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Computational studies exploring the extent to which differences in proximal axial ligands modulate structure, spectra, and function of peroxidases have been performed. To this end, three heme models of compound I were characterized differing only in the axial ligand. The axial ligands considered were L=ImH, Im^-, that are alternative protonation models for a typical peroxidase with an imidazole ligand such as horseradish peroxidase (HRP-I), and L=SCH- that is a model for an unsual peroxidase, chloroperoxidase (CPO-I). Density functional calculations (DFTs) were performed to determine the optimized geometries and electronic structure of each of these three species. Their electronic spectra were also calculated at the DFT optimized geometries, using the INDO/S/CI method. The results of these studies led to the following conclusions: (1) the presence of the nearby Asp in a typical peroxidase does indeed decrease the energy required to deprotonate the imidazole making the two forms essentially degenerate, (2) neither the state of protonation of the imidazole ligand nor the change in axial ligand from an imidazole in typical peroxidases such as HRP to a mercaptide in CPO significantly alters the characteristics of the lowest energy spin state or the electronic structure of compound I in a way that can obviously affect function, (3) both the Im^- and ImH forms of the peroxidase compound I (HRP-I) lead to the same dramatic reduction in intensity relative to the ferric resting form observed experimentally. However, only in the ImH form of HRP-I does the calculated relative shift of one component of the Soret bands relative to CPO-I agree with that observed in the transient spectra of HRP-I compared to CPO-I. These results taken together strongly indicate that factors other than the nature of the proximal axial ligand are the main determinants of function.

Aspects of protein engineering of cytochrome P450 (P450) and myoglobin (Mb) to construct selective oxygenation catalysts have been described. Heme enzymes are known as biocatalysts for various oxidations but the design of substrate specificity has still remained one of the significant challenges because of dynamic nature of enzyme-substrate interactions. In particular, P450s are the most interesting targets among the heme enzymes because they are able to catalyze many types of monooxygenations such as hydroxylation, epoxidation, and sulfoxidation with high selectivity. Thus, many researchers have made efforts to convert the selectivity for natural substrates into that for unnatural substrates by several protein engineering approaches. On the other hand, we have reported a rational design of Mb to convert its oxygen carrier function into that of peroxidase or peroxygenase. The Mb mutants prepared in our work afford oxo-ferryl porphyrin radical cation (compound I) as observable species in Mb for the first time. Furthermore, some of the mutants we have constructed are useful for enantioselective oxygenations by oxygen transfer from the Mb-compound I to substrates.

Catalases employ a tyrosinate-ligated ferric heme in order to catalyze the dismutation of hydrogen peroxide to O₂ and water. In the first half of the catalytic cycle, H₂O₂ oxidizes Fe(III) to the formally Fe(V) state commonly referred to as Compound I. The second half of the cycle entails oxidation of a second hydrogen peroxide molecule by Compound I to dioxygen. The present study employs density functional (DFT) calculations to examine the nature of this second step of the catalatic reaction. In order to account for the unusual choice of tyrosinate as an axial ligand in catalases, oxidation of hydrogen peroxide by an imidazole-ligated Compound I is also examined, bearing in mind that imidazole-ligated hemoproteins such as myoglobin or horseradish peroxidase tend to display little, if any, catalatic activity. Furthermore, in order to gauge the importance of the cation radical of Compound I in peroxide activation, the performance of Compound II (the one-electron reduced version of Compound I, formally Fe(IV)), is also examined. It is found that hydrogen peroxide oxidation occurs in a quasi-concerted manner, with two hydrogen-atom transfer reactions, and that the tyrosinate ligand is in no way required at this stage. We propose that the role of the tyrosinate is purely thermodynamic, in avoiding accumulation of the much less peroxide-reactive ferrous form in vivo – all in line with the predominantly thermodynamic role of the cysteinate ligands in enzymes such as cytochromes P450.

The mechanism of the reaction between ferric Caldariomyces fumago chloroperoxidase (CCPO) and meta-chloroperoxybenzoic acid (mCPBA) has been examined. It has previously been established that an Fe(IV)-oxo porphyrin radical species known as Compound I (Cpd I) is formed by two-electron oxidation of the native ferric enzyme by a variety of oxidants including organic peracids and hydroperoxides. Cpd I can return to the ferric state either by direct oxygen insertion into an organic substrate (e.g. a P450 oxygenase-like reaction), or by two consecutive one-electron additions, the first resulting in an intermediate Fe(IV)-oxo species known as Compound II (Cpd II). There has been much debate over the role of Cpd II and the details of its structure. In the present study, both CCPO Fe(IV)-oxo intermediates are formed, but unlike most CCPO reactions, Cpd I and Cpd II are formed using the same reactant, mCPBA. Thus, the peracid is used as an oxo donor to produce Cpd I and then as a reductant to reduce Cpd I to Cpd II, and finally, Cpd II to the ferric state. The observation of saturation kinetics with respect to mCPBA concentration for each step is consistent with the formation of CCPO-mCPBA complexes in each phase of the reaction. The original reaction mechanism for ferric CCPO with mCPBA was hypothesized to involve a scrambling mechanism with a unique Fe-OOO-C(O)R intermediate formed with no observed Cpd II intermediate. The data reported herein clearly demonstrate the formation of Cpd II in returning the oxidized enzyme back to its native ferric state.