NADPH reduction of cytochrome P-450 at different integrational levels of the enzyme system

The complex monooxygenatic enzyme exhibits different functional behaviour at different integrational levels, thus indicating distinct organizational states. The aerobic NADPH reduction of microsomes, solubilized and reconstituted systems follows a biphasic kinetics, the two phases are attributed to associated state (cluster) and random cytochrome P-450 reduction. States of different cytochrome P-450/reductase ratio (associates) could not be differentiated in rate. Detergents (Triton N-101, cholate) are capable of disintegrating the system, at last only monophasic slow reduction is observed. The hydroxylation activity follows the respective reduction behaviour. Sedimentation analysis proves the distinct structural states. Reconstitution of the system can be achieved by means of detergent dilution as well as by combining the constituents. The activity of the reconstituted system depends on the composition of the phospholipids as well as on its organizational state. The reassociation of the solubilized enzyme system at nearly microsomal components stoichiometry (Triton N-101 dilution) proves to be thermodynamically governed leading to self-organization of the system without matrix prerequisite. Individual step rate constants of the reduction reaction and other system parameters are accessible by means of a model treatment of the disintegrated system. Further application to mixed kinetics systems is in progress.