Resonance Raman studies of hydroperoxidase intermediates
Abstract
Heme proteins, containing iron protoporphyrin or related prosthetic groups, are key components in many different biological processes. The main factors that lead to the differences in the functional nature of these proteins include the nature and number of endogenous axial ligands presented to the heme by the associated polypeptide and the interactions between the heme peripheral substituents and the surrounding protein residues; these interactions manipulate the reduction potential and ligand binding properties of the heme. Peroxidases and catalases are members of the hydroperoxidase class of enzymes, utilizing H2 O2 as a primary substrate to catalyze a variety of oxidation reactions. Horseradish peroxidase (HRP) and other plant peroxidases employ heme b (protoheme), while the mammalian peroxidase, lactoperoxidase (LPO), utilize heme 1, as prosthetic groups; all of these enzymes are linked to histidine which acts as the fifth ligand. Bovine liver catalase (BLC) is a heme b containing catalase enzyme where the heme iron is coordinated to the phenolate of a tyrosyl residue, serving as the fifth ligand. Reaction of the ferric resting state peroxidases with H2 O2 generates a fleeting hydroperoxo species, which heterolytically cleaves to form several intermediates bearing highly oxidized heme groups, including the so-called Compound I and Compound II species, which act as the primary oxidants in reactions with substrates. While these intermediates have been stabilized and spectroscopically characterized in solutions, the initial hydroperoxo product decays too rapidly to reach detectable levels in solution phase reactions and must be studied using cryoradiolytic methods at 77K. In the present work resonance Raman spectroscopic methods are coupled with these cryoradiolytic techniques to provide a spectroscopic characterization of the hydroperoxo intermediates generated for the enzymes, with the spectra of the so-called Compound III (dioxygen adduct) precursors also being acquired. Attempts are also to acquire the RR spectra of intermediates following O-O bond cleavage that arises during controlled annealing procedures.
This paper has been withdrawn.