Mössbauer spectroscopy of protohaem and mesohaem cytochrome c peroxidases and their fluorides

Abstract

The Mössbauer spectra of cytochrome c peroxidase (CCP) containing protohaem and mesohaem have been observed in a variety of physical conditions. At very low temperature intricate paramagnetic spectra are observed, while at 195°K magnetic effects are almost completely suppressed and two quadrupole pairs appear. All spectra indicate the presence of both high- and low-spin components in agreement with susceptibility results. For the proto and meso forms in 0·2M pH 7 buffer the high-spin fractions at 4·2 °K are 0·25 and 0·4 respectively, while corresponding values at 195°K are 0·63 and 0·67. In both cases the low-spin component is in rough agreement with knowngvalues. The high-spin components are also consistent with electron spin resonance results, and the field dependence of the Mössbauer spectra indicate that the axial term in the spin Hamiltonian is 2D= 30 ± 6 cm^-1. The high-spin fraction increases with increasing buffer strength, and is freezing rate dependent at very low or zero buffer strength. Fluoride complexes of both enzymes also show low-temperature paramagnetic Mössbauer spectra. These appear to be entirely high spin with 2D= 14·6±0·6 cm^-1and a quadrupole interaction ½QV_zz= 0·85 mm s^-1. The zero-field spectra at low temperature are diffuse and consistent with the assumption of a large neighbour-nucleus interaction. At 195°K paramagnetic effects remain large and spectra are diffuse both with and without applied fields.