The Pool Function of Ubiquinone in the Mitochondrial Respiratory Chain: Role of Lateral Diffusion
The pool function of ubiquinone in the electron transfer chain was investigated by direct measurements of its lateral diffusion using a fluorescence quenching technique and by kinetic analysis of the catalytic activity in the quinone region either in reconstituted systems or in mitochondrial membranes after enrichment with soybean phospholipids. Lateral diffusion was measured exploiting dynamic fluorescence quenching of lipid-soluble probes by ubiquinones in model membranes and in mitochondria. The method was rigorously shown to measure short range diffusion and not microcollisions within the solvent cage in the nanosecond life span of the excited state of the fluorophores. The method has allowed us to calculate diffusion coefficients in the range of 10-6 cm2/s. The diffusion appears not to be affected by changing the viscosity of the outer medium, but is affected, as expected, by changing membrane viscosity by cholesterol incorporation. Very similar results were obtained in mitochondrial membranes.
The activation energies of the Q-enzymes and of integrated electron transfer are much higher (10 Kcal/mol) than those of membrane viscosity and ubiquinone lateral diffusion (2–4 Kcal/mol). The kcat/Km of ubiquinol cytochrome c reductase and the second order rate constants for ubiquinol oxidation by Complex III, directly measured, are at least three orders of magnitude less than the collisional frequencies of ubiquinone and Complex III in mitochondrial membranes, calculated from the diffusion coefficients by the Smoluchowski equation. Moreover increased membrane viscosity elicited by cholesterol incorporation retards Q diffusion but does not decrease the kcat/Km of the enzyme. Kinetic analysis of the integrated electron transport from NADH to cytochrome c in proteoliposomes, where the average distance between complexes I and III was varied increasing the phospholipid to protein ratio, or in phospholipid plus ubiquinone-enriched mitochondria, showed no decrease in the rate of electron transfer. The bulk of these observations is interpreted to mean that electron transfer is not diffusion controlled and the decrease of the electron transfer rate observed in the phospholipid-enriched membranes is a simple consequence of ubiquinone concentration not saturating the Q-enzymes according to the homogeneous pool equation.
KeywordsLateral Diffusion Reconstituted System Solvent Cage Membrane Viscosity Short Range Diffusion
Unable to display preview. Download preview PDF.
- 1.Rich, P.R. (1984) Biochim. Biophys. Acta 768, 53–79Google Scholar
- 4.Gutman, M. (1985) in: Coenzyme Q (G. Lenaz ed.) Wiley, London, pp 215–234Google Scholar
- 6.Cherry, R.J. (1979) Biochim. Biophys. Acta 559, 289–327Google Scholar
- 11.Schneider, H., Lemasters, J.J. and Hackenbrock, C.R. (1982) J. Biol. Chem. 25, 10789–10793Google Scholar
- 15.Casadio, R., Venturoli, G., Di Gioia, A., Castellani, P., Leonardi, L., and Melandri, B.A. (1984) J. Biol. Chem. 259, 9149–9157Google Scholar
- 17.Smoluchowski, V.M. (1917) Z. Phys. Chem. 92, 129–168Google Scholar
- 18.Blatt, E. and Sawyer, W.H. (1985) Biochim. Biophys. Acta 822, 43–62Google Scholar
- 19.Degli Esposti, M., Ferri, E. and Lenaz, G. (1981) Ital. J. Biochem. 30, 437–452Google Scholar
- 20.Lenaz, G. and Degli Esposti, M. (1985) in: Coenzyme Q (G. Lenaz ed.), Wiley, London, 83–105Google Scholar
- 21.Berg, H.C. (1983) Random Walks in Biology, Princeton University PressGoogle Scholar
- 27.Battino, M., Fahmy, T. and Lenaz, G. (1986) Biochim. Biophys. Acta 433, 133–148Google Scholar
- 35.Palmer, G., Tsai, A.L., Kauten, R., Degli Esposti, M. and Lenaz, G. (1985) in: Achievements and Perspectives in Mitochondrial Research (E. Quagliariello, E.C. Slater, F. Palmieri, C. Saccone and A.M. Kroon eds.) Elsevier, Amsterdam, 137–146Google Scholar
- 36.Lenaz G. and Parenti Castelli G. (1985) in: Structures and Properties of Cell Membranes (Gh. Benga ed.) Vol. 1 CRC Press Boca Raton FL. 93–136Google Scholar
- 39.Capaldi, R.A. (1982) Biochim. Biophys. Acta 694 291–306Google Scholar
- 40.Lenaz G. and Parenti Castelli G. (1984)Drugs Exp. Clin. Res. 10 481–490Google Scholar
- 43.Ragan, C.I. and Cottingham, I.R. (1985)Biochim. Biophys. Acta 811, 13–31Google Scholar
- 45.Poore, V.M., Ragan, C.I. (1982) in: Function of Quinones in Energy Conserving Systems (B.L. Trumpower, ed.) Academic Press, New York, 141–151Google Scholar
- 47.Sechi, A.M., Bertoli, E., Landi, L., Parenti Castelli, G., Lenaz, G., Curatola, G. (1973) Acta Vitamin. Enzymol. 27, 177–190Google Scholar