ELECTROCHEMICALLY INDUCED CONFORMATIONAL-CHANGES IN CYTOCHROME-C MONITORED BY FOURIER-TRANSFORM INFRARED DIFFERENCE SPECTROSCOPY - INFLUENCE OF TEMPERATURE, PH, AND ELECTRODE SURFACES

IR difference spectra between the oxidized and the reduced state of horse heart cytochrome c were obtained for different temperature and pH conditions at various surface-modified electrodes using an optically transparent thin-layer electrochemical cell. These difference spectra reflect changes in protein conformation, side-chain geometries, and protonation upon the redox transition. The IR difference spectra recorded in the 10-40-degrees-C temperature range showed thermally induced changes mainly in the amide-I (1700-1600 cm-1) and in the amide-II (ca. 1550 cm-1) spectral regions. Although the position of most of the signals remains unshifted, large differences in their relative amplitude were observed, leading in some cases to the masking and/or the disappearance of some IR signals. In the range 6.8-9.8, increasing pH of the samples led to a decrease in the reduction rate and to spectral changes which closely resemble those obtained by increasing the temperature. Both the thermal and the pH dependence of the reduced-minus-oxidized IR difference spectra reflect the transition of ferricytochrome c from the native to the alkaline form. An analysis of the IR difference spectra shows that the redox transition at neutral pH involves mainly beta-turns and beta-sheet segments of the cytochrome c molecule. However, once the ferricytochrome c alkaline transition is performed, the redox process is coupled to conformational changes involving alpha-helical segments. The shifts in tyrosine vibrational modes observed in the difference spectra obtained at neutral and slightly alkaline pH at high temperatures suggest an intermediate state of the ferricytochrome c in which the heme crevice is more accessible to the solvent. We propose that the influence of the electrode surface on the spectral changes observed may arise from the different nature of the interaction between the protein and the promoter molecules adsorbed on the electrode surface. The strength of this interaction plays an important role in the stabilization of the native structure of cytochrome c and thus delays the process of the loosening of the structure of the heme crevice. Either a metallic surface which is not fully covered or an excess of negative charges on the electrode surface may favor an opened or partially unfolded conformation of the protein.