Formation and Decay of Hydroperoxo-Ferric Heme Complex in Horseradish Peroxidase Studied by Cryoradiolysis

Abstract

Using radiolytic reduction of the oxy-ferrous horseradish peroxidase (HRP) at 77 K, we observed the formation and decay of the putative intermediate, the hydroperoxo-ferric heme complex, often called "Compound 0." This intermediate is common for several different enzyme systems as the precursor of the Compound I (ferryl-oxo -cation radical) intermediate. EPR and UV-visible absorption spectra show that protonation of the primary intermediate of radiolytic reduction, the peroxo-ferric complex, to form the hydroperoxo-ferric complex is completed only after annealing at temperatures 150 -180 K. After further annealing at 195-205 K, this complex directly transforms to ferric HRP without any observable intervening species. The lack of Compound I formation is explained by inability of the enzyme to deliver the second proton to the distal oxygen atom of hydroperoxide ligand, shown to be necessary for dioxygen bond heterolysis on the "oxidase pathway," which is nonphysiological for HRP. Alternatively, the physiological substrate H 2 O 2 brings both protons to the active site of HRP, and Compound I is subsequently formed via rearrangement of the proton from the proximal to the distal oxygen atom of the bound peroxide.Horseradish peroxidase (HRP) 1 is a highly characterized heme enzyme that has historically served as a paradigm for evaluating the rules of formation of redox active heme-oxygen intermediates, including the ferryl-oxo heme complexes commonly known as "Compound I" and "Compound II." A detailed mechanism for Compound I formation in the reaction of H 2 O 2 with peroxidases was formulated by Poulos and Kraut on the basis of structural analysis of cytochrome c peroxidase active center (1) and has been successfully applied to other enzymes (2-5), as shown in Reaction 1.The essential features of this mechanism also provide a framework for the analysis of new systems, which involve formation of redox-active heme-oxygen intermediates (6 -8). A crucial point in the formation of the ferryl-oxo species is the second protonation of the distal oxygen atom of the bound hydroperoxo species, followed by the heterolytic scission of O-O bond. The importance of these elementary steps has been confirmed by quantum chemical methods (9 -11). The vast amount of kinetic data obtained in mutagenesis studies of the distal pocket (5, 12-15) provide an additional support for the evaluation of the second protonation of the distal oxygen as the most important and, in some cases, the rate-limiting step in catalysis.Despite many efforts, experimental characterization of this step, as well as the detailed properties of the hydroperoxo-ferric heme (Fe 3ϩ P-OOH Ϫ ) complex in peroxidases, is still far from complete. One reason is the low stability of such complexes (16), because their transient character leads to a lack of accumulation during the reaction course. Several studies of these intermediates in wild-type and mutant HRP (17-19) and microperoxidase (20 -22) by means of stopped-flow methods have produced absorption spectra that could b...

Absence of a Detectable Intermediate in the Compound I Formation of Horseradish Peroxidase at Ambient Temperature*

“…Although the current data do not provide direct evidence for the Fe 3ϩ -HOOH species, the absence of the Fe 3ϩ -OOH species with a detectable lifetime is rather consistent with the recent proposals on the activation of H 2 O 2 by heme peroxidases (29,32). It is explained that the lifetime of the Fe 3ϩ -OOH species in heme peroxidases is short, because the distal His becomes an unstable imidazolium cation after accepting a proton from H 2 O 2 at the N⑀ site because of the conserved Asn residue (Asn-70 in HRP), stabilizing the N␦ proton (19ϳ21).…”

“…A clue to understand the conflicting information is proposed by Denisov et al (29) and Egawa et al (32). To explain the observation that the Fe 3ϩ -OOH species created by the cryoradiolysis of the ferrous oxygenbound HRP cannot convert to compound I, Denisov et al point out that the species lacks a proton required for the O-O bond heterolysis (29 (32,33), which was first identified in the reaction of H 2 O 2 with a mutant of metmyoglobin (34).…”

“…The subsequent theoretical study assigned the spectrum of the state to the ferric peroxide anion complex (Fe 3ϩ -OOH) (27). The cryoradiolysis experiment on the ferrous oxygen-bound form of HRP identifies the Fe 3ϩ -OOH species in the low spin form, which however, cannot convert to compound I and directly returns to the resting state upon the temperature increase (28,29). A similar cryoradiolysis experiment based on x-ray crystallographic analysis, however, detected the formation of compound I (30).…”

“…To explain the observation that the Fe 3ϩ -OOH species created by the cryoradiolysis of the ferrous oxygenbound HRP cannot convert to compound I, Denisov et al point out that the species lacks a proton required for the O-O bond heterolysis (29 (32,33), which was first identified in the reaction of H 2 O 2 with a mutant of metmyoglobin (34). They detected the Fe 3ϩ -OOH species only in alkaline conditions and explain that the absence of the N␦ proton from the distal His decelerates the conversion of the Fe 3ϩ -OOH species (Fig.…”

The role of Arginine 38 in horseradish peroxidase enzyme revisited: A computational investigation

“…The above residues are supposed to play a relevant role (3). As a matter of fact, Cpd0, a ferric-hydroperoxide anion, has been shown both experimentally (4)(5)(6)(7)(8)(9) and theoretically (10)(11)(12)(13)(14)(15) to be formed via deprotonation of H 2 O 2 by His42 acting as a base.…”