IDENTIFICATION OF PUTATIVE PEROXIDE INTERMEDIATES OF PEROXIDASES BY ELECTRONIC-STRUCTURE AND SPECTRA CALCULATIONS

The INDO/ROHF/CI quantum chemical method has been used to calculate th e electronic structure and spectra of two candidate peroxide intermedi ates of model peroxidases. In the enzymatic cycle of this family of ox idative metabolizing heme proteins, hydrogen peroxide is required to t ransform the ferric resting state to the catalytically active, ferryl Fe=O, compound I species. While a peroxide complex has been proposed a s a key intermediate in this reaction, this intermediate species is to o transient to have thus fat been definitively characterized. Electron ic spectra observed prior to compound I formation during the reaction of H2O2 with both wild type and the R38L mutant of horseradish peroxid ase C (HRP-C) have been attributed to this intermediate. There are, ho wever, significant qualitative differences in these spectra in the 300 -450-nm region, with a ''hyper-Soret'' observed in one and a normal So ret, not very different from the resting stale, found in the other. In the absence of any additional information, it is not possible from th ese reported spectra alone to identify the species that give rise to t hem or to understand these differences, In order to identify the origi n of these spectra and their differences, we have calculated the elect ronic structure and spectra of two possible forms of the peroxide inte rmediate of model peroxidases, one with a neutral peroxide and the oth er with an anionic form (OOH-) as the heme Fe ligand. Formation of the anion is possible by proton transfer to a nearby histidine residue, a lready implicated in compound I formation. A comparison of the calcula ted spectra for these two transient species indicates them to be quite distinct. Comparisons of the two spectra with those experimentally ob served suggest that the ''hyper-porphyrin'' spectrum observed in the w ild type (WT) HRP-C experiments originates from the OOH- form of this transient intermediate in a Tow-spin ground state, while tile normal S oret observed in the R38L HRP-C mutant experiment originates from the neutral peroxide form in a high-spin ground state. Thus by relating sp ecies to spectra, and by examining the consistency of calculated and o bserved spectra, a plausible identification has been made of the trans ient intermediate species in the pathway from the resting state to com pound I of peroxidases.