Biochemical studies have shown that cardiolipin is essential for the integrity and activity of the cytochrome bc(1) complex and many other membrane proteins. Recently the direct involvement of a bound cardiolipin molecule (CL) for proton uptake at center N, the site of quinone reduction, was suggested on the basis of a crystallographic study. In the study presented here, we probe the low frequency infrared spectroscopy region as a technique suitable to detect the involvement of the lipids in redox induced reactions of the protein. First the individual infrared spectroscopic features of lipids, typically present in the yeast membrane, have been monitored for different pH values in micelles and vesicles. The pK(a) values for cardiolipin molecule have been observed at 4.7 +/- 0.3 and 7.9 +/- 1.3, respectively. Lipid contributions in the electrochemically induced FTIR spectra of the bc(1) complex from yeast have been identified by comparing the spectra of the as isolated form, with samples where the lipids were digested by lipase-A(2). Overall, a noteworthy perturbation in the spectral region typical for the protein backbone can be reported. Interestingly, signals at 1159, 1113, 1039 and 980 cm(-1) have shifted, indicating the perturbation of the protonation state of cardiolipin coupled to the reduction of the hemes. Additional shifts are found and are proposed to reflect lipids reorganizing due to a change in their direct environment upon the redox reaction of the hemes. In addition a small shift in the alpha band from 559 to 556 nm can be seen after lipid depletion, reflecting the interaction with heme b(H) and heme c. Thus, our work highlights the role of lipids in enzyme reactivity and structure.
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