Ortiz de Montellano Pr

Peroxidation of a Specific Tryptophan of Metmyoglobin by Hydrogen Peroxide

Globin-centered radicals at tyrosine and tryptophan residues and a peroxyl radical at an unknown location have been reported previously as products of the reaction of metmyoglobin with hydrogen peroxide. The peroxyl radical is shown here to be localized on tryptophan through the use of recombinant sperm whale myoglobin labeled with 13C at the indole ring C-3. Peroxyl radical formation was not prevented by site-directed mutations that replaced all three tyrosines, the distal histidine, or tryptophan 7 with non-oxidizable residues. In contrast, mutation of tryptophan 14 prevents peroxyl radical formation, implicating tryptophan 14 as the specific site of the peroxidation.

An A245T mutation conveys on cytochrome P450eryF the ability to oxidize alternative substrates.

Cytochrome P450eryF (CYP107A1), which hydroxylates deoxyerythronolide B in erythromycin biosynthesis, lacks the otherwise highly conserved threonine that is thought to promote O–O bond scission. The role of this threonine is satisfied in P450eryF by a substrate hydroxyl group, making deoxyerythronolide B the only acceptable substrate. As shown here, replacement of Ala245 by a threonine enables the oxidation of alternative substrates using either H2O2 or O2/spinach ferredoxin/ferredoxin reductase as the source of oxidizing equivalents. Testosterone is oxidized to 1-, 11α-, 12-, and 16α-hydroxytestosterone. A kinetic solvent isotope effect of 2.2 indicates that the A245T mutation facilitates dioxygen bond cleavage. This gain-of-function evidence confirms the role of the conserved threonine in P450 catalysis. Furthermore, a Hill coefficient of 1.3 and dependence of the product distribution on the testosterone concentration suggest that two testosterone molecules bind in the active site, in accord with a published structure of the P450eryF-androstenedione complex. P450eryF is thus a structurally defined model for the catalytic turnover of multiply bound substrates proposed to occur with CYP3A4. In view of its large active site and defined structure, catalytically active P450eryF mutants are also attractive templates for the engineering of novel P450 activities.