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

Abstract

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 ψ₁, is stoichiometrically limited by the reductase in deficit: ψ₁, 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 ψ₁, Reductase cycling is proposed to occur in protein clusters of cytochrome P-450 and reductase in deficit.

5. The dissociation constant K_RP of the functionally active reductase—cytochrome P-450 complex has been determined by means of the amount of ψ₁, (DPLC) and the rate constant K^app₁ (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, K_RP in vesicles has been calculated to be about 3 orders of magnitude increased in comparison to DLPC-reconstituted systems.

6. V_max 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.