Structural characterization of the pH-denatured states of ferricytochrome-c by synchrotron small angle X-ray scattering

The ferricytochrome-c (cyt-c) shows a complex unfolding pathway characterized by a series of stable partially folded states. When titrated with HCl at low ionic strength, two transitions are detected. At pH 2, cyt-c assumes the U, unfolded state, whereas the successive addition of Cl- ion from either HCl or NaCl induces the recompaction to a molten globule conformation (A(1) and A(2) states, respectively). A second unfolded state (U-2) is also observed at pH 12. Recent data evidence different features for the local structure of the heme in the different states. To derive relationships between local and overall conformations, we analyzed the structural characteristics of the different states by synchrotron small angle x-ray scattering. The results show that in the acidic-unfolded U, form the protein assumes a worm-like conformation, whereas in the alkaline-unfolded U2 state, the cyt-c is globular. Moreover, the molten globule states induced by adding HCl or NaCl to U, appear structurally different: in the A(1) state cyt-c is dimeric and less compact, whereas in the A(2) form the protein reverts to a globular-like conformation. According to the local heme structure, a molecular model for the different forms is derived.