SELECTIVE CHEMICAL MODIFICATION OF CYS(264) WITH DIIODOFLUORESCEIN IODOACETAMIDE TO STUDY MEMBRANE TOPOLOGY OF CYTOCHROME P450SCC (CYP11A1)

Selective chemical modification of cytochrome P450scc (CYP11Al) with d iiodofluorescein iodoacetamide has been done. It is shown that Cys(264 ) of cytochrome P450scc undergoes selective modification. The labellin g influences the catalytic activity of soluble and membrane-bound cyto chrome P450scc in the cholesterol side-chain cleavage reaction in a re constituted system. The decrease of activity of cytochrome P450sce cor relates with the lowering of its affinity for the intermediate electro n transfer protein-adrenodoxin. Cytochrome P450scc incorporated into l iposomes is also accessible for modification, being cleaved by trypsin to form two main fragments, Fl and F2, which correspond to the N- and C-terminal sequences of cytochrome P450scc, respectively. These resul ts indicate that the fragment of cytochrome P450scc molecule containin g Cys(264) is exposed on the protein surface, is not inserted into the membrane, and appear to be involved in protein-protein interactions.