Protein conformational changes in tetraheme cytochromes detected by FTIR spectroelectrochemistry: Desulfovibrio desulfuricans Norway 4 and Desulfovibrio gigas cytochromes c3.

The conformational change coupled to the redox processes of two tetraheme cytochromes c3 from bacteria of the genus Desulfovibrio have been studied by UV-vis and FTIR difference spectroscopy combined with protein electrochemistry. Two pairs of equivalent hemes were found in Desulfovibrio desulfuricans Norway 4 cytochrome c3 by UV-vis spectroelectrochemical redox titration in an optically transparent thin-layer electrochemical cell. In contrast to this, Desulfovibrio gigas cytochrome c3 showed a UV-vis difference spectrum for the highest potential heme different from that of the others. The redox titrations were monitored by FTIR difference spectroscopy using the same spectroelectrochemical cell. They show that in both cytochromes the overall redox process from the fully oxidized (III4) to the fully reduced oxidation state (II4), III4<==>II4, proceeds via an intermediate oxidation stage (III2II2) which is formed after the second electron uptake. The small amplitude of the difference signals in the reduced-minus-oxidized FTIR difference spectra obtained for the overall redox process in both Desulfovibrio cytochromes indicates a very small conformational change induced by the redox transition. Nevertheless, by application of potential steps from the fully oxidized or reduced form to the midwave potential (as obtained from the UV-vis redox titrations), the reduced-minus-oxidized IR difference spectra corresponding to the intermediate redox transitions (III4<==>III2II2 and III2II2<==>II4) were obtained, reflecting separately the contributions of the high- and low-potential heme pairs to the overall redox-induced conformational change. The overall redox process and both intermediate redox transitions were fully reversible. In the spectral region between 1500 and 1200 cm-1 the IR difference spectra of both cytochromes show several signals previously observed in the reduced-minus-oxidized IR difference spectra of spinach cytochrome b559 and iron-protoporphyrin IX-bis(imidazole) model compounds [Berthomieu, C., Boussac, A., Mäntele, W., Breton, J., & Nabedryk, E. (1992) Biochemistry 31, 11460-11471]. Moreover, Raman spectra of Desulfovibrio vulgaris cytochrome c3 and cytochrome b5 show signals attributed to Raman active heme skeletal modes at nearly the same positions [Kitagawa, T., Kyogoyu, Y., Izuka, T., Ikeda-Saito, M., & Yamanaka, T. (1975) J. Biochem. 78, 719-728], thus allowing their assignment to signals arising from heme vibrational modes. Comparatively strong IR difference signals at 1618 cm-1, which are tentatively assigned to phenylalanine residues, were found in D. desulfuricans cytochrome c3. In the spectra of D. gigas cytochrome c3, IR signals at 1614 cm-1 were detected only for the first redox transition (III4<==>III2II2).(ABSTRACT TRUNCATED AT 400 WORDS)

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)

Redox-induced conformational changes in myoglobin and hemoglobin: electrochemistry and ultraviolet-visible and Fourier transform infrared difference spectroscopy at surface-modified gold electrodes in an ultra-thin-layer spectroelectrochemical cell.

Using suitable surface-modified electrodes, we have developed an electrochemical system which allows a reversible heterogeneous electron transfer at high (approximately 5 mM) protein concentrations between the electrode and myoglobin or hemoglobin in an optically transparent thin-layer electrochemical (OTTLE) cell. With this cell, which is transparent from 190 to 10,000 nm, we have been able to obtain electrochemically-induced Fourier-transform infrared (FTIR) difference spectra of both proteins. Clean protein difference spectra between the redox states were obtained because of the absence of redox mediators in the protein solution. The reduced-minus-oxidized difference spectra are characteristic for each protein and arise from redox-sensitive heme modes as well as from polypeptide backbone and amino acid side chain conformational changes concomitant with the redox transition. The amplitudes of the difference bands, however, are small as compared to the total amide I absorbance, and correspond to approximately 1% (4%) of the reduced-minus-oxidized difference absorbance in the Soret region of myoglobin (hemoglobin) and to less than 0.1% of the total amide I absorbance. Some of the bands in the 1560-1490-cm-1 spectral regions could be assigned to side-chain vibrational modes of aromatic amino acids. In the conformationally sensitive spectral region between 1680 and 1630 cm-1, bands could be attributed to peptide C = O modes because of their small (2-5 cm-1) shift in 2H2O. A similar assignment could be achieved for amide II modes because of their strong shift in 2H2O.(ABSTRACT TRUNCATED AT 250 WORDS)