[Analysis of protein-protein interactions in an enzymatically complete cholesterol hydroxylating system]

The interaction of the cholesterol side chain cleavage system components--adrenodoxin reductase, adrenodoxin, and cytochrome P-450scc was studied, using enzymatic conversion of the high spin form of cytochrome P-450scc to its low spin form and spectrophotometric analysis of various stoichiometric protein mixtures in the presence of pregnenolone and Tween 20. Upon reconstitution of the enzymatically complete system in the presence of NADPH the formation of a ternary protein complex was accompanied by a 2-10-fold increase of the Kd values as compared to the formation of binary adrenodoxin reductase . adrenodoxin and cytochrome P-450 . adrenodoxin complexes. The non-ionic detergent Tween 20 destabilized the adrenodoxin cytochrome P-450scc complex; as a result adrenodoxin reductase acquired the ability to replace adrenodoxin from the binary complex. At the same time this replacement was not observed in the presence of pregnenolone which does not change adrenodoxin affinity for cytochrome P-450scc thus indicating the formation of a ternary protein complex. Titration by adrenodoxin of different stoichiometric mixtures of adrenodoxin reductase and cytochrome P-450scc in the presence of Tween 20 demonstrated that the predominant formation of the binary adrenodoxin . adrenodoxin reductase complex is correlated with an increase of Kd for the cytochrome P-450scc . adrenodoxin complex and that at relatively low detergent concentrations adrenodoxin can interact both with adrenodoxin reductase and cytochrome P-450scc to form a ternary protein complex.