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

Thomas M. Makris,^‡ Konstanze von Koenig,^§ Ilme Schlichting,^§ and Stephen G. Sligar*^‡

Departments of Biochemistry and Chemistry and the School of Medicine, University of Illinois UrbanaChampaign, Urbana, Illinois 61801, and Max-Planck-Institut fr medizinische Forschung, Abt. Biomolekulare Mechanismen, Jahnstrasse 29, 69120 Heidelberg, Germany

Biochemistry, 2007, 46 (49), pp 14129–14140

DOI: 10.1021/bi7013695

Publication Date (Web): November 15, 2007

†

This work was supported by a merit award from the National Institutes of Health (R37 GM31756) to S.G.S.

‡

Department of Biochemistry, University of Illinois Urbana−Champaign.

Max-Planck-Institut für medizinische Forschung.

To whom correspondence should be addressed. Telephone: (217) 244-7395. Fax: (217) 265-4073. E-mail: s-sligar@uiuc.edu.

Department of Chemistry and the School of Medicine, University of Illinois Urbana−Champaign.

Abstract

Distal pocket water molecules have been widely implicated in the delivery of protons required in 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 results in the alteration of spin state equilibria, and has dramatic consequences on the catalytic rate, coupling efficiency, and kinetic solvent isotope effect parameters, highlighting an important role of the active-site hydration level on P450 catalysis. Cryoradiolysis of the mutant CYP101 oxyferrous complexes further indicates a specific perturbation of proton-transfer events required for the transformation of ferric-peroxo to ferric-hydroperoxo states. Finally, crystallography of the 248Val and 248Thr mutants in both the ferric camphor bound resting state and ferric−cyano adducts shows both the alteration of hydrogen-bonding networks and the alteration of heme geometry parameters. Taken together, these results indicate that the distal pocket microenvironment governs the transformation of reactive heme-oxygen intermediates in P450 cytochromes.

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