Format of Original
Journal of Inorganic Biochemistry
Original Item ID
Oxidation of the heme-thiolate enzyme chloroperoxidase (CPO) from Caldariomyces fumago with peroxynitrite (PN) gave the Compound II intermediate, which was photo-oxidized with 365 nm light to give a reactive oxidizing species. Cryo-solvents at pH ≈ 6 were employed, and reactions were conducted at temperatures as low as − 50 °C. The activity of CPO as evaluated by the chlorodimedone assay was unaltered by treatment with PN or by production of the oxidizing transient and subsequent reaction with styrene. EPR spectra at 77 K gave the amount of ferric protein at each stage in the reaction sequence. The PN oxidation step gave a 6:1 mixture of Compound II and ferric CPO, the photolysis step gave an approximate 1:1 mixture of active oxidant and ferric CPO, and the final mixture after reaction with excess styrene contained ferric CPO in 80% yield. In single turnover reactions at − 50 °C, styrene was oxidized to styrene oxide in high yield. Kinetic studies of styrene oxidation at − 50 °C displayed saturation kinetics with an equilibrium constant for formation of the complex of Kbind = 3.8 × 104 M− 1 and an oxidation rate constant of kox = 0.30 s− 1. UV–Visible spectra of mixtures formed in the photo-oxidation sequence at ca. − 50 °C did not contain the signature Q-band absorbance at 690 nm ascribed to CPO Compound I prepared by chemical oxidation of the enzyme, indicating that different species were formed in the chemical oxidation and the photo-oxidation sequence.