Selective Chemical Modification of Cys²⁶⁴ with Diiodofluorescein Iodoacetamide to Study Membrane Topology of Cytochrome P450scc (CYP11A1)

A. A. Chernogolov,¹ D. Schwarz,² and S. A. Usanov^1,3

¹Institute of Bioorganic Chemistry, Academy of Sciences of Belarus, Zhodinskaya 5/2, Minsk, 220141 Belarus; fax: (0172) 63-72-74; E-mail: usanov@eco2.iasnet.com

²Max Delbruck Center for Molecular Medicine, Robert Roessle st. 10, Berlin-Buch, 13122 Germany; fax: (030) 949-41-61.

³To whom correspondence should be addressed.

Submitted April 28, 1996; revision submitted August 7, 1996.

Selective chemical modification of cytochrome P450scc (CYP11A1) with diiodofluorescein iodoacetamide has been done. It is shown that Cys²⁶⁴ of cytochrome P450scc undergoes selective modification. The labelling influences the catalytic activity of soluble and membrane-bound cytochrome P450scc in the cholesterol side-chain cleavage reaction in a reconstituted system. The decrease of activity of cytochrome P450scc correlates with the lowering of its affinity for the intermediate electron transfer protein--adrenodoxin. Cytochrome P450scc incorporated into liposomes is also accessible for modification, being cleaved by trypsin to form two main fragments, F1 and F2, which correspond to the N- and C-terminal sequences of cytochrome P450scc, respectively. These results indicate that the fragment of cytochrome P450scc molecule containing Cys²⁶⁴ is exposed on the protein surface, is not inserted into the membrane, and appear to be involved in protein--protein interactions.

KEY WORDS: cytochrome P450scc (CYP11A1), domains, membrane topology, chemical modification, bovine adrenal cortex.

---