Mutation of trimethyllysine 72 to alanine enhances his79-heme-mediated dynamics of iso-1-cytochrome c

Trimethyllysine 72 (Tml72) of yeast iso-1-cytochrome c lies across the surface of the heme crevice loop (Ω-loop D, residues 70-85) like a brace. Lys72 is oriented similarly in horse cytochrome c (Cytc). To determine whether this residue affects the dynamics of opening the heme crevice loop, we have studied the effect of a Tml72 to Ala substitution on the formation of the His79-heme alkaline conformer near neutral pH using a variant of iso-1-Cytc including K72A and K79H mutations. Guanidine hydrochloride denaturation shows that the Tml72 to Ala substitution within error does not affect the global stability of the protein. The effect of the Tml72 to Ala substitution on the thermodynamics of the His79-heme alkaline transition is also small. However, pH-jump kinetic studies of the His79-heme alkaline transition show that both the forward and backward rates of conformational change are increased by the Tml72 to Ala substitution. The barrier for opening the heme crevice is reduced by 0.5 kcal/mol and for closing the heme crevice by 0.3 kcal/mol. The ability of Tml72 to modulate the heme crevice dynamics may indicate a crucial role in regulating function, such as in the peroxidase activity seen in the early stages of apoptosis.