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

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• 作者：Bartalesi ; Ilaria ; Bertini ; Ivano ; Ghosh ; Kaushik ; Rosato ; Antonio ; Turano ; Paola
• 关键词：cytochrome c ; protein unfolding ; NMR spectroscopy ; iron coordination ; Gram positive ; Bpcytc ; oxidized fragment 22&ndash ; 92 of Bacillus pasteurii cytochrome c₅₅₃ ; CPMG ; Carr&ndash ; Purcell&ndash ; Meiboom&ndash ; Gill ; EXSY ; exchange spectroscopy ; Gd
• 刊名：Journal of Molecular Biology
• 出版年：2002
• 出版时间：August 23, 2002
• 年：2002
• 卷：321
• 期：4
• 页码：693-701
• 全文大小：284 K

文摘

The reversible unfolding of oxidized Bacillus pasteurii cytochrome c₅₅₃ 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 (LS₁) species, depending on the pH value. In the LS₁ 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 α₃, the C-terminal region of helix α₄, and helix α₅ maintain low exchangeability of the amide protons in the LS₁ 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.