DYNAMIC INTERACTIONS OF RABBIT LIVER CYTOCHROMES P450IA2 AND P450IIB4WITH CYTOCHROME B(5) AND NADPH-CYTOCHROME P450 REDUCTASE IN PROTEOLIPOSOMES

Purified liver microsomal cytochrome P450IA2 or P450IIB4 was co-recons tituted with cytochrome b(5) or NADPH-cytochrome P450 reductase in cho line-phosphatidylethanolmine-phosphatidylserine vesicles at a lipid to P450 weight ratio of 2 by cholate dialysis procedures. The proteolipo somes catalyzed drug oxidation. Rotational-diffusion of cytochrome P45 0 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 anisotro py decayed within 1 ms to a time-independent value, r(3). Different ro tational mobility for the two cytochrome P450s was observed. Though 20 % of cytochrome P450IA2 was immobile, all cytochrome P450IIB4 molecule s were rotating. The rotational relaxation time, phi, of the mobile po pulation was 237 mu s for cytochrome P450IA2 and 160 mu s for cytochro me P450IIB4. The two cytochrome P450s have shown very different intera ctions with cytochrome bs and NADPH-cytochrome P450 reductase. By the presence of the redox partner, the mobile population of cytochrome P45 0IA2 was increased significantly from 80% to 96% (plus cytochrome b(5) ) and to 89% (plus NADPH-cytochrome P450 reductase) due to dissociatio n of P450 oligomers. On the other hand, the mobility of cytochrome P45 0IIB4 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 imp ly that cytochrome P450IA2 forms a transient association with cytochro me b(5) and NADPH-cytochrome P450 reductase. Taking together biochemic al experiments, it is suggested that cytochrome P450IIB4 would associa te transiently with cytochrome b(5) and that cytochrome P450IIB4 would diffuse independently of NADPH-cytochrome P450 reductase. Further ana lysis showed that the tilt angle of the heme plane from the membrane p lane was either 47 degrees or 63 degrees for cytochrome P450IA2 and 55 degrees for cytochrome P450IIB4.