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.(ABSTRACT TRUNCATED AT 250 WORDS)