Although a single binary functional complex between cytochrome P450 and cytochrome P450 reductase (CPR) has been generally accepted in the literature, this simple model failed to explain experimentally observed catalytic activity of recombinant P450 2E1 in dependence on the total concentration of added CPR-K56Q mutant. Our rejection of the simplest 1:1 binding model was based on two independent lines of experimental evidence. First, under the assumption of the 1:1 binding model, separate analyses of titration curves obtained while varying either P450 or CPR concentrations individually produced contradictory results. Second, an asymmetric Job plot suggested the existence of higher order molecular complexes. To identify the most probable complexation mechanism, we generated a comprehensive data set where the concentrations of both P450 and P450 were varied simultaneously, rather than one at a time. The resulting two-dimensional data were globally fit to 32 candidate mechanistic models, involving the formation of binary, ternary, and quaternary P450:CPR complexes, in the absence or presence or P450 and CPR homodimers. Of the 32 candidate models (mechanisms), two models were approximately equally successful in explaining our experimental data. The first plausible model involves the binary complex P450•CPR, the quaternary complex (P450) 2 • (CPR) 2 , and the homodimer (P450) 2 . The second plausible model additionally involves a weakly bound ternary complex (P450) 2 •CPR. Importantly, only the binary complex P450•CPR seems catalytically active in either of the two most probable mechanisms.Protein-ligand and protein-protein interactions are fundamental to biological processes such as signal transduction, chemical transformations, and electron transport. An understanding of the role of these processes in biological function requires the identification of the detailed interaction mechanisms. These details provide a framework in which to understand how the process of molecular recognition maintains proper homeostasis, or leads to deleterious conditions. In this study, we characterize the details of protein-protein interactions involving cytochrome P450 1 (P450) and the cytochrome P450 reductase (CPR). Traditional experimental protocols and data-analytic methods applied to this two-component system failed to provide a clear answer regarding the binding stoichiometry. We describe an alternative data-analytic approach applicable to any multi-component system. The method is based on a general numerical analysis of simultaneous biochemical equilibria, without any restriction on the number of component molecular species or the number of complexes they form (1). Our results Microsomal P450 enzymes are major catalysts in the oxidative transformation of a structurally diverse class of compounds including steroids, fatty acids, hormones, antibiotics, and a wide variety of artificially produced chemicals (xenobiotics), such as drugs, food additives, and environmental contaminants (2). P450s convert lipid-soluble molecules t...
