Protein rotation study of cytochrome P-450 in submitochondrial particles: effect of KCl and intermolecular...

The rotational diffusion of cytochrome P-450 in submitochondrial particles (SMP) of bovine adrenocortical mitochondria was measured by detecting 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 the effect of KCl on intermolecular interactions involving cytochrome P-450 and to investigate the interactions of cytochrome P-450 with other redox partners. The rotational diffusion of cytochrome P-450 was significantly dependent on KCl concentration. When the KCl concentration was increased from 0 to 1,000 mM, the mobile population of cytochrome P-450 was increased from 33 to 82%. After removing the KCl, the mobile population of cytochrome P-450 returned to the original 33%. These results suggest that nonspecific protein aggregates are dissociated by the presence of KCl, possibly due to the change in electrostatic interactions, resulting in mobilization of cytochrome P-450. SMP were observed to be nearly free from adrenodoxin and adrenodoxin reductase. The addition of adrenodoxin to SMP increased the mobile population of cytochrome P-450 from 35 to 54%. Further addition of adrenodoxin reductase to SMP containing adrenodoxin immobilized cytochrome P-450 by 6%. The addition of only adrenodoxin reductase to SMP, however, did not immobilize cytochrome P-450. The present results are consistent with our previous observations [Ohta, Y., Mitani, F., Ishimura, Y., Yanagibashi, K., Kawamura, M., & Kawato, S. (1990) J. Biochem. 107, 97-104] that cholesterol-bearing P-450SCC forms a transient ternary association with adrenodoxin and adrenodoxin reductase.