Resonance Raman spectroscopy of chloroperoxidase compound II provides direct evidence for the existence of an iron(IV)-hydroxide.

Abstract

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 (2)H)-sensitive band at nu(Fe-O) = 565 cm(-1). Preparation of CPO-II in H(2)O using H(2)(18)O(2) results in a red-shift of 22 cm(-1), while preparation of CPO-II in (2)H(2)O using H(2)O(2) 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-A Fe-O bond reported for CPO-II. A Badger's rule analysis of this distance provides an Fe-O stretching frequency of nu(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(III)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.