludes possible two-electron alternatives (as occur in the related diiron bacterial alkane hydroxylases and fatty acyl desaturases). in the F208Y site-directed mutant of R2, Y208 is hydroxylated (a two-electron oxidation) in preference to the normal reaction [Aberg, A., Ormo, M., Nordlund, P., & Sjoberg, B. M. (1993) Biochemistry 32, 9845-9850], implying that this substitution blocks electron injection or (more likely) introduces an endogenous reductant (Y208) that effectively competes. Here we demonstrate that O-2 activation in the F208Y mutant of R2 partitions between these two-electron (Y208 hydroxylation) and one-electron (Y122 radical production) outcomes and that the latter becomes predominant under conditions which favor electron injection (Namely, high concentration of the reluctant ascorbate), Moreover, we show that the sensitivity of the partition ratio to ascorbate concentration is strictly dependent an the integrity of a hydrogen-bund network involving the near surface residue W48: when this residue is substituted with F, Y208 hydroxylation predominates irrespective of ascorbate concentration. These data suggest that the hydrogen-bond network involving W48 is a specific electron-transfer pathway between the cofactor site and the protein surface.