European Molecular Biology Laboratory, Heidelberg, Germany.
A long-standing puzzle in structure-function studies of cytochrome P450cam is how the substrate, camphor, reaches the buried active site. The crystal structure shows no channel from the surface to the active site large enough for substrate to pass through. Recent experiments indicate that access of the rather nonpolar substrate to the active site is controlled by electrostatic interactions and may involve rupture of the two salt links to Asp251 [Deprez, E., Gerber, N. C., Di Primo, C., Douzou, P., Sligar, S. G., Hui Bon Hoa, G. (1994) Biochemistry 33, 14464-14468]. Consequently, we have computed the electrostatic strength of 53 ionic pairs, including 32 salt links, in cytochrome P450cam by numerical solution of the finite-difference linearized Poisson-Boltzmann equation. The calculated electrostatic free energies, delta Gtot, of the salt links range from -9 to +6 kcal/mol with approximately 60% of the salt links being energetically favorable and 40% being unfavorable with respect to mutation to their uncharged, nonpolar isosteres. Strikingly, of the four most stable salt links in the protein (delta Gtot -6 kcal/mol), two involve the propionate groups of the heme and the other two involve Asp251. In the modeled D251N mutant, for which electrostatic effects on substrate binding are diminished, the latter two salt links lose their stability (delta Gtot > -2.4 kcal/mol). Thus it appears that cytochrome P450cam has evolved four unusually strong salt bridges, stabilized by surrounding charged and polar groups in the protein, to keep its heme cofactor in place and to regulate substrate binding.
Biochemistry 1997 May 6;36(18):5402-5417
PMID: 9154922, UI: 97299773