Horseradish peroxidases, porphyrin

Figure 5. Comparison of the optical absorption spectra of cobalt(III) porphyrin ir-cation radical species with those of catalase compound / and horseradish peroxidase compound L The ground states of the bromide and perchlorate species are 2A lu and 2Agu, respectively.

FIGURE 4. Scheme for the photooxidation of porphyrin (P) by Mg horseradish peroxidase... 

Peroxidases are haem proteins that are activated from the ferric state to one-electron oxidants by H202. They play a significant role in the generation of radicals from xenobiotics. The compound I state contains one oxidising equivalent as an oxoferryl-haem entity and the second as a porphyrin -radical cation. Upon the oxidation of a substrate the porphyrin radical is repaired, giving the compound II. Reduction of the oxoferryl haem back to the ferric state by a second substrate molecule completes the enzyme cycle. In addition to the classical peroxidases, several other haem proteins display pseudo-peroxidase activity. The plant enzyme horseradish peroxidase (HRP) is often employed in model systems. 

The peroxidase activity of PGHS is comparable to that of better known peroxidases such as horseradish peroxidase (HRP). The catalytic cycle of HRP is shown in Figure 5 [9], Its first step is the formation of an intermediate very often found in hemoproteins by transfer of an oxygen atom from various oxygen atom donors to the Fe(III) heme (Eq. 6). It is a high-valent iron-oxo species, at least formally a Fe(V)=0 complex. In fact, the detailed electronic structure of this intermediate depends on the environment of the heme provided by the protein. In HRP, this intermediate (called compound I) is a (porphyrin radical-cation)-Fe(IV)=0 complex, as shown by many spectroscopic techniques [9],... 

Miller VP, Goodin DB, Friedman AE et al (1995) Horseradish peroxidase Phel72Tyr mutant. Sequential formation of Compound I with a porphyrin radical cation and a protein radical. J Biol Chem 270 181413-181419... 

Rutter R, Valentine M, Hendrich MP et al (1983) Chemical nature of the porphyrin 7i cation radical in horseradish peroxidase Compound I. Biochemistry 22 4769—4774... 

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... 

Finally, the spontaneous liberation of free radicals via the unimolecular decay of CIII is feasible, since the peroxyl radical is not covalently bound to the porphyrin (pathway 2). This assumption is supported by experimental evidence, which demonstrates that in the presence of excess H202 and no reductant, CIII decays irreversibly into GS and superoxide species in lignin peroxidase [46], horseradish peroxidase [104], myeloperoxidase [51, 105, 106]. 

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]. 

Mossbauer spectra has been extensively used to probe the structure of the iron nucleus in biological FeIV=0 compounds. These include horseradish peroxidase compoundl[134,180,181], horseradish peroxidase compound II [182,183], horseradish peroxidase compound X [181], Japanese-radish peroxidase compounds I and II [184], chloroperoxidase compound I [185], cytochrome c peroxidase compound I [186] and ferryl myoglobin [183]. Examples of Mossbauer spectra attributed to non-porphyrin-bound FeIV are only available from synthetic model compounds. These include compounds with [130] and without [4-8] an FeIV=0 bond. 

Unlike the case of optical or MCD spectroscopy, the presence of a nearby free radical (porphyrin or amino acid) has only a small effect on the Mossbauer spectra of ferryl iron (by contrast the nature of the axial ligand appears to have a greater effect on the Mossbauer spectra). Thus in the absence of a magnetic field there is little difference between the Mossbauer spectra of horseradish peroxidase compounds I and II [134,181,183]. Due to... 

One of the variables in the structures of the porphyrins present in heme proteins is the presence or absence of vinyl substituents on the periphery of the macrocycle. For example, b hemes have vinyl substituents whereas c hemes do not. Because of the sensitivity of such vinyl substituents during synthetic transformations, it has often been desirable to use octa-alkyl porphyrins in model studies of the spectroscopic properties of heme systems. The development of improved methods for the preparation of octa-alkyl porphyrins has likewise increased the availability of such porphyrins for model studies (20, 21). To assess the effect that replacement of the two vinyl substituents in protoporphyrin IX with alkyl (ethyl) groups has on the MCD properties of the heme system, an extensive and systematic study of the MCD properties of mesoheme IX-reconstituted myoglobin and horseradish peroxidase in comparison with the spectra of the native protoheme-bound proteins has been carried out (22). The structures of these two porphyrins are shown in Figure 3. 

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. 

---