Effect of an Ala81His mutation on the Met80 loop dynamics of iso-1-cytochrome c.

An A81H variant of yeast iso-1-cytochrome c is prepared to test the hypothesis that the steric size of the amino acid at sequence position 81 of cytochrome c, which has evolved from Ala in yeast to Ile in mammals, slows the dynamics of the opening of the heme crevice. The A81H mutation is used both to increase steric size and to provide a probe of the dynamics of the heme crevice through measurement of the thermodynamics and kinetics of the His81-mediated alkaline conformational transition of A81H iso-1-cytochrome c. Thermodynamic measurements show that the native conformer is more stable than the His81-heme alkaline conformer for A81H iso-1-cytochrome c. ΔGu°(H2O) is approximately 1.9 kcal/mol for formation of the His81-heme alkaline conformer. By contrast, for K79H iso-1-cytochrome c, the native conformer is less stable than the His79-heme alkaline conformer. ΔGu°(H2O) is approximately −0.34 kcal/mol for formation of the His79-heme alkaline conformer. pH jump and gated electron transfer kinetics demonst...