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Impedance Spectroscopy of Bacterial Membranes: Coenzyme-Q Diffusion in a Finite Diffusion Layer Lars J. C. Jeuken,*,† Sophie A. Weiss,‡ Peter J. F. Henderson,† Stephen D. Evans,‡ and Richard J. Bushby§ Institute of Membrane and Systems Biology, School of Physics and Astronomy, and Centre for Self-Organising Molecular Systems, University of Leeds, Leeds LS2 9JT, U.K. The inner membrane of Escherichia coli, overexpressing an ubiquinol oxidase, cytochrome bo3 (cbo3), was “tethered” in a planar configuration to a gold electrode. Electron transfer to cbo3 was achieved via native ubiquinol-8 or added ubiquinol-10, and impedance spectroscopy was used to characterize the diffusion properties of the ubiquinol/ubiquinone in the tethered membrane system. Spectra were obtained at varying direct current (DC) potentials covering the potential window in which the voltammetric catalytic wave of cbo3 is visible. These spectra were compared to those obtained after addition of a potent inhibitor of cbo3, cyanide, and the difference in impedance was analyzed using a derived equivalent circuit, which is similar to that of open finite-length diffusion (OFLD) or the finite Warburg circuit, but with the boundary conditions modified to account for the fact that ubiquinol reoxidation is limited by enzyme activity. Analysis of the impedance spectra of the tethered membrane system gave kinetic parameters that are consistent with values obtained using cyclic voltammetry. Importantly, the diffusion rate of ubiquinone (10-13-10-12 cm2/s) was found to be orders of magnitude lower than accepted values for lateral diffusion (10-8-10-7 cm2/ s). It is hypothesized that this result represent perpendicular diffusion of quinone across the membrane, corresponding to a “flip” time between 0.05 and 1 s. The function of coenzyme-Q (quinone) in electron and proton transport (the “redox loop”) has been undisputed since the chemiosmotic theory was generally accepted, about a decade after it was initially proposed by Mitchell.1 However, many properties of this lipophilic quinone, like its exact position and orientation in the membrane, remain debated. The same is true for our understanding of the antioxidant role that has more recently been attributed to quinones.2 It is now generally accepted that the polyisoprene chains of quinones lie in the hydrophobic midplane or the fatty-acid chain region of the membrane,3-10 mostly parallel * Corresponding author. E-mail:
[email protected]. Fax: 0044-(0)1133433900. † Institute of Membrane and Systems Biology. ‡ School of Physics and Astronomy. § Centre for Self-Organising Molecular Systems. (1) Mitchell, P. Nature 1961, 191, 144–148. (2) Bentinger, M.; Brismar, K.; Dallner, G. Mitochondrion 2007, 7S, S41S50. (3) Ulrich, E. L.; Girvin, M. E.; Cramer, W. A.; Markley, J. L. Biochemistry 1985, 24, 2501–2508.
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with the membrane surface. However, it is debated whether this is in a linear or bend conformation. The position of the polar headgroup is either located in the midplane10,11 or at the tail-headgroup interface of the phospholipids.12 Alternatively, is has been suggested that the quinone ring oscillates between both sites13 while the isoprenoid chain anchors the coenzyme to the midplane. The reported diffusion rates of quinones also vary. Fluorescent quenching techniques usually report values in the order of 10-7 cm2/s.13-16 Fluorescence recovery after photobleaching (FRAP) and electrochemical techniques using hybrid bilayers give values 1 order of magnitude lower.4,17 Apart from the properties of the quinones themselves, the interaction between quinones and quinone enzymes also pose many unanswered questions. Quinones are substrates for a wide variety of enzymes, most of which belong the class of oxidoreductases and are either integral or peripheral membrane enzymes. The lipophilic properties of both the coenzymes and the enzymes increase the complexity of these questions. Traditional enzyme assays have often taken one of two approaches. Water soluble analogues of coenzyme-Q can be utilized. These offer experimental advantages as the substrate can be titrated into the assay solution to study the substrate concentration profile. However, these analogues have been shown to have different positions and orientations in the membrane3,13,18 and slightly higher diffusion (4) Chazotte, B.; Wu, E. S.; Hackenbrock, C. R. Biochim. Biophys. Acta-Bioenerg. 1991, 1058, 400–409. (5) Hauss, T.; Dante, S.; Haines, T. H.; Dencher, N. A. Biochim. Biophys. ActaBioenerg. 2005, 1710, 57–62. (6) Sderhll, J. A.; Laaksonen, A. J. Phys. Chem. B 2001, 105, 9308–9315. (7) Lenaz, G. J. Membr. Biol. 1988, 104, 193–209. (8) Samori, B.; Lenaz, G.; Battino, M.; Marconi, G.; Domini, I. J. Membr. Biol. 1992, 128, 193–203. (9) Lenaz, G. FEBS Lett. 2001, 509, 151–155. (10) Gomez-Fernandez, J. C.; Llamas, M. A.; Aranda, F. J. Eur. J. Biochem. 1999, 259, 739–746. (11) Castresana, J.; Alonso, A.; Arrondo, J. L. R.; Goni, F. M.; Casal, H. Eur. J. Biochem. 1992, 204, 1125–1130. (12) Fiorini, R.; Ragni, L.; Ambrosi, S.; Littarru, G. P.; Gratton, E.; Hazlett, T. Photochem. Photobiol. 2008, 84, 209–214. (13) Lenaz, G.; Samori, B.; Fato, R.; Battino, M.; Castelli, G. P.; Domini, I. Biochem. Cell Biol. 1992, 70, 504–514. (14) Di Bernardo, S.; Fato, R.; Casadio, R.; Fariselli, P.; Lenaz, G. FEBS Lett. 1998, 426, 77–80. (15) Blackwell, M. F.; Gounaris, K.; Zara, S. J.; Barber, J. Biophys. J. 1987, 51, 735–744. (16) Blackwell, M. F.; Whitmarsh, J. Biophys. J. 1990, 58, 1259–1271. (17) Marchal, D.; Boireau, W.; Laval, J. M.; Moiroux, J.; Bourdillon, C. Biophys. J. 1998, 74, 1937–1948. (18) Roche, Y.; Peretti, P.; Bernard, S. Biochim. Biophys. Acta-Biomembr. 2006, 1758, 468–478. 10.1021/ac8015856 CCC: $40.75 2008 American Chemical Society Published on Web 10/29/2008
constants.17 Additional complications arise when interpreting results of studies with detergent-solubilized enzymes. Alternatively, assays are performed with membrane extracts using the intrinsic quinone levels or the extracts are depleted from quinones using organic solvents. Here, the physiological conditions are more closely realized but the resulting kinetic analyses can show high degree of scatter. Finally, the interpretation of these results could be further complicated by the existence of an enzyme supercomplex in these extract. For a long time, it was believed that quinone-enzymes have a random organization in the membranes (e.g., refs 19 and 20), but now, the hypothesis of enzyme supercomplexes is favored again.21,22 We have recently developed a system in which the inner membrane of Escherichia coli is tethered to a gold electrode modified with cholesterol groups to bind the membrane in a planar configuration.23 In this supported membrane system, the quinone pool in the membrane can be electrochemically oxidized and reduced24 while the quinone still interacts with the oxidoreductases as was shown by the catalytic activity of cytochrome bo3 (cbo3), an ubiquinol oxidase of E. coli.23 In this thin layer system, the quinone diffusion in the finite layer can, in principle, be studied with impedance spectroscopy. Here, we describe a theoretical approach to describe an equivalent circuit for our system and have applied this theory to impedance spectra of the supported membranes. METHODS Materials. EO3-cholestol was made as previously described.25 6-Mercaptohexanol (6MH, Sigma), 2-propanol, methanol (HPLC grade, Fisher), and E. coli “polar” lipid extract (Avanti) were used as received. All electrochemical experiments were performed in 20 mM (3-(N-morpholino)propanesulfonic acid (MOPS) buffer (Sigma) with 30 mM Na2SO4 adjusted to pH 7.4 with NaOH at 20 °C. Vesicles were prepared by mixing 5 mg lipid with 0.05 mg ubiquinone-10 (Sigma) in 50/50 methanol/chloroform and drying for 2 h under nitrogen, vortexing in 1 mL buffer, and extruding 11 times through 200 nm track-etched nucleopore membranes (Avanti). Gold Electrodes. Template-stripped gold (TSG) surfaces were prepared according to Stamou et al.26 In short, 150 nm of gold (Goodfellows) was evaporated on a polished silicon wafer (Rockwood Riddings Wafer Reclaim) using an Edwards Auto 306 evaporator at
