Document Type

Article

Language

eng

Format of Original

8 p.

Publication Date

11-2010

Publisher

Elsevier

Source Publication

Journal of Inorganic Biochemistry

Source ISSN

0162-0134

Original Item ID

doi: 10.1016/j.jinorgbio.2010.07.004

Abstract

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.

Comments

NOTICE: this is the author’s version of a work that was accepted for publication in Journal of Inorganic Biochemistry. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Journal of Inorganic Biochemistry, Vol. 104, No. 11 (November 2010): 1156-1163. DOI. © 2010 Elsevier. Used with permission.

Brian Bennett was affiliated with the Medical College of Wisconsin at the time of publication.

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