Compound I of horseradish peroxidase

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. 

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... 

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-... 

The reaction chemistry and electronic characterization of the green adduct (2) are consistent with an oxygen atom covalently bound to an iron(II)-porphyrin radical center [P-OFe (0) ]. The latter has the spectral, magnetic, and redox characteristics of Compound I of horseradish peroxidase (HRP), and the selective stereospecific oxygenase character of the reactive intermediate for cytochrome P-450. Reduction of the green species by one-electron equivalent yields a red species (3, Scheme 1), which has the spectral characteristics and reactivity of Compound II of HRP. 

The results of recent investigations S of model systems provide compelling evidence that stabilized atomic oxygen is present in Compound I and Compound II of horseradish peroxidase. Thus, the combination of tetrakis(2,6-dichlorophenyl)-porphinato-iron(ni) perchlorate (6, Scheme 4-4) with pentafluoro-iodosobenzene, zn-chloroperbenzoic add, or ozone in acetonitrile at -35°C yields a green porphyrin-oxene adduct (7). This species, which has been characterized by spectroscopic, magnetic, and electrochemical methods, cleanly and stereospecifically epoxidizes olefins (>99% exo-norbornene-oxide). 

The debate on whether the pJCa of 8.6 for compound II of horseradish peroxidase (HRP-II) represents the ionization of an iron-bound water molecule continues. Dunford adduces further kinetic evidence that it does while Schejter provides additional n.m.r. evidence that there is no water molecule in the inner co-ordination sphere of the iron(m) atom however, the latter interpretation of the data has been questioned by Vuk-Pavlovi6 and Benko, who suggest that the sixth ligand site of the metal contains what they term a sedentary water molecule. The formation of HRP-I from the reaction of native horseradish peroxidase with HgOa h a rate constant of 2.5 x 10 1 mol s at 25 °C (pH 7.10), with an activation energy of 3.5 1.0 kcal mol". The kinetics of cyanide and fluoride binding to turnip peroxidases Pi and P7 have also been measured. "... 

According to Reichl et al. (2000), the oxidation of pholasin by compound I or II of horseradish peroxidase induces an intense light emission, whereas native horseradish peroxidase shows only a small effect. The luminescence of pholasin by native myeloperoxidase (verdoperoxidase) is diminished by preincubation with catalase, which is interpreted as the result of the removal of a trace amount of naturally occurring H2O2 in the buffer (about 10-8 M) that forms compound I... 

Baek, H.K. and van Wart, H.E., Elementary steps in the reaction of horseradish peroxidase with several peroxides kinetics and thermodynamics of formation of compound 0 and compound I, J. Am. Chem. Soc., 114, 718-725, 1992. 

Figure 4.3. The catalytic cycle of horseradish peroxidase with ferulate as reducing substrate. The rate constants Ki, K2, and K3 represent the rate of compound I formation, rate of compound I reduction, and rate of compound II reduction, respectively.

The rate constants of kn and kx obtained using Eq. (24) reveal that (i) the activity of Fem-TAMLs in bleaching Safranine O (k ) increases more than 10-fold when the tail ethyl groups of la are replaced by fluorine atoms in lk. The rate constant kn for lk equals l(rM 1s 1 at 25°C, a value that corresponds to those found for the reactivity of horseradish peroxidase Compound II... 

Mechanisms of catalase and peroxidase catalysis. Attention has been focused on a series of strikingly colored intermediates formed in the presence of substrates. When a slight excess of H202 is added to a solution of horseradish peroxidase, the dark brown enzyme first turns olive green as compound I is formed, and then pale red as it turns into compound II. The latter reacts slowly with substrate AH2 or with another H202 molecule to regenerate the original enzyme. This sequence of reactions is indicated by the colored arrows in Fig. 16-14, steps a-d. 

Hayashi Y, Yamazaki I (1979) The oxidation-reduction potentials of compound I/compound II and compound II/ferric couples of horseradish peroxidases A2 and C. J Biol Chem 254 101-106... 

Baek HK, Van Wart HE (1989) Elementary steps in the formation of horseradish peroxidase Compound I Direct observation of Compound 0, a new intermediate with a hyperporphyrin spectrum. Biochemistry 28 5714-5719... 

Shintaku M, Matsuura K, Yoshioka S et al (2005) Absence of a detectable intermediate in the Compound I formation of horseradish peroxidase at ambient temperature. J Biol Chem 280 40934-40938... 

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... 

Roberts JE, Hoffman BM, Rutter R et al (1981) Electron double resonance of horseradish peroxidase Compound I. Detection of the porphyrin 7i-cation radical. J Biol Chem 256 2118-2121... 

Penner-Hahn JE, Eble KS, McMurry TJ et al (1986) Structural characterization of horseradish peroxidase using EXAFS spectroscopy. Evidence for Fe=0 ligation in Compounds I and II. J Am Chem Soc 108 7819-7825... 

Filizola, M. and Loew, G.H. (2000) Role of protein environment in horseradish peroxidase compound I formation molecular dynamic stimulation of horseradish peroxidase-HOOH complex, J. Am. Chem. Soc. 122,18-25. 

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