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Low temperature photo-oxidation of chloroperoxidase Compound II. J Inorg Biochem 2010 Nov;104(11):1156-63

Date

08/03/2010

Pubmed ID

20674981

Pubmed Central ID

PMC2963949

DOI

10.1016/j.jinorgbio.2010.07.004

Scopus ID

2-s2.0-79954561206 (requires institutional sign-in at Scopus site)   3 Citations

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 77K 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 K(bind)=3.8 x 10(4)M(-1) and an oxidation rate constant of k(ox)=0.30s(-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.

Author List

Yuan X, Sheng X, Horner JH, Bennett B, Fung LW, Newcomb M

Author

Brian Bennett D.Phil. Professor and Chair in the Physics department at Marquette University




MESH terms used to index this publication - Major topics in bold

Chloride Peroxidase
Cold Temperature
Electron Spin Resonance Spectroscopy
Epoxy Compounds
Heme
Hydrogen-Ion Concentration
Iron
Kinetics
Oxidants
Oxidation-Reduction
Peroxynitrous Acid
Photochemical Processes
Photolysis
Reactive Oxygen Species
Solvents
Spectrophotometry, Ultraviolet
Styrene
Temperature