COMPARISON OF THERMODYNAMIC AND KINETIC EFFECTS BETWEEN THE LEU32-]NORVALINE AND LEU35-]NORVALINE SUBSTITUTIONS OF THE 3-FRAGMENT COMPLEX OF CYTOCHROME-C

The three fragment complex (1-25)H.(28-38).(39-104) of horse cytochrom e c (e.g., (1-25)H, the heme fragment containing residues 1 to 25) clo sely resembles the native protein except for residues 39 to 55, which are flexible. We have investigated how the Leu35-->Nva (norvaline) sub stitution differs from the Leu32-->Nva in the perturbation of the stab ility of this complex. The side chains of Leu32 and Leu35 are adjacent in the well-packed hydrophobic core of tuna cytochrome c (T. Takano a nd R. E. Dickerson (1981) J. Mol. Biol. 153, 95-115). We measured the effects of the substitutions on (i) the binding of (28-38) with ferri- and ferrocomplexes on the right side of the heme (Dickerson's orienta tion); (ii) the heat stability of the 695-nm band which monitors the F e-S bond on the left side of the heme (cf. Takano and Dickerson, 1981) ; and (iii) the rate constant for the direct dissociation of (39-104) of the ferrous complex as a function of temperature. The results sugge st that the Leu32-->Nva is unique in that the stabilizing energy assoc iated with the ground state is markedly more perturbed in the Leu32--> Nva than in the Leu35-->Nva. This is true despite the fact that both s ubstitutions introduce no stereochemical conflict and remove only the gamma-methyl groups of the Leu side chains which are presumably positi oned adjacently with respect to each other in the core. Furthermore, t he effect of the perturbation of the structure imposed by the removal of the Leu32 gamma-methyl group propagates itself perhaps through the core and affects the stability of the Fe-S bond and the binding streng th of (39-104). These properties resemble the core domain-domain inter action (A. Fisher and H. Taniuchi (1992) Arch. Biochem. Biophys. 296, 1-16). (C) 1994 Academic Press, Inc.