Resonance Raman Spectroscopy of Chloroperoxidase Compound II Provides Direct Evidence for the Existence of an Iron(IV)-Hydroxide

We report direct evidence for the existence of an iron(IV)-hydroxide. Resonance Raman measurements on chloroperoxidase compound II (CPO-II) reveal an isotope ($^{18}O$and ²H)-sensitive band at$\upsilon_{Fe-O} = 565 cm^{-1}$. Preparation of CPO-II in H₂O using$H_{2}^{18}O_{2}$results in a red-shift of 22 cm⁻¹, while preparation of CPO-II in ²H₂0 using H₂O₂ results in a red-shift of 13 cm⁻¹. These values are in good agreement with the isotopic shifts predicted (23 and 12 cm⁻¹, respectively) for an Fe-OH harmonic oscillator. The measured Fe-O stretching frequency is also in good agreement with the 1.82-Å Fe-O bond reported for CPO-II. A Badger's rule analysis of this distance provides an Fe-O stretching frequency of$\upsilon_{Badger} = 563 cm^{-1}$. We also present X-band electron nuclear double resonance (ENDOR) data for cryoreduced CPO-II. Cryogenic reduction (77 K) of the EPR-silent Fe(IV)OH center in CPO-II results in an EPR-active Fe(lIII)OH species with a strongly coupled (13.4 MHz) exchangeable proton. Based on comparisons with alkaline myoglobin, we assign this resonance to the hydroxide proton of cryoreduced CPO-II.