To investigate the role of the heme axial ligand in the conformational
stability of c-type cytochrome, we constructed M58C and M58H mutants of the
red alga Porphyra yezoensis cytochrome c6 in which the sixth heme iron
ligand (Met58) was replaced with Cys and His residues, respectively. The
Gibbs free energy change for unfolding of the M58H mutant in water
(ΔGounf=1.48 kcal/mol) was lower than that of the wild-type (2.43
kcal/mol), possibly due to the steric effects of the mutation on the
apoprotein structure. On the other hand, the M58C mutant exhibited a
ΔGounf of 5.45 kcal/mol, a significant increase by 3.02 kcal/mol
compared with that of wild-type. This increase was possibly responsible for
the sixth heme axial bond of M58C mutant being more stable than that of
wild-type according to the heme-bound denaturation curve. Based on these
observations, we propose that the sixth heme axial ligand is an important
key to determine the conformational stability of c-type cytochromes, and
the sixth Cys heme ligand will give stabilizing effects.