Determination of "active" cytochrome P-450 from relaxation kinetics of product formation.

We estimate the "active" part of cytochrome P-450, which is involved in a special substrate transformation, by measuring the initial change of the production rate as a function of the relaxation transitions between two different steady states of the reaction cycle of cytochrome P-450 using the light-reversibility of the carbon monoxide inhibition. The kinetic data of such relaxations are interpreted within a model cycle, which reduces the reaction cycle to three steps. The estimation of the rate constant of the first reduction step, derived from model simulation of the production rate, is confirmed by independent experimental study of the reduction kinetics. An application of our model to the O-deethylation of 7-ethoxycoumarin reveals that--in a time average--10%-15% of the spectroscopically detectable cytochrome P-450 is involved in that transformation.