Haeme peroxidases, reaction

The haem peroxidases are a superfamily of enzymes which oxidise a broad range of structurally diverse substrates by using hydroperoxides as oxidants. For example, chloroperoxidase catalyses the regioselective and stereoselective haloge-nation of glycals, the enantioselective epoxidation of distributed alkenes and the stereoselective sulfoxidation of prochiral thioethers by racemic arylethyl hydroperoxides [62]. The latter reaction ends in (i )-sulfoxides, (S)-hydroperoxides and the corresponding (R)-alcohol, all In optically active forms. 

Peroxidases [19] are by far the best-characterised of the enzymes that utilise ferryl iron (especially by spectroscopists, see section 4). Fig. 2 shows the now well-established reaction cycle of the haem peroxidases and catalases. Ferric... 

Fig. 2. Mechanism of haem peroxidases and catalases. Main reaction pathways and oxidation state of iron in haem peroxidases and catalases. Enzymes illustrated LPO, lactoperoxidase MPO, myeloperoxidase TPO, thyroid peroxidase Cat., catalase CCP, cytochrome c peroxidase HRP, horseradish peroxidase. Fe(III), Ferric (met) form of enzyme Compounds I, II and III enzyme intermediates.

Haem peroxidases are globular proteins with an iron-porphyrin complex as a prosthetic group. These enzymes are widespread among prokaryotes and eukaryotes. They catalyze the oxidation of substrates by organic peroxides or hydrogen peroxide. During the past decades, considerable scientific effort has been put into elucidation ofthe mechanisms of reactions catalyzed by these enzymes. Pulse radiolysis technique has made an important contribution by providing information on the redox states of the enzymes and their interconversion, as well as on the properties ofthe free radical intermediates involved [23]. 

The best known and most nsefnl of the chemiluminescent reactions involving electron transfer is the oxidation of luminol (3.100) or its derivatives in alkaline medium. The oxidant can be hydrogen peroxide, sodium ferricyanide or hypochlorite, usually with a catalyst that can be a transition metal ion, such as Cu " Co +, Fe + and Mtf+, or haem and haemproteins, e.g. peroxidases. The reaction mode is shown in Figure 3.22.4"... 

Cyt c is associated with the outer surface of the inner mitochondrial membrane. Phospholipids induce conformational changes in the protein and, in certain instances, the haem can convert to the high spin (S = 5/2) form, indicative of a weakening of the ligand field caused by displacement of the sixth ligand (Met-80). This has been associated with the detection of lipid radicals by direct EPR (at 11 K).65 Indeed, peroxidase-type activity is also evident in the reaction of cyt c with lipid hydroperoxides, as studied by spin trapping in conjunction with HPLC and MS.66... 

Some haem proteins undergo a peroxidase cycle like that in Fig. 2, but very slowly, e.g. myoglobin converting to ferryl myoglobin. In this case the reaction is presumed not to have a physiological role. Indeed, it can have deleterious consequences (see section 5). 

Haem proteins that react with oxygen also utilise ferryl intermediates. Fig. 4 compares the (proposed) reaction mechanisms of cytochrome oxidase and cytochrome P-450 with those of peroxidases and catalases. As can be seen, several of the reaction intermediates have the same oxidation states (although the protonation steps and stage at which H2O is released may be different). However, in contrast to peroxidases, oxidases must react with molecular oxygen, and this requires a reaction cycle that includes Fe11. 

With ferryl myoglobin, in contrast to peroxidases, the reactions of the protein free radicals and that of the ferryl haem can be considered as uncoupled from each other. The protein has not been designed to form a cation radical for a specific reaction therefore not only is more than one cation free radical generated, but there is no control over their subsequent reactions. A similar situation can be observed in cytochrome c peroxidase mutants that have lost tryptophan-191. A different amino-acid free radical is still formed that is less stable. Indeed, even in the presence of tryptophan-191, small amounts of other free radicals are formed [237] this is further evidence that even in enzymes it is difficult to exclusively control free radical reactions. 

Cytochrome C peroxidase (CCP) is a 294-residue haem-containing enzyme which catalyses the reduction of peroxides by ferrocytochrome c. The exact function of CCP is somewhat obscure but it probably serves to protect yeast mitochondria from the toxic build-up of peroxides. The overall scheme for the enzymatic reaction is given below (Pelletier and Kraut, 1992). In step 1, Eq. (6) CCP reacts with hydrogen peroxide to... 

Figure 13.9(b) also illustrates analogies between CcO and peroxidases and catalases, which we discuss next, in terms of both oxygen—oxygen bond cleavage chemistry and the nature of the products of the reactions. In CcO, the enzyme extracts three electrons from metals in the active site — two from haem 03 as it goes from the +2 to the +4 state and one from Cub as it is oxidized from cuprous to cupric — and one electron from a redox-active protein side... 

The reactions of peroxidases have been mimicked, using synthetic iron(III) and manganese(III) porphyrins catalysts as models for the haem prosthetic group, and these simplified systems have been used to obtain mechanistic information about the oxidations. " At York we have been investigating the oxidation of phenols by oxoiron(IV) and oxomanganese(IV) porphyrin models for HRP II in aqueous solution. This research has shown that, at pH 7.6, oxidation proceeds by hydrogen atom abstraction by the oxometallo(IV) species from the phenol rather than electron transfer from the phenolate anion. Preliminary studies with commercial azo dyes has revealed that these are also substrates for oxoiron(IV)porphyrins. In this paper we report the results from our investigations of the mechanism of oxidation of l-phenylazo-2-naphthol-6-sulfonates by oxoiron(IV) tetra(2,6-dichloro-3-sulfonatophenyl)porphyrin (1) in aqueous solution. 

Trace metals, particularly copper, cobalt, and iron, greatly increase the rate of LO and influence the direction of peroxide decomposition [72], These metals function both to reduce the induction period and increase reaction rate by decomposing hydroperoxides. Trace levels of these catalysts, e.g., as little as 0.3 ppm iron or 0.01 ppm copper, will result in prooxidant effects [73]. Iron may exist in foods in the free form or as a part of an enzyme (contain organically bound haem, Fe+ or haemin, Fe+ ). Enzymes containing haematin compounds include catalase and peroxidase (plant tissues) and haemoglobin, myoglobin, and cytochrome C (animal tissues). While heat treatment results in denaturation of the enzymes, it frees the iron to greatly enhance its catalytic properties. This is particularly relevant in the formation of warmed-over off-flavor in cooked meats. 

So far, examples to illustrate experimental methods for following the time course of the approach to steady states and of their kinetic interpretation have been restricted to enzymes which do not have a natural chromophore attached to the protein although reference has been made to the classic studies of Chance with peroxidase (see p. 142). Qearly the application of these techniques to the study of enzymes with built in chromophores, such as the prosthetic groups riboflavine, pyridoxal phosphate or haem, contributed considerably to the elucidation of reaction mechanisms. However, the progress in the identification of the number and character of intermediates depended more on the improvements of spectral resolution of stopped-flow equipment than on any kinetic principles additional to those enunciated above. This is illustrated, for instance, by the progress made between the first transient kinetic study of the flavoprotein xanthine oxidase by Gutfreund Sturtevant (1959) and the much more detailed spectral analysis of intermediates by Olson et al. (1974) and Porras, Olson Palmer (1981). 

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