On the role of phospholipids in the reconstituted cytochrome P ‐450 system

The difference in pentoxyresorufin O ‐dealkylating activity observed in a reconstituted system containing dilauroylglycerophosphocholine (Lau 2 Gro P Cho) or distearoylglycerophosphocholine (Ste 2 Gro P Cho) was used as a model to study the role of phospholipids in the reconstituted cytochrome P ‐450b (IIB1) system. The hypotheses proposed in the literature for the role of phospholipids in the reconstituted cytochrome P ‐450 system, mainly based on the comparison of systems without phospholipid and with Lau 2 Gro P Cho, were either validated or shown to be unlikely when tested by comparing reconstituted systems with different phosphatidylcholines. The higher activity in the Lau 2 Gro P Cho system as compared to the Ste 2 Gro P Cho system cannot be ascribed to (a) an increased affinity of cytochrome P ‐450 for the NADPH‐cytochrome reductase in the Lau 2 Gro P Cho system, also not to (b) a Lau 2 Gro P Cho‐dependent dissociation of protein multimers, nor to (c) a change in the spin state of the heme. We found a different apparent K m for pentoxyresorufin in the Lau 2 Gro P Cho system compared with the Ste 2 Gro P Cho system. Furthermore, we found a difference between the cytochrome P ‐450b tryptophan fluorescence polarization of the Lau 2 Gro P Cho system and the Ste 2 Gro P Cho system as well as with a system without phosphatidylcholine. From these observations it is concluded that the higher activity of the Lau 2 Gro P Cho system compared with the Ste 2 Gro P Cho system or with a system without additional phosphatidylcholine may at least in part be caused by a difference in the conformation of the cytochrome P ‐450 molecules in these systems. Furthermore, the different effects of both phosphatidylcholines on the K m and V for pentoxyresorufin not only suggest a role of phospholipids in the binding of the substrate to the active site of the cytochrome P ‐450 molecule, but also on the efficiency of electron transfer from NADPH‐cytochrome reductase to cytochrome P ‐450.