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 oxidiation state (II4), III4 double-line arrow pointing left and right 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 double-line arrow pointing left and right III2II2 and III2II2 double-line pointing left and right 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., Mantele, 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 double-line arrow pointing left and right III2II2). The IR reduced-minus-oxidized difference spectra of D. gigas cytochrome c3 corresponding to both redox transitions (III4 double-line arrow pointing left and right III2II2 and III2II2 double-line arrow pointing left and right II4) show a small and sharp signal at 1512 cm-1 which is tentatively assigned to tyrosine residues.