Redox-linked structural changes in ribonucleotide reductase.

Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides. Class I RNRs are composed of two homodimeric proteins, alpha2 and beta2. The class Ia E. coli beta2 contains dinuclear, antiferromagnetically coupled iron centers and one tyrosyl free radical, Y122*/beta2. Y122* acts as a radical initiator in catalysis. Redox-linked conformational changes may accompany Y122 oxidation and provide local control of proton-coupled electron transfer reactions. To test for such redox-linked structural changes, FT-IR spectroscopy was employed in this work. Reaction-induced difference spectra, associated with the reduction of Y122* by hydroxyurea, were acquired from natural abundance, (2)H(4) tyrosine, and (15)N tyrosine labeled beta2 samples. Isotopic labeling led to the assignment of a 1514 cm(-1) band to the upsilon19a ring stretching vibration of Y122 and of a 1498 cm(-1) band to the upsilon7a CO stretching vibration of Y122*. The reaction-induced spectra also exhibited amide I bands, at 1661 and 1652 cm(-1). A similar set of amide I bands, with frequencies of 1675 and 1651 cm(-1), was observed when Y* was generated by photolysis in a pentapeptide, which matched the primary sequence surrounding Y122. This result suggests that reduction of Y122* is linked with structural changes at nearby amide bonds and that this perturbation is mediated by the primary sequence. To explain these data, we propose that a structural perturbation of the amide bond is driven by redox-linked electrostatic changes in the tyrosyl radical aromatic ring.