Identification of a hydroxide ligand at the iron center of ribonucleotide reductase by resonance Raman spectroscopy

Abstract

The resonance Raman spectrum of protein B2 of ribonucleotide reductase from Escherichia coli shows several features related to its oxo-bridged binuclear iron center. A peak at 492 cm-1 is assigned to the symemtric stretch of the Fe.sbd.O-Fe moiety on the basis of its 13-cm-1 shift to lower energy upon 18O substitution. The 18O species shows an additional peak at 731 cm-1, which is a good candidate for the asymmetric stretch of the Fe-O-Fe moiety. Its exact location in the 16O species is obscured by the presence of a protein tryptophan vibration at 758 cm-1. A third resonance-enhanced peak at 598 cm-1 is identified as an Fe-OH vibration on the basis of its 24-cm-1 shift to lower energy in H218O, its 2-cm-1 shift to lower energy in D2O, and its pH-dependent intensity. A hydrogen-bonded .mu.-oxo bridge similar to that in hemerythrin is suggested by the unusually low frequency for the Fe-O-Fe symmetric strech and the 3-cm-1 shift to higher energy of .nu.s (Fe-O-Fe) in D2O. From the oxygen isotope dependence of .nu.s (Fe-O-Fe), an Fe-O-Fe angle of 138.degree. can be calculated. This small angle suggests that the iron center consists of a tribridged core as in hemerythrin. A model for the binuclear iron center of ribonucleotide reductase is presented in which the hydroxide ligand sites provide an explanation for the half-of-sites reactivity of the enzyme.