Role of lipid in the electron transfer between NADPH-cytochrome P-450 reductase and cytochrome P-450 from mammalian liver cells.

1. The anaerobic NADPH-reduction of the isozymes cytochrome P-450 LM2 and LM4 was used as a functional tool to study the component interaction in reconstituted monooxygenase systems in dependence on different phospholipids. 2. The isozymes were shown to exhibit similar lipid interaction. The lipids generally favour a catalytically active 1:1 complex formation between reductase and cytochrome P-450 as the rate-determining unit in electron transfer. 3. The cytochrome P-450 reduction proceeds in a biphasic reaction. In dilauroyl phosphatidylcholine (DLPC)-reconstituted systems the amount of the fast reduction psi 1 is stoichiometrically limited by the reductase in deficit: psi 1 corresponds to the 1:1 complex formation capability of the reductase. 4. In vesicle-reconstituted systems an 'overstoichiometric' reductase cycling is observed which gives rise to a significantly increased amount of fast reduction psi 1. Reductase cycling is proposed to occur in protein clusters of cytochrome P-450 and reductase in deficit. 5. The dissociation constant KRP of the functionally active reductase-cytochrome P-450 complex has been determined by means of the amount of psi 1 (DPLC) and the rate constant kapp 1 (vesicles) of the fast reduction as a measure of the complex formation in dependence on the protein molar ratio. Taking into account the actual protein concentration in the vesicular lipid phase, KRP in vesicles has been calculated to be about 3 orders of magnitude increased in comparison to DLPC-reconstituted systems. 6. Vmax data reveal almost the same catalytic activity of both reconstitution modes, which justifies DLPC-reconstitution in model investigations. The vesicle-specific increased accumulation of reduced cytochrome P-450 in the steady state as originated by reductase cycling may offer the physiological advantage of an increased capacity of cytochrome P-450 for synergistic substrate conversion via cytochrome b5.