Resonance Raman Spectroscopy Reveals pH-Dependent Active Site Structural Changes of Lactoperoxidase Compound 0 and Its Ferryl Heme O–O Bond Cleavage Products

The first step in the enzymatic cycle of mammalian peroxidases, including lactoperoxidase (LPO), is binding of hydrogen peroxide to the ferric resting state to form a ferric-hydroperoxo intermediate designated as Compound 0, the residual proton temporarily associating with the distal pocket His109 residue. Upon delivery of this “stored” proton to the hydroperoxo fragment, it rapidly undergoes O–O bond cleavage, thereby thwarting efforts to trap it using rapid mixing methods. Fortunately, as shown herein, both the peroxo and the hydroperoxo (Compound 0) forms of LPO can be trapped by cryoradiolysis, with acquisition of their resonance Raman (rR) spectra now permitting structural characterization of their key Fe–O–O fragments. Studies were conducted under both acidic and alkaline conditions, revealing pH-dependent differences in relative populations of these intermediates. Furthermore, upon annealing, the low pH samples convert to two forms of a ferryl heme O–O bond-cleavage product, whose ν(Fe═O) frequencies reflect substantially different Fe═O bond strengths. In the process of conducting these studies, rR structural characterization of the dioxygen adduct of LPO, commonly called Compound III, has also been completed, demonstrating a substantial difference in the strengths of the Fe–O linkage of the Fe–O–O fragment under acidic and alkaline conditions, an effect most reasonably attributed to a corresponding weakening of the trans-axial histidyl imidazole linkage at lower pH. Collectively, these new results provide important insight into the impact of pH on the disposition of the key Fe–O–O and Fe═O fragments of intermediates that arise in the enzymatic cycles of LPO, other mammalian peroxidases, and related proteins

Resonance Raman Studies of Hydroperoxidase Intermediates

Heme proteins, which are ubiquitous in physiological systems, are key components involved in many biological processes, having several different biological functions. For example, myoglobin (Mb) and hemoglobin (Hb) are heme proteins whose biological functions are to reversibly bind an oxygen molecule in order to store and transport molecular oxygen within blood and muscle tissues; cytochromes P-450 (CYPs) play a critical role in activation and cleavage of molecular oxygen to generate redox intermediates which catalyze hydroxylation or epoxidation of their substrates 4; peroxidases mainly participate in an oxidation reaction of organic and inorganic compounds by utilizing hydrogen peroxide as a primary substrate; catalases (CATs) serve a key biological role to eliminate toxic hydrogen peroxide by catalyzing the conversion of hydrogen peroxide to water and oxygen molecules..

Resonance Raman studies of hydroperoxidase intermediates

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