Calculations of the structure and spectra of the putative transient peroxide intermediates of peroxidases

摘要

Peroxidases are oxiclativc metabolizing heme proteins that require hydrogen peroxide to he transformed to the catalylically active Compound I species from the Ferric resting state. Although a peroxide complex with the heme iron has been proposed as a key intermediate in this reaction, this intermediate is too transient to have thus far been definitively characterized. While electronic spectra attributed to it have been observed, in the absence of any addition information, it is not possible from these reported spectra alone to identify the transient species that give rise to them. Results of previous molecular dynamic (MD) simulations of a peroxide complex with eytoehronic C pcroxidase (CCP) indicated that peroxide does indeed form a stable complex with the heme unit and hinds in a nonsyinmetric end-on mode in which the oxygen atoms systematically exchange places as ligands lor the heme iron. To further assess this mode of binding, ab initio quantum chemical methods have been used to determine the optimized geometry and stability of a peroxide complex with a model heme pemxidase. The results obtained provide confirmation of the important characteristics of this transient intermediate found in the previous MD simulations. Specifically, two equivalent stable minima were identified with a binding energy of about lOkcal mole"' in which the peroxide hinds in an end-on mode with alternative oxygen atoms as the Fc ligand and a small energy barrier between them. To further explore evidence for this species, the semicmpirieal INDO/ROHF/CI quantum chemical method has been used to calculate its electronic: spectra and the results were compared with the observed spectra for the transient species of horse radish pcroxidase and a mutant of it. Comparison of the calculated spectra of the neutral (HOOH) and anionic (OOH) form of the peroxide intermediate with the two experimental data, indicated that both had been observed, the unionic form in the wild type and the neutral form in the mutant horse radish pcroxidase. Thus, these calculations, by providing the missing correspondence 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 intermediate.