Peroxidase hydrogen peroxide complex

The cytochrome-c peroxidase test offers the advantages of stability of the peroxidase-hydrogen peroxide complex and high specificity for its hydrogen donor, cytochrome c. 

Fig. 1. This illustrates the zero-order reaction kinetics of the oxidation of ascorbic acid by horseradish peroxidase-hydrogen peroxide complex. The disappearance of hydrogen peroxide upon the addition of 3 mM ascorbic acid is recorded by means of the platinum microelectrode (Expt. 574e).

Peroxidases are found in milk and in leukocytes, platelets, and other tissues involved in eicosanoid metabolism (Chapter 23). The prosthetic group is protoheme. In the reaction catalyzed by peroxidase, hydrogen peroxide is reduced at the expense of several substances that will act as electron acceptors, such as ascorbate, quinones, and cytochrome c. The reaction catalyzed by peroxidase is complex, but the overall reaction is as follows ... 

Catalase was found to form an intermediate compound in the presence of hydrogen peroxide (Chance, 69). The spectrum was measured from 380-430 nqi and is slightly shifted toward the visible as compared with free catalase. The complex shows no similarities to cyan-catalase or the compound formed when peroxide is added to azide catalase. Its formation is very rapid, the bimolecular velocity constant having a value of about 3 X 107 M.-1 sec.-1. In the absence of added hydrogen donors, the complex decomposes slowly according to a first order reaction with a velocity constant of about 0.02 sec.-1. This catalase complex thus resembles the green primary hydrogen peroxide complex of peroxidase. 

In a more recent series of experiments, George (97) has shown that the secondary hydrogen peroxide complexes of horseradish peroxidase and cytochrome-c peroxidase can be titrated with ferroevanide or ferrous ions and also appear to take part in a one oxidizing equivalent reduction to the ferric form of the enzyme. In this important chemical property they thus resemble the metmyoglobin complex in spite of marked spectroscopic differences in the visible region of the spectrum (Keilin and Hartree, 48). If the peroxide molecule is not a component part of the structure it... 

In an earlier study Brown" reported that both horseradish peroxidase and beef liver catalase were electroinactive polarographically under anaerobic conditions. However, under conditions which gave rise to the primary hydrogen peroxide complexes of these proteins, a reductive response was observed. Brown attributed these responses to the direct reduction of the respective hydrogen peroxide complexes of peroxidase and catalase." In one other report catalase was reported to be electroinactive at the dropping mercury electrode. ... 

The reactions of the lactoperoxidase-hydrogen peroxide complexes with ascorbic acid and with p3rrogallol were investigated by Chance (95) and allow us to compare the h values of two different peroxidases with the same donor. Some of the rate constants of the peroxidase-HtOt S3rstems are listed in Table IX. 

In the most common method for chemiluminescent immunoassay (GLIA), after the immunological reaction and any necessary separation steps, the labeled compounds or complexes react with an oxidizer, eg, hydrogen peroxide, and an enzyme, eg, peroxidase, or a chelating agent such as hemin or metal... 

A method of detecting herbicides is proposed the photosynthetic herbicides act by binding to Photosystem II (PS II), a multiunit chlorophyll-protein complex which plays a vital role in photosynthesis. The inhibition of PS II causes a reduced photoinduced production of hydrogen peroxide, which can be measured by a chemiluminescence reaction with luminol and the enzyme horseradish peroxidase (HRP). The sensing device proposed combines the production and detection of hydrogen peroxide in a single flow assay by combining all the individual steps in a compact, portable device that utilises micro-fluidic components. 

Heme (C34H3204N4Fe) represents an iron-porphyrin complex that has a protoporphyrin nucleus. Many important proteins contain heme as a prosthetic group. Hemoglobin is the quantitatively most important hemoprotein. Others are cytochromes (present in the mitochondria and the endoplasmic reticulum), catalase and peroxidase (that react with hydrogen peroxide), soluble guanylyl cyclase (that converts guanosine triphosphate, GTP, to the signaling molecule 3, 5 -cyclic GMP) and NO synthases. 

In the reaction of luminol, hydrogen peroxide, and horse radish peroxidase 122> the chemiluminescence intensity is proportional to the square of luminol radical concentration. The lifetime of these luminol radicals was found by ESR techniques to be about 10 sec. Titration studies revealed that luminol acts as two-electron donor during the reduction of a hydrogen peroxide-horseradish peroxidase complex. The enzyme is not involved in the reaction step leading directly to light emission. This step is formulated as... 

How does nature prevent the release of hydrogen peroxide during the cytochrome oxidase-mediated four-electron reduction of dioxygen It would appear that cytochrome oxidase behaves in the same manner as other heme proteins which utilize hydrogen peroxide, such as catalase and peroxidase (vide infra), in that once a ferric peroxide complex is formed the oxygen-oxygen bond is broken with the release of water and the formation of an oxo iron(IV) complex which is subsequently reduced to the ferrous aquo state (12). Indeed, this same sequence of events accounts for the means by which oxygen is activated by cytochromes P-450. 

Answers to these questions were initiated over a decade ago during our studies on catalase (CAT) and horseradish peroxidase (HRP) (30). Both native enzymes are ferric hemoproteins and both are oxidized by hydrogen peroxide. These oxidations cause the loss of two electrons and generate active enzymatic intermediates that can be formally considered as Fe + complexes. 

In order to relate structure and function at a more direct level, it is necessary to focus on systems which have better characterized structure than the complex membrane bound proteins like cyt c oxidase. One particularly useful paradigm in this context is the cytochrome c-cytochrome c peroxidase couple [18]. Cep is not involved in electron transport, per se its apparent function [19] is detoxification of hydrogen peroxide via the sequence H2O2 -I- cep Fe(III) -> H2O -I- cep Fe(IV) O (protein) compound ES ... 

The ability of HRP to degrade the plant hormone indole-3-acetic acid (lAA) in the absence of hydrogen peroxide was noted as early as 1955 (136). Plant peroxidases are now known to be of major importance in the metabolism of lAA (137) (note that they are often referred to as indole acetic acid oxidases in the older literature). The mechanism of lAA oxidation by HRP C is complex and has been studied experimentally in great detail by several groups (23, 137). Reaction products include indole-3-methanol, indole-3-aldehyde, and 3-methylene oxin-dole, which is probably a nonenzymatic conversion product of indole-3-methylhydroperoxide. The most important developments in this area have been reviewed (23). 

---