Optical biosensor studies on the productive complex formation between the components of cytochrome p450scc dependent monooxygenase system

The formation of individual complexes between the components of cholesterol side chain cleavage system - cytochrome P450scc, adrenodoxin (Ad) and adre nodoxin reductase (AdR) was kinetically characterized and their association and dissociation rate constants were measured by optical biosensor. The do minant role of interprotein electrostatic interactions in productive comple x formation was demonstrated. Despite of the fact that P450scc and AdR comp ete for the binding with the same or closely placed negatively charged grou ps on the surface of immobilized Ad, the formation of the AdR/P450scc/Ad te rnary complex upon AdR immobilization on dextran was registered. It is show n, that Ad does not bind to AdR immobilized via amino groups AdR(im) but it is possible only after the preliminary binding of P450scc to AdR(im). The life time of such ternary complex, about 15 s, is sufficient for the realiz ation of 5-8 catalytic cycles.