Effect of the tyrosyl radical on the reduction and structure of the Escherichia coli ribonucleotide reductase protein R2 diferric site as probed by EPR on the mixed-valent state.

It was recently shown by EPR that high yields of a sterically constrained mixed-valent species may be formed in radical free protein metR2 of Escherichia coli ribonucleotide reductase by gamma-irradiation at 77 K [Davydov, R., Kuprin, S., Gräslund, A., & Ehrenberg, A. (1994) J. Am. Chem. Soc. 116, 11120]. This species, with S = 1/2, essentially retains the ligand geometry of the original diferric center and should be a sensitive probe for structural changes in the diferric centers. Here we apply this probe and demonstrate that there is a structural difference between the diferric iron center of the complete site of protein R2, with a tyrosyl radical, and that of metR2, without radical. The EPR spectrum of the mixed-valent species of metR2 shows pure axial symmetry, while complete sites show rhombic distortion and a shifted high-field turning point. Differences also remain in the EPR of the first S = 9/2 species obtained by annealing at 165 K, but disappear after relaxation at 200 K. In addition, the diferric center of a complete site is not reduced radiolytically until the associated tyrosyl radical has been reduced, indicating that an electron first reaching the iron center may be transferred to the radical. This route of electron transfer and the influence of the radical on the structure of the iron center are likely to have functional roles for the formation of the proposed substrate radical and regulation of redox processes within the enzyme. The sensitivity of the structure of the iron site to the structure of the Tyr-122 site is also demonstrated by the strong influence the mutation Y122F has on the EPR spectra of the corresponding mixed-valent species.