Alteration of P450 Distal Pocket Solvent Leads to Impaired Proton Delivery and Changes in Heme Geometry

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• 作者：Thomas M. Makris ; Konstanze von Koenig ; Ilme Schlichting ; Stephen G. Sligar
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：December 11, 2007
• 年：2007
• 卷：46
• 期：49
• 页码：14129 - 14140
• 全文大小：399K
• 年卷期：v.46,no.49(December 11, 2007)
• ISSN：1520-4995

文摘

Distal pocket water molecules have been widely implicated in the delivery of protons requiredin O-O bond heterolysis in the P450 reaction cycle. Targeted dehydration of the cytochrome P450cam(CYP101) distal pocket through mutagenesis of a distal pocket glycine to either valine or threonine resultsin the alteration of spin state equilibria, and has dramatic consequences on the catalytic rate, couplingefficiency, and kinetic solvent isotope effect parameters, highlighting an important role of the active-sitehydration level on P450 catalysis. Cryoradiolysis of the mutant CYP101 oxyferrous complexes furtherindicates a specific perturbation of proton-transfer events required for the transformation of ferric-peroxoto ferric-hydroperoxo states. Finally, crystallography of the 248Val and 248Thr mutants in both the ferriccamphor bound resting state and ferric-cyano adducts shows both the alteration of hydrogen-bondingnetworks and the alteration of heme geometry parameters. Taken together, these results indicate that thedistal pocket microenvironment governs the transformation of reactive heme-oxygen intermediates in P450cytochromes.