Resonance Raman and electronic absorption spectra of horseradish peroxidase isozyme A2: evidence for a quantum-mixed spin species

The resonance Raman (RR) and electronic absorption spectra of ferric horseradish peroxidase isozyme A2 (HRPA2) and of its complex with fluoride are reported. The data show the presence of a five-coordinate quantum-mixed spin (QS) species, resulting from the admixture of intermediate-spin, S=3/2, and high-spin, S=5/2, configurations, coexisting with a five- and a six-coordinate high-spin species. Fluoride binds almost completely, giving rise to a six-coordinate high-spin heme. In the RR spectra of both the resting enzyme and fluoride complex two ν(C=C) stretching modes of the vinyl substituents are observed at 1622 and 1631 cm-1. A comparison of the data obtained for HRPA2 with the electronic and RR spectra of soybean and barley peroxidases, all belonging to class III of the ‘superfamily of plant peroxidases,’ shows that the QS species is common to all these proteins. Furthermore, it has allowed us to infer that the QS heme is characterized by very high wavenumbers of the core-size marker bands and an electronic absorption spectrum blue-shifted with respect to those of the high-spin hemes. © 1998 John Wiley & Sons, Ltd.