A study on the role of evolutionarily invariant leucine 32 of cytochrome c.
To investigate the role of evolutionarily invariant leucine 32 of horse cytochrome c, analogs of residues 28-38, (28-38), each containing a substituted amino acid at positions 32 or 35 were synthesized using Merrifield's method. Position 35 is leucine in horse cytochrome c but replaced by nonpolar amino acids in some species. The ability of the analogs to bind to the two-fragment complex of ferri- or ferro heme fragment (1-25)H and apofragment (39-104) was measured using gel filtration and equilibrium dialysis. Replacement of leucine 32 with isoleucine, for example, increased the dissociation constant by more than 400-fold for the ferrous complex. In contrast, replacement of leucine 35 with isoleucine seems to increase it only by a small degree. Since both leucine 32 and leucine 35 are completely buried within the structure, hydrophobic interaction would not explain this striking difference. However, thermodynamic analyses and absorption spectra of the ferric complex have indicated that replacement with norvaline of leucine 32 increases both delta H and delta S (more positive) associated with formation of an intermediate three-fragment complex and decreases both delta H and delta S (more negative) associated with transformation from the intermediate to the ground state, resulting in weakening the methionine 80--S--heme-Fe bond formed in the latter step. Taking the results together with the fragment exchange studies on the ferrous complex and available evidence, we suggest that the interaction involving leucine 32 would be coupled not only with the methionine 80--S--heme-Fe bond but also with the energy state of other distant residues such as tryptophan 59, generating extra energy for modulating the binding of the complex, i.e. the force of folding. In contrast, leucine 35 would be less important even if it were involved in such coupling.
D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D003574:Cytochrome c Group; D003956:Dialysis; D006736:Horses; D007930:Leucine; D008433:Mathematics; D008956:Models, Chemical; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D003574:Cytochrome c Group; D007930:Leucine