The Unfolding of Oxidized c-Type Cytochromes: The Instructive Case of Bacillus pasteurii

Abstract

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 α 3, the C-terminal region of helix α 4, and helix α 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.