CALCULATIONS OF THE STRUCTURE AND SPECTRA OF THE PUTATIVE TRANSIENT PEROXIDE INTERMEDIATES OF PEROXIDASES

Peroxidases are oxidative metabolizing heme proteins that require hydr ogen peroxide to be transformed to the catalytically active Compound I species from the ferric resting state. Although a peroxide complex wi th the heme iron has been proposed as a key intermediate in this react ion, this intermediate is too transient to have thus far been definiti vely characterized. While electronic spectra attributed to it have bee n observed, in the absence of any addition information, it is not poss ible from these reported spectra alone to identify the transient speci es that give rise to them. Results of previous molecular dynamic (MD) simulations of a peroxide complex with cytochrome C peroxidase (CCP) i ndicated that peroxide does indeed form a stable complex with the heme unit and binds in a nonsymmetric end-on mode in which the oxygen atom s systematically exchange places as ligands for the heme iron. To furt her assess this mode of binding, ab initio quantum chemical methods ha ve been used to determine the optimized geometry and stability of a pe roxide complex with a model heme peroxidase. The results obtained prov ide confirmation of the important characteristics of this transient in termediate found in the previous MD simulations. Specifically, two equ ivalent stable minima were identified with a binding energy of about 1 0 kcal mole(-1) in which the peroxide binds in an end-on mode with alt ernative oxygen atoms as the Fe ligand and a small energy barrier betw een them. To further explore evidence for this species, the semiempiri cal INDO/ROHF/CI quantum chemical method has been used to calculate it s electronic spectra and the results were compared with the observed s pectra for the transient species of horse radish peroxidase and a muta nt of it. Comparison of the calculated spectra of the neutral (HOOH) a nd anionic (OOH-) form of the peroxide intermediate with the two exper imental data, indicated that both had been observed, the anionic form in the wild type and the neutral form in the mutant horse radish perox idase, Thus, these calculations, by providing the missing corresponden ce between species and spectra, have allowed the identification of the transient intermediate species in the pathway from the resting state to Compound I of peroxidases as the long proposed peroxide intermediat e. (C) 1997 Elsevier Science B.V.