Dual role of phospholipid in the reconstitution of cytochrome P-450 LM2-dependent activities.

The effect of dilauroylphosphatidylcholine (DLPC) concentration on cytochrome P-450 LM2 (LM2)-dependent reduction and monooxygenase activities was examined as a function of preincubation time. Purified NADPH-cytochrome P-450 reductase (reductase) and LM2 were reconstituted at different DLPC to LM2 ratios by preincubation of the proteins in the presence of DLPC for either 5 min or 2 hr at room temperature. After preincubation was complete, the samples were assayed for either monooxygenase activity or first-electron transfer activity. When preincubated for 5 min, overall monooxygenase activity was dependent on the [DLPC]:[LM2] ratio, beginning at a low level in the absence of phospholipid and increasing to a maximum at a 160:1 ratio. At [DLPC]:[LM2] ratios above 160:1, the rate was decreased to 80% of the maximum rate. When the samples were preincubated for 2 hr, again low monooxygenase activities were obtained in the absence of DLPC, which increased to a maximum at 160:1 [DLPC]:[LM2] ratio. Above this [DLPC]:[LM2]ratio, the rate was decreased to less than 50% of the maximum value. These changes in overall activities appear to be related to changes in the amount of functional reductase-LM2 complex formed. Similar results were found when LM2 reduction was examined. When preincubated for 5 min, LM2 reduction was shown to be diminished as the DLPC to LM2 ratio decreased below 160:1. The DLPC-dependent effect on reduction was primarily characterized by alterations in the fraction of LM2 reduced in the first phase, with the first-phase rate constant and the slow phase parameters being largely unaffected. Below a 16:1 ratio [( DLPC]:[LM2]), no phospholipid stimulation of LM2 reduction was observed. When the [DLPC]:[LM2] ratio was increased above a 160:1 ratio, only a small effect on the kinetic constants was observed, which was characterized by a 20% decrease in the fraction of LM2 reduced in the first phase. LM2 reduction was more sensitive to DLPC concentration after longer preincubations (2 hr), with a 50% decrease in the fraction of reduction in the first phase being observed at [DLPC]:[LM2] ratios above 160:1. The results are consistent with a dual role for phospholipid in the stimulation of LM2-dependent activities. First, DLPC facilitates the association of reductase and LM2 and, second, DLPC provides a matrix for the incorporation of LM2 and reductase. Facilitation of the protein association appears to be a relatively rapid process, occurring after a 5-min preincubation, whereas a 2-hr preincubation altered the protein interactions in a manner consistent with incorporation of the LM2 and reductase into the phospholipid.