EXCEPTIONALLY STABLE SALT BRIDGES IN CYTOCHROME P450CAM HAVE FUNCTIONAL ROLES

A long-standing puzzle in structure-function studies of cytochrome P45 0cam is how the substrate, camphor, reaches the buried active site. Th e crystal structure shows no channel from the surface to the active si te large enough for substrate to pass through. Recent experiments indi cate that access of the rather nonpolar substrate to the active site i s 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 strengt h of 53 ionic pairs, including 32 salt links, in cytochrome P450cam by numerical solution of the finite-difference linearized Poisson-Boltzm ann equation, The calculated electrostatic free energies, Delta G(tot) of the salt links range from -9 to +6 kcal/mol with approximately 60% of the salt links being energetically favorable and 40% being unfavor able with respect to mutation to their uncharged, nonpolar isosteres. Strikingly, of the four most stable salt links in the protein (Delta G (tot) < -6 kcal/mol), two involve the propionate groups of the heme an d 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 G(tot) > -2.4 kcal/mol). T hus it appears that cytochrome P450cam has evolved four unusually stro ng salt bridges, stabilized by surrounding charged and polar groups in the protein, to keep its heme cofactor in place and to regulate subst rate binding.