PROTEIN CONFORMATIONAL-CHANGES IN TETRAHEME CYTOCHROMES DETECTED BY FTIR SPECTROELECTROCHEMISTRY - DESULFOVIBRIO-DESULFURICANS NORWAY-4 ANDDESULFOVIBRIO-GIGAS CYTOCHROMES-C3

The conformational change coupled to the redox processes of two tetrah eme cytochromes C3 from bacteria of the genus Desulfovibrio have been studied by UV-vis and FTIR difference spectroscopy combined with prote in electrochemistry. Two pairs of equivalent hemes were found in Desul fovibrio desulfuricans Norway 4 cytochrome c3 by UV-vis spectroelectro chemical redox titration in an optically transparent thin-layer electr ochemical cell. In contrast to this, Desulfovibrio gigas cytochrome c3 showed a UV-vis difference spectrum for the highest potential heme di fferent from that of the others. The redox titrations were monitored b y FTIR difference spectroscopy using the same spectroelectrochemical c ell. 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 secon d electron uptake. The small amplitude of the difference signals in th e reduced-minus-oxidized FTIR difference spectra obtained for the over all redox process in both Desulfovibrio cytochromes indicates a very s mall conformational change induced by the redox transition. Neverthele ss, by application of potential steps from the fully oxidized or reduc ed form to the midwave potential (as obtained from the UV-vis redox ti trations), the reduced-minus-oxidized IR difference spectra correspond ing to the intermediate redox transitions (III4 double-line arrow poin ting left and right III2II2 and III2II2 double-line pointing left and right II4) were obtained, reflecting separately the contributions of t he high- and low-potential heme pairs to the overall redox-induced con formational change. The overall redox process and both intermediate re dox 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 d ifference spectra of spinach cytochrome b559 and iron-protoporphyrin I X-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 c ytochrome b5 show signals attributed to Raman active heme skeletal mod es 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 mo des. Comparatively strong IR difference signals at 1618 cm-1, which ar e tentatively assigned to phenylalanine residues, were found in D. des ulfuricans cytochrome c3. In the spectra of D. gigas cytochrome c3, IR signals at 1614 cm-1 were detected only for the first redox transitio n (III4 double-line arrow pointing left and right III2II2). The IR red uced-minus-oxidized difference spectra of D. gigas cytochrome c3 corre sponding to both redox transitions (III4 double-line arrow pointing le ft and right III2II2 and III2II2 double-line arrow pointing left and r ight II4) show a small and sharp signal at 1512 cm-1 which is tentativ ely assigned to tyrosine residues.