The unfolding of oxidized c-type cytochromes: the instructive case of Bacillus pasteurii.

The reversible unfolding of oxidized Bacillus pasteurii cytochrome c(553) by guanidinium chloride under equilibrium conditions has been monitored by NMR and optical spectroscopy. The results obtained indicate that unfolding takes place through a mechanism involving the detachment from heme iron coordination of the sulfur of the Met71 axial ligand and yielding either a high spin (HS) or a low spin (LS(1)) species, depending on the pH value. In the LS(1) form the Met71 is replaced by another protein ligand, possibly Lys. The ligand exchange reaction does not reach completion until the protein backbone reaches a largely unfolded state, as monitored through 1H-15N NMR experiments, thus demonstrating that there is a significant correlation between formation of the Fe-S bond and native structure stability. 1H/2H exchange data, however, show that helix alpha(3), the C-terminal region of helix alpha(4), and helix alpha(5) maintain low exchangeability of the amide protons in the LS(1) form. This finding most likely implies that these regions maintain some ordered non-covalent structure, in which the amide moieties are involved in H-bonds. Finally, a folding mechanism is proposed and discussed in terms of analogies and differences with the larger mitochondrial cytochrome c proteins. It is concluded that the thermodynamic stability of the region around the metal cofactor is determined by the chemical nature of the residues around the axial methionine residue.