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 the electronic structure and spectra of two candidate peroxide intermediates of model peroxidases. In the enzymatic cycle of this family of oxidative metabolizing heme proteins, hydrogen peroxide is required to transform the ferric resting state to the catalytically active, ferryl Fe=O, compound I species. While a peroxide complex has been proposed as a key intermediate in this reaction, this intermediate species is too transient to have thus fat been definitively characterized. Electronic spectra observed prior to compound I formation during the reaction of H2O2 with both wild type and the R38L mutant of horseradish peroxidase C (HRP-C) have been attributed to this intermediate. There are, however, significant qualitative differences in these spectra in the 300-450-nm region, with a ''hyper-Soret'' observed in one and a normal Soret, not very different from the resting stale, found in the other. In the absence of any additional information, it is not possible from these reported spectra alone to identify the species that give rise to them or to understand these differences, In order to identify the origin of these spectra and their differences, we have calculated the electronic structure and spectra of two possible forms of the peroxide intermediate of model peroxidases, one with a neutral peroxide and the other with an anionic form (OOH-) as the heme Fe ligand. Formation of the anion is possible by proton transfer to a nearby histidine residue, already implicated in compound I formation. A comparison of the calculated spectra for these two transient species indicates them to be quite distinct. Comparisons of the two spectra with those experimentally observed suggest that the ''hyper-porphyrin'' spectrum observed in the wild type (WT) HRP-C experiments originates from the OOH- form of this transient intermediate in a Tow-spin ground state, while the normal Soret observed in the R38L HRP-C mutant experiment originates from the neutral peroxide form in a high-spin ground state. Thus by relating species to spectra, and by examining the consistency of calculated and observed spectra, a plausible identification has been made of the transient intermediate species in the pathway from the resting state to compound I of peroxidases.