1990 年 107 巻 1 号 p. 97-104
Rotation of cytochrome P-450 was examined in bovine adrenocortical mitochondria before and after an enzymatic transformation of cholesterol into pregnenolone by cytochrome P-450scc in the presence of malate. Rotational diffusion was measured by observing the decay of absorption anisotropy, r(t), after photolysis of the heme•CO complex by a vertically polarized laser flash. Analysis of r(t) was based on a “rotation-about-membrane normal” model. The measurements were used to investigate substrate-dependent intermolecular interactions of cytochrome P-450 with other redox components. Rotational mobility of cytochrome P-450 was significantly dependent on the decrease in cholesterol content by side chain cleavage reaction catalyzed by cytochrome P-450scc. In a typical experiment, the observed value for the normalized time-independent anisotropy r(∞)/ r(0) was decreased from 0.78 in control mitochondria to 0.60 after conversion of 21% of cholesterol to pregnenolone, while no significant change was observed for the a verage rotational relaxation time φ of about 700 μs. Significantly high values of r(∞)/r(0)=0.78 and 0.60 imply co-existence of mobile and immobile populations of cytochrome P-450. Since we observed that the heme angle tilted 55° from membrane plane, 22% (control mitochondria) and 40% (after conversion of cholesterol to pregnenolone) of cytochrome P-450 in mitochondria are calculated to be mobile in the preparation. The significant mobilization of cytochrome P-450scc molecules caused by the conversion of cholesterol to pregnenolone is likely due to changes in protein-protein interactions with its redox partners, since the lipid fluidity was kept unchanged by the cholesterol depletion. Over several independent experiments the extent of mobilization for cytochrome P-450 showed a good correlation with the amounts of depleted cholesterol and of cholesterol-depleted P-450scc A model explaining the observed mobilization of the cytochrome in terms of dissociation of a transient multienzyme complex of cytochrome P-450scc with adrenodoxin and adrenodoxin reductase induced by cholesterol depletion is discussed based on the above results.
