Thermal stability of hydrophobic heme pocket variants of oxidized cytochrome c

Microcalorimetry has been used to measure the stabilities of mutational var iants of yeast iso-1 cytochrome c in which F82 and L85 have been replaced b y other hydrophobic amino acids. Specifically, F82 has been replaced by Y a nd L85 by A. The double mutant F82Y, L85A iso-1 has also been studied, and the mutational perturbations are compared to those for the two single mutan ts, F82Y iso-1 and L85A iso-1. Results are interpreted in terms of known cr ystallographic structures. The data show that (I) the destabilization of th e mutant proteins is similar in magnitude to that which is theoretically pr edicted by the more obvious mutation-induced structural effects; (2) the fr ee energy of destabilization of the double mutant, F82Y, L85A iso-1, is les s than the sum of those of the two single mutants, almost certainly because , in the double mutant, the -OH group of Y82 is able to protrude into the c avity formed by the L85A substitution. The more favorable structural accomm odation of the new -OH group in the double mutant leads to additional stabi lity through (1) further decreases in the volumes of internal cavities and (2) formation of an extra protein-protein hydrogen bond.