Kinetic and equilibrium studies of acrylonitrile binding to cytochrome c peroxidase and oxidation of acrylonitrile by cytochrome c peroxidase compound I

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• 作者：Diana Chinchilla ; Heather Kilheeney ; Lidia B. Vitello ; James E. Erman
• 关键词：ACN ; acrylonitrile ; CEO ; 2-cyanoethylene oxide ; CcP ; generic abbreviation for cytochrome c peroxidase whatever the source ; yCcP ; authentic yeast cytochrome c peroxidase isolated from S. cervisiae ; rCcP ; recombinant cytochrome c peroxidase expressed in E. coli ; which has an identical amino acid sequence to that of yCcP ; CcP-I ; cytochrome c peroxidase compound I ; metMb ; metmyoglobin
• 刊名：Biochemical and Biophysical Research Communications
• 出版年：3 January, 2014
• 年：2014
• 卷：443
• 期：1
• 页码：200-204
• 全文大小：435 K

文摘

Ferric heme proteins bind weakly basic ligands and the binding affinity is often pH dependent due to protonation of the ligand as well as the protein. In an effort to find a small, neutral ligand without significant acid/base properties to probe ligand binding reactions in ferric heme proteins we were led to consider the organonitriles. Although organonitriles are known to bind to transition metals, we have been unable to find any prior studies of nitrile binding to heme proteins. In this communication we report on the equilibrium and kinetic properties of acrylonitrile binding to cytochrome c peroxidase (CcP) as well as the oxidation of acrylonitrile by CcP compound I. Acrylonitrile binding to CcP is independent of pH between pH 4 and 8. The association and dissociation rate constants are 0.32 卤 0.16 M^鈭? s^鈭? and 0.34 卤 0.15 s^鈭?, respectively, and the independently measured equilibrium dissociation constant for the complex is 1.1 卤 0.2 M. We have demonstrated for the first time that acrylonitrile can bind to a ferric heme protein. The binding mechanism appears to be a simple, one-step association of the ligand with the heme iron. We have also demonstrated that CcP can catalyze the oxidation of acrylonitrile, most likely to 2-cyanoethylene oxide in a 鈥減eroxygenase鈥?type reaction, with rates that are similar to rat liver microsomal cytochrome P450-catalyzed oxidation of acrylonitrile in the monooxygenase reaction. CcP compound I oxidizes acrylonitrile with a maximum turnover number of 0.61 min^鈭? at pH 6.0.