Mechanism of rate control of the NADPH‐dependent reduction of cytochrome P‐450 by lipids in reconstituted phospholipid vesicles

The NADPH‐supported reduction of cytochrome P‐450 LM2 (liver microsomal isozyme 2) in reconstituted phospholipid vesicles in general exhibits two‐exponential kinetics. The physiologically relevant rapid partial reaction is favoured in amount with increasing reductase/P‐450 ratio. A lipid specificity was observed in that negatively charged lipids favour that process, too. The rate constant increases concomitantly. The data are consistent with the formation of a reactive 1:1 complex the amount of which determines the rate constant. The dissociation constants amount to 0.048 μM for a microsomal lipid extract, 0.051 μM for a 3:1 (w/w) mixture of dioleoylglycerophosphoethanolamine and phosphatidylserine, and 0.47 μM for dioleoylglycerophosphocholine, respectively, in the respective reconstituted systems. At low reductase, P‐450 ratio the amount of the rapidly reduced P‐450 exceeds the equilibrium concentration of a 1:1 complex. Preformed 1:1 associates, therefore, cannot fit the derived mechanism. Instead, a cluster model based on P‐450 association does correspond to the data.