Comparison of thermodynamic and kinetic effects between the Leu32-->norvaline and Leu35-->norvaline substitutions of the three-fragment complex of cytochrome c.
The three fragment complex (1-25)H.(28-38).(39-104) of horse cytochrome c (e.g., (1-25)H, the heme fragment containing residues 1 to 25) closely resembles the native protein except for residues 39 to 55, which are flexible. We have investigated how the Leu35-->Nva (norvaline) substitution differs from the Leu32-->Nva in the perturbation of the stability of this complex. The side chains of Leu32 and Leu35 are adjacent in the well-packed hydrophobic core of tuna cytochrome c (T. Takano and 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 orientation); (ii) the heat stability of the 695-nm band which monitors the Fe-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 suggest that the Leu32-->Nva is unique in that the stabilizing energy associated 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 substitutions introduce no stereochemical conflict and remove only the gamma-methyl groups of the Leu side chains which are presumably positioned adjacently with respect to each other in the core. Furthermore, the 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 strength of (39-104). These properties resemble the core domain-domain interaction (A. Fisher and H. Taniuchi (1992) Arch. Biochem. Biophys. 296, 1-16).
Picur
B
B
Laboratory of Chemical Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Lisowski
M
M
Taniuchi
H
H
Poerio
E
E
D000595:Amino Acid Sequence; D000818:Animals; D003574:Cytochrome c Group / Q000737:chemistry*; D006418:Heme / Q000737:chemistry; D006736:Horses; D066298:In Vitro Techniques; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D017434:Protein Structure, Tertiary; D013057:Spectrum Analysis; D013816:Thermodynamics; D014633:Valine / Q000031:analogs & derivatives / Q000737:chemistry
D003574:Cytochrome c Group; D010446:Peptide Fragments; D006418:Heme; C005313:norvaline; D007930:Leucine; D014633:Valine