Redox-Linked Structural Changes in Ribonucleotide Reductase

Abstract

Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides. Class I RNRs are composed of two homodimeric proteins, α2 and β2. The class Ia E. coli β2 contains dinuclear, antiferromagnetically coupled iron centers and one tyrosyl free radical, Y122•/β2. 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, [superscript 2]H[subscript 4] tyrosine, and [superscript 15]N tyrosine labeled β2 samples. Isotopic labeling led to the assignment of a 1514 cm[superscript −1] band to the υ19a ring stretching vibration of Y122 and of a 1498 cm[superscript −1] band to the υ7a CO stretching vibration of Y122•. The reaction-induced spectra also exhibited amide I bands, at 1661 and 1652 cm[superscript −1]. A similar set of amide I bands, with frequencies of 1675 and 1651 cm[superscript −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.