Flavin-binding and protein structural integrity studies on NADPH-cytochrome P450 reductase are consistent with the presence of distinct domains.

NADPH-cytochrome P450 reductase (reductase) contains FMN and FAD in 1:1 stoichiometry as tightly bound cofactors. Electrons from NADPH are transferred to cytochrome P450 through the intermediacy of reductase. A knowledge of the interactions which must occur to allow the intermolecular and intramolecular transfer of electrons is not only of intrinsic interest but is necessary to understand the regulation of the overall oxidation-reduction processes in which cytochromes P450 participate in the endoplasmic reticulum of many organs. In the present study, urea has been employed as a chaotropic agent to study the dissociation of flavins from NADPH-cytochrome P450 reductase. The results show that dissociation of FMN occurs at concentrations of urea between 0 and 1 M and that, as the concentrations of urea approach 1 M, the intrinsic protein fluorescence increases, indicating a change in protein conformation. Above 2 M urea protein fluorescence increases, reaching a plateau at 3 M urea, and FAD begins to dissociate from the enzyme. In the range of 0-1 M urea, a completely reversible dissociation of FMN occurs and, at 3 M urea, the fluorescence values representing flavin dissociation and protein conformation changes have reached a maximum. Thus, the definition of various states of the flavoprotein with both, one, or no flavins bound and the ability to remove the flavins reversibly under specific conditions have permitted the construction of a simple model to explain the various unfolding intermediates of this enzyme. Our experiments suggest that reductase is composed of distinct domains which can be examined independently by the application of chaotropic agents.