How does heme axial ligand deletion affect the structure and the function of cytochrome b(562)?

We have recently generated a new mutant of cytochrome b(562) (cytb(562)) in which Met7, one of the axial heme ligands, is replaced by Ala (M7A cytb(56 2)). The M7A cytb(562) can bind heme and the UV-Visible absorption spectrum is of a typical high-spin ferric heme. To investigate the effect of the la ck of Met7 ligation on the structural integrity of cytb(562), thermal trans ition analyses of M7A cytb(562) were conducted. From the thermodynamic para meters obtained, it is concluded that the folding of M7A cytb(562) is compa rable to the apoprotein despite the presence of heme. On the other hand, ex ogenous ligands such as cyanide and azide ions are readily bound to the hem e iron, indicating that the axial coordination site is available for substr ate binding. The peroxidase activity of this mutant is thus examined to eva luate new enzymatic function at this site and M7A cytb(562) was found to ca talyze an oxidation reaction of aromatic substrates with hydrogen peroxide. These observations demonstrate that the Met7/His102 bis-ligation to the he me iron is crucial for the stable folding Of cytb(562), whereas the functio nal conversion of cytb(562) is successfully achieved by the loose folding t ogether with the open coordination site.