Spectro-Electrochemical Investigation of the bc1 Complex from the yeast Saccharomyces cerevisiae using Surface Enhanced B-Band Resonance Raman Spectroscopy

The ubiquinol-cytochrome c oxidoreductase (bc1 complex), also denoted complex III of the respiratory chain, is present in the inner mitochondrial membrane of eukaryotic organisms and many aerobic bacteria. It catalyzes electron transfer from ubiquinol to cytochrome c coupled to the electrogenic translocation of protons across the membrane. The proton electrochemical gradient thus generated is used for the synthesis of ATP. The crystal structure of the bacterial and the mitochondrial bc1 complex was determined by X-ray diffraction.(Xia, Yu et al. 1997; Hunte, Koepke et al. 2000; Lange and Hunte 2002; Palsdottir, Lojero et al. 2003; Berry, Huang et al. 2004; Solmaz and Hunte 2008) Both are present as a dimer, the mitochondrial complex differs from the bacterial one by a larger number of subunits extending into the aqueous phase. The elements essential for the function of the enzyme such as the location and orientation of the redox centers, however, are very similar. Three of the four prosthetic groups of the bc1 complex, heme bl and bh and cytochrome c1, are metalloporphyrins.. Vibrational modes of metalloporphyrins have been extensively studied by Resonance Raman (RR) spectroscopy, since specific modes are very sensitive to the redox state of the heme structure.(Kitagawa, Kyogoku et al. 1975; Spiro 1975; Kitagawa, Ozaki et al. 1978; Spiro 1978) RR spectra were obtained mostly from B-band (Soret) excitations,(Spiro 1988) whereas Q-band excited spectra are less intensive and informative at low protein concentrations.(Le Moigne, Schoepp et al. 1999) Soret excitation and Q-band resonance was applied for investigations of the bacterial (Hobbs, Kriauciunas et al. 1990; Le Moigne, Schoepp et al. 1999) and mitochondrial bc1 complex (Gao, Qin et al. 1998), respectively. In these cases the redox state of the hemes was altered by adding soluble redox compounds such as ascorbate and sodium hydrosulphite to the protein, which was present in the detergent-solubilized form. By contrast in the present investigation we use electrochemistry to direct electron transfer (ET) into the enzyme reconstituted into a lipid bilayer, a method that we had introduced successfully in the case of cytochrome c