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Characterization of Peroxide‐Bound Heme Species Generated in the Reaction of Thermally Tolerant Cytochrome c552 with Hydrogen Peroxide
Author(s) -
Ichikawa Yusuke,
Nakajima Hiroshi,
Watanabe Yoshihito
Publication year - 2006
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200600135
Subject(s) - heme , chemistry , hydrogen peroxide , photochemistry , ascorbic acid , cytochrome , peroxide , homolysis , protonation , moiety , radical , stereochemistry , organic chemistry , enzyme , food science , ion
Abstract Peroxide‐bound heme species have been considered difficult to detect under physiological conditions because of their intrinsically transient properties. Cytochrome c552 (cyt c552), from Thermus thermophirus HB8, bearing a mutation to an alanine at Met69 (M69A) reacts with hydrogen peroxide (H 2 O 2 ) to generate a detectable hydroperoxo‐ferric heme ([Fe 3+ OOH]) species at ambient temperature. EPR measurements during appropriate reaction periods reveal that the [Fe 3+ OOH] species is in a preequilibrium state between the resting form of the cyt c552 variant and a subsequent intermediate, compound II with a protein radical. Addition of ascorbic acid to the reaction mixture of the cyt c552 variant and H 2 O 2 does not affect the formation of the [Fe 3+ OOH] species,a result suggesting that the species is incompetent for the oxidation of even an oxidatively fragile substrate such as ascorbic acid. Another variant bearing an additional mutation to aspartic acid at Val49 (V49D/M69A) reveals that a highly hydrophobic heme cavity in cyt c552 accounts for the generation of the durable [Fe 3+ OOH] species. The less polar environment inside the cavity is expected to prevent H 2 O from approaching the cavity. This would suppress protonation of the distal oxygen atom of the [Fe 3+ OOH] species and retard subsequent dissociation of H 2 O from the OOH moiety.