Cooperativity in rabbit cytochrome P450 1A2 pyrene oxidation: Evidence for a multiple binding site model

BIOL 29

Christal D. Sohl, c.sohl@Vanderbilt.Edu1, Emre M. Isin1, Glenn A. Marsch2, and F. Peter Guengerich, f.guengerich@vanderbilt.edu1. (1) Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University, 642 Robinson Research Building, 23rd and Pierce Aves, Nashville, TN 37232, (2) Department of Physics, Grove City College, Grove City, PA
Cytochrome P450s are a family of heme-thiolate monooxygenases, oxidizing drugs, carcinogens, and steroids. Most P450 reactions display Michaelis-Menten kinetics, but in some oxidations by certain P450s sigmoidal curves indicative of cooperativity are observed. We previously identified non-Michaelis-Menten behavior in oxidation of 4-alkoxynitrobenzenes by rabbit P450 1A2 and proposed a two-binding site model. Expansion of these studies showed that P450 1A2 oxidizes pyrene to 1-hydroxypyrene, displaying striking sigmoidal behavior. Stopped-flow fluorescence kinetic studies demonstrated that excimers form in P450 1A2 following a fast pyrene monomer binding step, suggesting simultaneous occupancy of the active site with two pyrene molecules. When the iron ligand 1,4-phenylenediisocyanide is premixed with P450 1A2, pyrene binding rates are unaffected but the magnitude of the absorbance change increases. These studies are consistent with a multiple ligand binding site model for the cooperativity seen in pyrene oxidation by P450 1A2. (Supported by USPHS R37 CA090426, T32 ES007028).
 

Frontiers in Chemical Biology
5:00 PM-7:00 PM, Sunday, August 19, 2007 BCEC -- Exhibit Hall - B2, Poster

Division of Biological Chemistry

The 234th ACS National Meeting, Boston, MA, August 19-23, 2007