Site-Directed Mutagenesis of Cytochrome P450scc (CYP11A1). Effect of Lysine Residue Substitution on Its Structural and Functional Properties

G. I. Lepesheva^1*, T. N. Azeva¹, N. V. Strushkevich¹, A. A. Gilep², and S. A. Usanov²

¹Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, ul. Zhodinskaya 5/2, Minsk, 220141 Belarus; fax: 375 (0172) 63-7274; E-mail: lepesh@ns.iboch.ac.by

²Present address: Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX, 75235-9038, USA; E-mail: susano@biochem.swmed.edu

^* To whom correspondence should be addressed.

Received April 18, 2000

---

Our previous chemical modification and cross-linking studies identified some positively charged amino acid residues of cytochrome P450scc that may be important for its interaction with adrenodoxin and for its functional activity. The present study was undertaken to further evaluate the role of these residues in the interaction of cytochrome P450scc with adrenodoxin using site-directed mutagenesis. Six cytochrome P450scc mutants containing replacements of the surface-exposed positively charged residues (Lys103Gln, Lys110Gln, Lys145Gln, Lys394Gln, Lys403Gln, and Lys405Gln) were expressed in E. coli cells, purified as a substrate-bound high-spin form, and characterized as compared to the wild-type protein. The replacement of the surface Lys residues does not dramatically change the protein folding or the heme pocket environment as judged from limited proteolysis and spectral studies of the cytochrome P450 mutants. The replacement of Lys in the N-terminal sequence of P450scc does not dramatically affect the activity of the heme protein. However, mutant Lys405Gln revealed rather dramatic loss of cholesterol side-chain cleavage activity, efficiency of enzymatic reduction in a reconstituted system, and apparent dissociation constant for adrenodoxin binding. The present results, together with previous findings, suggest that the changes in functional activity of mutant Lys405Gln may reflect the direct participation of this amino acid residue in the electrostatic interaction of cytochrome P450scc with its physiological partner, adrenodoxin.

---

KEY WORDS: cytochrome P450scc, site-directed mutagenesis, heterologous expression, protein-protein interactions

---