pH-Dependent Local Structure of Ferricytochrome c Studied by X-Ray Absorption Spectroscopy

We have studied, using x-ray absorption spectroscopy by synchrotron radiation, the native state of the horse heart cytochrome c (N), the HCl denatured state (U 1 at pH 2), the NaOH denatured state (U 2 at pH 12), the intermediate HCl induced state (A 1 at pH 0.5), and the intermediate NaCl induced state (A 2 at pH 2). Although many results concerning the native and denatured states of this protein have been published, a site-specific structure analysis of the denatured and intermediate solvent induced states has never been attempted before. Model systems and myoglobin in different states of coordination are compared with cytochrome c spectra to have insight into the protein site structure in our experimental conditions. New features are evidenced by our results: 1) x-ray absorption near edge structure (XANES) of the HCl intermediate state (A 1) presents typical structures of a pentacoordinate Fe(III) system, and 2) local site structures of the two intermediate states (A 1 and A 2) are different.