Detection of a Tryptophan Radical as an Intermediate Species in the Reaction of Horseradish Peroxidase Mutant (Phe-221 → Trp) and Hydrogen Peroxide

Abstract

The crucial reaction intermediate in the reaction of peroxidase with hydrogen peroxide (H 2 O 2 ), compound I, contains a porphyrin -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 3 Trp). Although the rapid spectral scanning and stoppedflow experiments confirmed that the F221W mutant reacts with H 2 O 2 to form the porphyrin -cation radical at the same rate as for the wild-type enzyme, the characteristic spectral features of the porphyrin -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 -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.

Activation of the cytochrome c peroxidase of Pseudomonas aeruginosa. The role of a heme-linked protein loop: A mutagenesis studies

“…The H71G form of the protein presents an absorption spectrum which has previously been associated with the formation of an oxy-ferryl heme centre coupled to an adjacent Trp radical in a number of peroxidase systems [26][27][28][29]. The loop mutant produces a Soret spectrum for the product of reaction with hydrogen peroxide typical of an oxy-ferryl heme centre coupled to a porphyrin cation radical previously observed in a number of other peroxidase proteins [30][31][32][33].…”

Understanding the roles of strictly conserved tryptophan residues in O2producing chlorite dismutases

“…These nonbonded interactions are thought to be important conduits for radical migration away from the porphyrin. 8 …”

Crystal structure of aldoxime dehydratase and its catalytic mechanism involved in carbon-nitrogen triple-bond synthesis

“…In this strain, we identified a unique hemoprotein that catalyzes the dehydration of aliphatic aldoximes to the corresponding nitriles (25), naming it aliphatic aldoxime dehydratase (OxdA); it has been approved as a unique enzyme by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB): EC 4.99.1.5 (www.chem.qmul.ac.uk/iubmb/enzyme/EC4/99/1/5.html). The reaction mechanisms of heme-containing enzymes have received the close attention of a large number of researchers (26)(27)(28)(29)(30). The reaction mechanism of OxdA is also very attractive, because the OxdA reaction is very unique and intriguing in the following aspects: (i) dehydration of the substrate even in an aqueous solution; (ii) utilization of the favorable hydrophobic environment of the heme pocket for catalysis of dehydration; (iii) synthesis of a carbon-nitrogen triple bond, which is a highly toxic functional group; and (iv) direct binding of the substrate to the heme iron in contrast to other heme-containing enzymes (e.g., horseradish peroxidase).…”