Cytochrome P450 (P450) 3A4, the major catalyst involved in human drug oxidation, displays substrate-and reaction-dependent homotropic and heterotropic cooperative behavior. Although several models have been proposed, these mainly rely on steadystate kinetics and do not provide information on the contribution of the individual steps of P450 catalytic cycle to the observed cooperativity. In this work, we focused on the kinetics of substrate binding, and the fluorescent properties of bromocriptine and ␣-naphthoflavone allowed analysis of an initial ligand-P450 3A4 interaction that does not cause a perturbation of the heme spectrum. The binding stoichiometry for bromocriptine was determined to be unity using isothermal titration calorimetry and equilibrium dialysis methods, suggesting that the ligand bound to the peripheral site during the initial encounter dissociates subsequently. A three-step substrate binding model is proposed, based on absorbance and fluorescence stopped-flow kinetic data and equilibrium binding data obtained with bromocriptine, and evaluated using kinetic modeling. The results are consistent with the substrate molecule binding at a site peripheral to the active site and subsequently moving toward the active site to bind to the heme and resulting in a low to high spin iron shift. The last step is attributed to a conformational change in the enzyme active site. The later steps of binding were shown to have rate constants comparable with the subsequent steps of the catalytic cycle. The P450 3A4 binding process is more complex than a two-state system, and the overlap of rates of some of the events with subsequent steps is proposed to underlie the observed cooperativity.Cytochrome P450 (P450) 2 enzymes are found throughout nature, from bacteria to humans. These enzymes generally catalyze mixed function oxidation reactions that have similar chemistry or else utilize parts of the general catalytic mechanism for reductions and rearrangements (2, 3). The wide diversity of substrates of these enzymes and the basis of catalytic selectivity is a topic of considerable interest in the context of both basic biochemistry and practical applications (4). P450 3A4 is one of the most widely studied of the 57 human P450s (5), mainly due to its role in the metabolism of more than one-half of the drugs on the market as well as various endogenous and exogenous molecules (6, 7). In addition, P450 3A4 is the major P450 expressed in liver (8) and in the intestine (9). Recently solved P450 3A4 crystal structures (10 -12) demonstrate the presence of a large active site, consistent with the broad range of substrates that P450 3A4 can accommodate (7, 13), including (in order of increasing size) acetaminophen (14) (M r 151), testosterone (15) (M r 288), bromocriptine (16) (M r 655), and cyclosporin (17) (M r 1201). Despite its seemingly flexible substrate selectivity, P450 3A4 displays a high degree of regio-and stereoselectivity in many substrate oxidations (18,19).One of the important features of P450 3A4 is its cooperativ...