Formation of P450 · P450 complexes and their effect on P450 function

Abstract

Cytochromes P450 (P450) are membrane-bound enzymes that catalyze the monooxygenation of a diverse array of xenobiotic and endogenous compounds. The P450s responsible for foreign compound metabolism generally are localized in the endoplasmic reticulum of the liver, lung and small intestine. P450 enzymes do not act alone but require an interaction with other electron transfer proteins such as NADPH-cytochrome P450 reductase (CPR) and cytochrome b(5). Because P450s are localized in the endoplasmic reticulum with these and other ER-resident proteins, there is a potential for protein-protein interactions to influence P450 function. There has been increasing evidence that P450 enzymes form complexes in the ER, with compelling support that formation of P450 · P450 complexes can significantly influence their function. Our goal is to review the research supporting the formation of P450 · P450 complexes, their specificity, and how drug metabolism may be affected. This review describes the potential mechanisms by which P450s may interact, and provides evidence to support each of the possible mechanisms. Additionally, evidence for the formation of both heteromeric and homomeric P450 complexes are reviewed. Finally, direct physical evidence for P450 complex formation in solution and in membranes is summarized, and questions directing the future research of functional P450 interactions are discussed with respect to their potential impact on drug metabolism.

Figure 1

Experimental strategy used to demonstrate that both P450s must reside in the same membrane in order to interact. (reproduced from J. Biol. Chem. (2010) 285, 8942–8952)

Model 1. Simple Competition between Two P450 Enzymes for CPR

In this model, each of the P450 enzymes has its own inherent ability to associate with CPR (defined by the Michaelis constants K_Da and K_Db) as well as their rates of substrate turnover (defined by the rate constants k_a and k_b). Neither the K_D nor the rate constant for either P450 are affected by the presence of the second P450 enzyme; however, at subsaturating CPR levels both P450s will compete for the available CPR with overall substrate turnover being dependent on these constants. The species labeled in blue are capable of converting substrate to product.

Model 2. Formation of a P450•P450 complex that affects CPR binding

In this model, P450_a and P450_b are capable of forming a physical complex that affects the ability of CPR to bind. P450_a and P450_b monomers each have their characteristic abilities to bind and metabolize substrate as described in Model 1. However, formation of the P450_a•P450_b complex can affect the ability of either moiety of the complex to associate with CPR (i.e. K_Dcba or K_Dcab). According to this model, the rate constants for substrate turnover are not affected. At subsaturating CPR concentrations the reaction rate can either be stimulated or inhibited by altering the fraction of CPR bound to a particular P450 enzyme. At saturating CPR concentrations, an additive effect would be observed because all of the P450s would be complexed with CPR. The forms capable of substrate turnover include the monomeric P450s (shown in blue), the P450_a•P450_b complexes associated with a single CPR (shown in brown), and the CPR•P450_a•P450_b•CPR complex (shown in green). The latter complex would only be expected to accumulate at saturating CPR levels or in the event of a significant cooperative effect. The dissociation constants affected by P450•P450 complex formation are shown in red.

Model 3. Formation of a P450•P450 complex that affects the efficiency of substrate turnover

This model is similar to Model 2 in that P450_a and P450_b can form a physical complex. However, rather than the P450_a•P450_b complex affecting CPR binding affinity, the rate constants for substrate turnover are affected. This would cause either an inhibiton or synergistic stimulation of substrate turnover that would be observed at both subsaturating and saturating CPR concentrations. The CPR-bound P450 moieties that would contribute to substrate turnover are the monomers (blue), the complexes with a single CPR (brown), and the complexes with CPR on each of the P450s (green). The rate constants affected by P450•P450 complex formation are shown in red. ⁱ Potentially, P450s in separate membranes could interact; however, our data shows that they do not. These results support the idea that the membrane helps to orient the proteins in a manner that facilitates these P450•P450 interactions.