Compound I of peroxidase

Fujii, H., T. Yoshimura, and H. Kamada (1997). Imidazole, and p-nitrophenolate complexes of oxo-iron(IV) porphyrin-cation radicals as models for compounds I of peroxidase and catalase. Inorg. Chem. 36,6142-6143. 

Fig. 2. Different routes for the generation of activated bleomycin. The formal oxidation state (V) of the bleomycin-iron-oxo species (perferryl complex) is two oxidant equivalents above BLM-Fe ", but one oxidant equivalent might be located on the ligand, as in Compound I of peroxidases, with an iron" -oxo-ligand radical cation structure.

On the basis of many spectroscopic measurements, the species was characterized to be an oxo-ferryl porphyrin K-cation radical (1), identical to compound I of peroxidases and catalases. Because of its high reactivity, introduction of sterically hindered groups at the ortho-positions of the phenyl rings are required to observe 1 as relatively stable species at low temperature. Alternatively, Nakamoto has reported the formation of compound I of Fe(OEP) and Fe(TPP) by laser irradiation to oxy forms of the complexes in oxygen matrices at 30 K [51, 52]. Because of the formation of compound I in the matrices, characterization of less sterically hindered porphyrin complexes can be made. 

N—Fe(IV)Por complexes. Oxo iron(IV) porphyrin cation radical complexes, [O—Fe(IV)Por ], are important intermediates in oxygen atom transfer reactions. Compound I of the enzymes catalase and peroxidase have this formulation, as does the active intermediate in the catalytic cycle of cytochrome P Q. Similar intermediates are invoked in the extensively investigated hydroxylations and epoxidations of hydrocarbon substrates cataly2ed by iron porphyrins in the presence of such oxidizing agents as iodosylbenzene, NaOCl, peroxides, and air. 

Mn-dependent peroxidase differs from lignin peroxidase in that it utilizes Mn (II) as the main substrate (74). The oxidized manganese ion, Mn (III), carries out the oxidation of organic molecules. Compound I of Mn-dependent peroxidase is able to oxidize Mn (II) to Mn (III) as well as some phenolic compounds the compound II can only oxidize Mn (II) (14) ... 

On the contrary, in cytochrome c peroxidase, the intermediate is a (porphy-rin)Fe(IV)=0 complex with a free radical derived from the one-electron oxidation of an amino acid residue in the vicinity of the heme [10], The second step of the HRP catalytic cycle is the one-electron oxidation by compound I of HRP substrates that are generally electron-rich aromatic compounds. This leads to the second intermediate, called compound II, of the catalytic cycle, which is a (porphyrin) Fe(IV)=0 complex. The one-electron reduction of compound II by HRP substrates regenerates HRP in its resting iron(III) state (Figure 5a). 

Hydroperoxo-ferric intermediate, termed also Compound 0, is the immediate precursor of the main catalytic intermediate Compound I in peroxidase enzymatic cycle. Attempts to study this intermediate directly in reactions of hydrogen peroxide with HRP using fast kinetic methods have been inconclusive, possibly because it is not accumulated in sufficient concentrations.90,91 However, Compound 0 could be prepared and studied by EPR and optical absorption spectroscopy via cryoreduction of... 

Dolphin D, Forman A, Borg DC et al (1971) Compounds I of catalase and horseradish peroxidase 7i-cation radicals. Proc Natl Acad Sci USA 68 614—618... 

Fig. 3. Nature of free radical associated with compound I in peroxidases/catalases. Structure of first intermediate, following peroxide addition to ferric peroxidases and catalases. Boxes denote porphyrin ring. The amino-acid free radicals are depicted as protonated (tryptophan) and deprotonated (tyrosine), although this is yet to be conclusively determined.

Fig. 8. MCD spectra of ferryl iron. Low-temperature (50 or 100K) MCD spectra of ferryl iron in different proteins HRPCII, horse-radish peroxidase compound II HRPCX, horse-radish peroxidase compound X YCCP, yeast cytochrome c peroxidase compound I PsCCP, compound I of the dihaem cytochrome c peroxidase from Pseudomonas aeruginosa-, Mb pH 3.5, ferryl myoglobin formed at pH 3.5 MbpD9.0, the same compound found at pD9.0. Note the similarity of all the spectra with the exception of the alkaline form of ferryl myoglobin. Reprinted with permission from Cheesman, M.R., Greenwood, C. and Thomson, A.J. (1991) Adv. Inorg. Chem. 36, 201-255.

As with the optical spectra the presence of a porphyrin radical cation dominates the MCD spectra of compound I of catalase [177] and horseradish peroxidase [29,177], These spectra are quite distinct from that shown in Fig. 8, but vary similar to each other. This suggests that their radical cations must have similar structures [10]. 

On reaction with a stoichiometric amount of hydroperoxide, catalase and horseradish peroxidase are converted to a green colored intermediate. Compound I (5). The chemical nature of Compound I has been extensively debated since its discovery by Theorell 59). Recently, Dolphin et al. 60) have demonstrated that upon one-equivalent oxidation several metalloporphyrins are converted to stable porphyrin jr-cation radicals, the absorption spectra of which possess the spectral characteristics of Compound I, namely, a decreased Soret w-n transition and an appearance of the 620-670-nm absorption bands. Since Moss et al. 61) proposed the presence of Fe(IV) in Compound I of horseradish peroxidase from Mossbauer spectroscopic measurements, it is attractive to describe Compound I as Fe(IV)-P, where P is a porphyrin w-cation radical. Then, Compound I and Compound ES become isoelectronic. Both contain Fe(IV) and a radical the former as a porphyrin radical (P ) and the latter as a protein radical (R ). Then the reaction cycles of horseradish and cytochrome c peroxidases may be compared as shown in Fig. 4. 

HRP exhibits the typical peroxidase fold and active site stractme as shown in Figme 6(b). As with CcP, the native resting form of HRP-C contains a five-coordinate, high-spin Fe + heme. Compound I of HRP contains two oxidizing equivalents, one as oxyferryl (Fe" +-0) and the other as porphyrin radical. " A transient Trp r-cation radical has been detected in the Phe221Trp mutant of HRP-C compound... 

Cytochrome P-450, which is the most extensively studied of the monooxygenase proteins, has a heme-iron active center with an axial thiol ligand (a cysteine residue). However, most chemical model investigations use simple iron(III) porphyrins without thiolate ligands. As a result, model mechanisms for cytochrome P-450 invoke a reactive intermediate that is formulated to be equivalent to Compound I of horseradish peroxidase, (por+-)Fe =0, with a high-potential porphyrin cation radical. Such a species would be reduced by thiolate, and therefore is an unreasonable formulation for the reactive center of cytochrome P-450. 

To clarify the mechanism of reaction of P-450, it is crucial to characterize the reactive intermediates in the rate-determining step. Definitive evidence for an electron-transfer mechanism (C in Scheme 2) for the 7V-demethylation of N,N-dimethylanilines has been obtained by direct observation of the reduction of the high-valent species responsible for P-450 catalysis [96]. For peroxidase, an oxoferryl porphyrin 7r-radical cation, compound I ([(P)Fe =0] "), has been well characterized as the species equivalent to the proposed active intermediate of P-450 [97-103]. Compound I of horseradish peroxidase (HRP) can be readily generated by chemical oxidation of HRP [100-103]. The involvement of the electron-transfer process of compound I in the oxidation of several amines catalyzed by HRP was... 

This is usually isolated from yeast, and has a molecular weight of 53 000 with one heme b. It catalyzes the oxidation of ferrocytochrome c by hydrogen peroxide. It is the first peroxidase for which the structure has been determined. The imidazole axial ligand is His-174, while Arg-48, Trp-51 and His-52 provide distal catalytic groups. The mechanism involves nucleophilic attack of peroxide on the Fe, loss of the ROOH proton to the imidazole of His-52, and transfer of this proton to the leaving RO group. Compound I of cytochrome c peroxidase is red, and differs from HRPI in that the additional oxidizing equivalent is on a protein residue. ESR and ENDOR spectra have been interpreted in terms of a methionine-centred free radical. One possibility is that a ferryl porphyrin cation radical is formed with cytochrome c peroxidase (it is attractive to assume that this would be common to all peroxidases), but that cytochrome c peroxidase has a readily oxidizable substrate which reduces the porphyrin radical. ... 

Fig. 4.78. Role of the distal histidine in the "pull" mechanism for the cleavage of the dioxygen bond and creation of the high-valent iron-oxo porphyrin it cation radical (Compound I) in peroxidases.

Altschul et al. (1, 2) originally discovered that cytochrome c peroxidase reacts with a stoichiometric amount of hydroperoxide to form a red peroxide compound, which will be referred to hereafter as Compound ES. It has a distinct absorption spectrum, as shown in Fig. 2. The formation of Compound ES from the enzyme and hydroperoxides is very rapid (fci > 10 10 sec"M. No intermediate, which precedes Compound ES, has been thus far detected. In the absence of reductants, or S2, Compound ES is highly stable. The rate constant of its spontaneous decay is of the order of 10 sec 22). The primary peroxide compound (Compound I) of horseradish peroxidase decays much faster at a rate of 10 sec (6). This unusual stability of Compound ES allows one to determine various physical and chemical parameters quantitatively and reliably. Titrations of Compound ES with reductants such as ferrocjHio-chrome c Iff, 20) and ferrocyanide 18, 34) have established that Compound ES is two oxidizing equivalents above the original ferric nnzyme. The absorption spectrum of Compound ES is essentially identical to that of Compound II of horseradish peroxidase which contains one oxidizing equivalent per mole in the form of Fe(IV). In addition, EPR examinations have revealed that Compound ES contains a stable free radical, the spin concentration of which is approximately one equivalent per mole (Fig. 3). Therefore, it is reasonable to conclude that two oxidiz-... 

Both oxidizing equivalents of the hydroperoxide are incorporated into compound I, through an oxygen-atom transfer process ". A free radical is generated elsewhere in the molecule on amino acid residue(s) in the case of yeast cytochrome c peroxidase" and at a site strongly coupled to the iron in horseradish peroxidase . (Compound I of yeast cytochrome c peroxidase is called complex ES in earlier literature.) EPR results on horseradish peroxidase are interpreted in terms of a porphyrin rr-cation radical for compound I . Thus, EPR data prove that one oxidizing equivalent obtained from the hydroperoxide is a free radical species"" ... 

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