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

The spectroscopic and functional properties of the single Met80Ala and double Tyr67His/Met80Ala mutants have been investigated in order to gain better insight into the role of Met80-Fe ligation and of the hydrogen bonding network, which also involves Tyr67, in the structure and function of human cyt c. Structural modifications are observed in the two mutants. In the ferric form at pH 7.0, both mutants display an aquo high spin and a low spin species. The latter corresponds to an OH- ligand in Met80Ala but to a His in the double mutant. The existence of these two species is also reflected in the functional behavior of the mutants, which show a biphasic reaction pattern with an exogenous ligand, such as sodium azide. A 10-fold increase in the peroxidase activity has been observed for the Met80Ala mutant. A slight increase is still present in the double mutant, unlike the single Tyr67His mutant reported previously (Tognaccini et al., J. Inorg. Chem. 155, 56 2016), suggesting that mutations of neighboring non-axial residues affect the peroxidase activity of cyt c. On the other hand, while in the ferrous form of Met80Ala there is a mixture of two species, a five-coordinated high spin and a hexa-coordinated low spin (whose internal ligand nature is unclear), in the double mutant the situation is closely similar to wild type cyt c, with His67 likely coordinating ligand. However, this Fe-His67 bond is much weaker than the native Fe-Met80, being displaced by CO in the double mutant.