RESONANCE RAMAN STUDIES OF COMPOUNDS I AND II OF ARTHROMYCES RAMOSUS PEROXIDASE - CLOSE SIMILARITIES IN THEIR RAMAN-SPECTRA BUT DISTINCT OXYGEN EXCHANGEABILITY OF THE FE=O HEME

Simultaneous measurements of resonance Raman and absorption spectra we re performed for intermediates generated upon addition of hydrogen per oxide to ferric Arthromyces ramosus peroxidase (ARP) using the microci rculating system constructed in this laboratory, which enables generat ion of desirable intermediates under steady-state conditions. Compound I of ARP generated at neutral pH was stable over tens of minutes in t he absence of laser illumination with this circulation system, but was gradually degraded under laser illumination, giving rise to a new irr eversible species with an iron-ore heme. Such photosensitivity was not observed for compound II in the steady state at alkaline pH. Surprisi ngly, the Raman spectrum of compound I of ARP in the high-frequency re gion, where characteristic frequency shifts are expected upon oxidatio n of the macrocycle, was quite close to that of compound II, despite t he fact that the reduced Soret absorption indicated the formation of a pi-cation radical. The Fe = O stretching (nu(Fe=O)) frequency of comp ound I was observed at 781 cm(-1) for the O-16 derivative but appeared as a doublet at 744 and 731 cm(-1) for the O-18 derivative. The isoto pe sensitivity of the nu(Fe=O) mode of compound I was seen upon (H2O)- O-16/(H2O)-O-18 solvent substitution but not upon (H2O2)-O-16/(H2O2)-O -18 peroxide substitution in (H2O)-O-16 at neutral pH. This directly i ndicates the occurrence of an oxygen atom exchange between the oxo-hem e and bulk water, providing the first example of such exchange in comp ound I of peroxidases. The oxygen exchange was abolished for compound II at alkaline pH, for which the nu(Fe=O) mode was seen at 787/749 cm( -1) only upon (H2O2)-O-16/(H2O2)-O-18 peroxide substitution. The oxyge n exchangeability seems to depend on protonation of a nearby residue w ith pK(a) similar to 9 and to correlate with stability of compound I. (C) 1996 John Wiley & Sons, Inc.