Abstract
The crucial reaction intermediate in the reaction of peroxidase with hydrogen peroxide (H2O2), compound I, contains a porphyrin pi-cation radical in horseradish peroxidase (HRP), which catalyzes oxidation of small organic and inorganic compounds, whereas cytochrome c peroxidase (CcP) has a radical center on the tryptophan residue (Trp-191) and oxidizes the redox partner, cytochrome c. To investigate the roles of the amino acid residue near the heme active center in discriminating the function of the peroxidases in these two enzymes, we prepared a CcP-like HRP mutant, F221W (Phe-221 --> Trp). Although the rapid spectral scanning and stopped-flow experiments confirmed that the F221W mutant reacts with H2O2 to form the porphyrin pi-cation radical at the same rate as for the wild-type enzyme, the characteristic spectral features of the porphyrin pi-cation radical disappeared rapidly, and were converted to the compound II-type spectrum. The EPR spectrum of the resultant species produced by reduction of the porphyrin pi-cation radical, however, was quite different from that of compound II in HRP, showing typical signals from a Trp radical as found for CcP. The sequential radical formation from the porphyrin ring to the Trp residue implies that the proximal Trp is a key residue in the process of the radical transfer from the porphyrin ring, which differentiates the function of peroxidases.
Highlights
Peroxidases catalyze one-electron oxidation of various substrates by using H2O2 as an oxidant [1]
To investigate the control mechanism for the position of the radical center in the reaction intermediates, some mutagenic studies focused on Trp-191 in cytochrome c peroxidase (CcP) have been carried out and shown that a “horseradish peroxidase (HRP)-type” CcP mutant, in which the proximal Trp was replaced with Phe (Trp-191 3 Phe), yields an oxyferryl porphyrin -cation radical intermediate similar to that of HRP compound I, suggesting that the proximal Trp controls the position of the radical center [8, 9, 13]
These spectroscopies, demonstrating formation of the Trp radical during the oxidation of the mutant by hydrogen peroxide after the porphyrin -cation radical formation, provide strong evidence that the radical center on the porphyrin ring can be transferred to the Trp residue near the heme in peroxidase, indicating that the Trp residue is the key amino acid residue to differentiate the function of peroxidases
Summary
Peroxidases catalyze one-electron oxidation of various substrates by using H2O2 as an oxidant [1]. To investigate the control mechanism for the position of the radical center in the reaction intermediates, some mutagenic studies focused on Trp-191 in CcP have been carried out and shown that a “HRP-type” CcP mutant, in which the proximal Trp was replaced with Phe (Trp-191 3 Phe), yields an oxyferryl porphyrin -cation radical intermediate similar to that of HRP compound I, suggesting that the proximal Trp controls the position of the radical center [8, 9, 13]. NMR, EPR, and ICP emission spectroscopies were used in this study to characterize the heme environmental structure of the mutant These spectroscopies, demonstrating formation of the Trp radical during the oxidation of the mutant by hydrogen peroxide after the porphyrin -cation radical formation, provide strong evidence that the radical center on the porphyrin ring can be transferred to the Trp residue near the heme in peroxidase, indicating that the Trp residue is the key amino acid residue to differentiate the function of peroxidases
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