Tryptophan fluorescence of mitochondrial complex III reconstituted in phosphatidylcholine bilayers.
The tryptophan intrinsic fluorescence of mitochondrial complex III reconstituted in phosphatidylcholine bilayers was examined at different temperatures. Absorption and emission maxima occur at 277 and 332 nm, irrespective of temperature or lipid:protein ratio even if there are indications (from fluorescence quenching) of protein conformational changes as a function of lipid:protein ratio. Low values of Trp fluorescence quantum yield in complex III (0.008-0.010) are probably due to the neighborhood of the heme groups. The temperature-dependent decrease of fluorescence intensity is nonlinear; the corresponding Arrhenius plots show "breaks" or discontinuities that could be interpreted as thermally dependent changes in protein conformation. However, no temperature-dependent changes in fluorescence quenching have been observed that may be related to protein conformational changes. In addition, Arrhenius plots of the fluorescence intensity of simple molecules, such as Trp or 1-anilino-8-naphthalene sulfonate in the presence of aqueous phospholipid dispersions, also show breaks in the same temperature range. Stern-Volmer plots of acrylamide and iodide quenching were also nonlinear, indicating large differences in quenching constants for the various tryptophanyl residues. The quenching results also suggest that, at high lipid:protein ratios, the microviscosity of the protein matrix is higher than that in lipid-poor systems. Comparison of quenching efficiencies of iodide and acrylamide suggest that no significant fraction of the fluorophores occurs in the neighborhood of charged residues.
Valpuesta
J M
JM
Department of Biochemistry, Faculty of Science, University of the Basque Country, Bilbao, Spain.
Goñi
F M
FM
Macarulla
J M
JM
D000818:Animals; D002417:Cattle; D014450:Electron Transport Complex III / Q000378:metabolism*; D007700:Kinetics; D008051:Lipid Bilayers; D008929:Mitochondria, Heart / Q000201:enzymology; D010713:Phosphatidylcholines; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D014364:Tryptophan; D014450:Electron Transport Complex III