NMR investigation of ferricytochrome c unfolding: Detection of an equilibrium unfolding intermediate and residual structure in the denatured state

Horse ferricytochrome c (cyt c) undergoes exchange of one of its axial heme ligands (Met-80) for one or more non-native ligands under denaturing condi tions. We have used H-1 NMR spectroscopy to detect two conformations of par amagnetic cyt c with non-native heme ligation through a range of urea conce ntrations. One nonnative form is an equilibrium unfolding intermediate obse rved under partially denaturing conditions and is attributed to replacement of Met-so with one or more Lys side chains. The second non-native form, in which the native Met ligand is replaced by a His, is observed under strong ly denaturing conditions. Thermodynamic analysis of these data indicates a relatively small Delta G (17 kJ/mol) for the transition from native to the Lys-ligated intermediate and a significantly larger Delta G (47 kJ/mol) for the transition from native to the His-ligated species. Although CD and flu orescence data indicate that the equilibrium unfolding of cyt c is a two-st ate process, these NMR results implicate an intermediate with His-Lys ligat ion.