The present study was undertaken to evaluate the role of positively charged amino
acid residues proposed to reside on the proximal surface of bovine cytochrome P450
cholesterol side chain cleavage (P450scc, CYP11A1) and to determine which residues
may be involved in protein-protein interactions with the electron carrier adrenodoxin
(Adx). In previous studies, nine different lysine residues were identified by chemical
and immunological cross-linking experiments as potentially interacting with Adx, while
in the present study, two arginine residues have been identified from sequence alignments.
From these 11 residues, 13 different P450scc mutants were made of which only seven
were able to be expressed and characterized. Each of the seven mutants were evaluated
for their ability to bind Adx, to be reduced, and for their enzymatic activity. Among
these, K403Q and K405Q showed a consistent decrease in Adx binding, the ability to
be reduced by Adx, and enzymatic activity, with K405Q being affected to a much greater
extent. More dramatic was the complete loss of Adx binding by R426Q, while still retaining
its ability to be chemically reduced and bind carbon monoxide. Independently, a homology
model of P450scc was constructed and docked with the structure of Adx. Four potential
sites of interaction were identified: P450scc:K403 with Adx:D76, P450scc:K405 with
Adx:D72; P450scc:R426 with Adx:E73, and P450scc:K267 with Adx:E47. Thus, the biochemical
and molecular modeling studies together support the hypothesis that K267, K403, K405,
and R426 participate in the electrostatic interaction of P450scc with Adx.