DYNAMIC INTERACTIONS OF RABBIT LIVER CYTOCHROMES P450IA2 AND P450IIB4 WITH CYTOCHROME B(5) AND NADPH-CYTOCHROME P450 REDUCTASE IN PROTEOLIPOSOMES

Purified liver microsomal cytochrome P450IA2 or P450IIB4 was co-reconstituted with cytochrome b(5) or NADPH-cytochrome P450 reductase in phosphatidylcholine-phosphatidylethanolmine-phosphatidylserine vesicles at a lipid to P450 weight ratio of 2 by cholate dialysis procedures. The proteoliposomes catalyzed drug oxidation. Rotational-diffusion of cytochrome P450 was measured by observing the decay of absorption anisotropy, r(t), after photolysis of the heme . CO complex. Analysis of r(t) was based on a ''rotation-about-membrane normal'' model. The absorption anisotropy decayed within 1 ms to a time-independent value, r(3). Different rotational mobility for the two cytochrome P450s was observed. Though 20% of cytochrome P450IA2 was immobile, all cytochrome P450IIB4 molecules were rotating. The rotational relaxation time, phi, of the mobile population was 237 mu s for cytochrome P450IA2 and 160 mu s for cytochrome P450IIB4. The two cytochrome P450s have shown very different interactions with cytochrome bs and NADPH-cytochrome P450 reductase. By the presence of the redox partner, the mobile population of cytochrome P450IA2 was increased significantly from 80% to 96% (plus cytochrome b(5)) and to 89% (plus NADPH-cytochrome P450 reductase) due to dissociation of P450 oligomers. On the other hand, the mobility of cytochrome P450IIB4 was not considerably affected by the presence of cytochrome b(5) or NADPH-cytochrome P450 reductase as judged by little difference in phi and r(3), keeping the mobile population of 100%. These results imply that cytochrome P450IA2 forms a transient association with cytochrome b(5) and NADPH-cytochrome P450 reductase. Taking together biochemical experiments, it is suggested that cytochrome P450IIB4 would associate transiently with cytochrome b(5) and that cytochrome P450IIB4 would diffuse independently of NADPH-cytochrome P450 reductase. Further analysis showed that the tilt angle of the heme plane from the membrane plane was either 47 degrees or 63 degrees for cytochrome P450IA2 and 55 degrees for cytochrome P450IIB4.