Effect of an Ala81His Mutation on the Met80 Loop Dynamics of Iso-1-cytochromec

Abstract

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. ΔGuo(H2O) is about 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. ΔGuo(H2O) is about −0.34 kcal/mol for formation of the His79-heme alkaline conformer. pH jump and gated electron transfer kinetics demonstrate that this stabilization of the native conformer in A81H iso-1-cytochrome c arises primarily from a decrease in the rate constant for formation of the His81-heme alkaline conformer, kf,His81, relative to kf,His79 for formation of the His79-heme alkaline conformer, which forms by a similar mechanism to that observed for the His81-heme alkaline conformer. The result is discussed in terms of the effect of global protein stability on protein dynamics and in terms of optimization of the sequence of cytochrome c for its role as a peroxidase in the early stages of apoptosis in higher eukaryotes.

Naturally Occurring I81N Mutation in Human Cytochrome c Regulates Both Inherent Peroxidase Activity and Interactions with Neuroglobin

“…Thus, Cyt c can oxidize the lipids such as cardiolipin on the mitochondrial membrane to enhance the membrane permeability, which promotes the release of Cyt c to the cytoplasm and subsequently facilitates the assembly of the apoptosome. 10 , 11 …”

Disruption of a hydrogen bond network in human versus spider monkey cytochrome c affects heme crevice stability

“…The peroxidase activity of Cyt c is linked to loss of the Met80-heme bond [5, 6]. Thus, there has been considerable interest recently in the factors that control the stability of the heme crevice [10, 11]. …”

The Met80Ala and Tyr67His/Met80Ala mutants of human cytochrome c shed light on the reciprocal role of Met80 and Tyr67 in regulating ligand access into the heme pocket

“…Recently, we have shown that Tyr67 plays a crucial role in maintaining this structure in human cyt c [14]. However, although the amino acid sequence of human and yeast cyt c share about 70% identity [10], major differences between these two cytochromes are found for the surface amino acid residues involved in cardiolipin binding and modulation of the peroxidase activity [23]. Therefore, caution must be used when comparing conformational stability and peroxidase activity between various cyt c proteins.…”

“…The release of cyt c from mitochondria into the cytosol upon CL peroxidation is an important apoptotic trigger [7,8]. The identification of these alternative conformations and characterization of the structural features that determine the flexibility of the cyt c-CL complex, which confer to the protein important roles in "life" and "death" processes, continue to be a major challenge [9,10].…”

“…Indeed, recent findings on various cyt c proteins from higher eukaryotes reveal the presence of more sterically bulky amino acids in the heme crevice loop with respect to yeast cyt c. These features may be responsible for the slow opening of the heme crevice loop and reduced loss of Met80 ligation in human cyt c, suggesting that the heme crevice loop has evolved to minimize undesirable peroxidase activity [10,16,17].…”