SELECTIVE CHEMICAL MODIFICATION OF CYS(264) WITH DIIODOFLUORESCEIN IODACETAMIDE AS A TOOL TO STUDY THE MEMBRANE TOPOLOGY OF CYTOCHROME P450SCC (CYP11A1)

Cys(264) of cytochrome P450scc (CYP11A1) was selectively labelled with diiodofluorescein iodacetamide in solution and in proteoliposomes. Th e labelling affected the interaction of P450scc with adrenodoxin and s ignificantly inhibited the side-chain cleavage activity of the soluble and membrane-bound hemeprotein in the reconstituted system. In proteo liposomes both the labelled and unlabelled hemepoteins were susceptibl e to trypsin and split into F1 and F2, two fragments corresponding to the two main domains of P450scc. These results suggest that the hinge connecting the two domains in the region Arg(250)-Asn(257) is exposed to the surface of the membrane and involved in the interaction of P450 scc with adrenodoxin.