How does heme axial ligand deletion affect the structure and the function of cytochrome b₅₆₂?

We have recently generated a new mutant of cytochrome b₅₆₂ (cytb₅₆₂) in which Met7, one of the axial heme ligands, is replaced by Ala (M7A cytb₅₆₂). The M7A cytb₅₆₂ can bind heme and the UV-visible absorption spectrum is of a typical high-spin ferric heme. To investigate the effect of the lack of Met7 ligation on the structural integrity of cytb₅₆₂, thermal transition analyses of M7A cytb₅₆₂ were conducted. From the thermodynamic parameters obtained, it is concluded that the folding of M7A cytb₅₆₂ is comparable to the apoprotein despite the presence of heme. On the other hand, exogenous ligands such as cyanide and azide ions are readily bound to the heme iron, indicating that the axial coordination site is available for substrate binding. The peroxidase activity of this mutant is thus examined to evaluate new enzymatic function at this site and M7A cytb₅₆₂ was found to catalyze an oxidation reaction of aromatic substrates with hydrogen peroxide. These observations demonstrate that the Met7/His102 bis-ligation to the heme iron is crucial for the stable folding of cytb₅₆₂, whereas the functional conversion of cytb₅₆₂ is successfully achieved by the loose folding together with the open coordination site.