Abstract

The mechanism of energy converting NADH:ubiquinone oxidoreductase (complex I) is still unknown. A current controversy centers around the question whether electron transport of complex I is always linked to vectorial proton translocation or whether in some organisms the enzyme pumps sodium ions instead. To develop better experimental tools to elucidate its mechanism, we have reconstituted the affinity purified enzyme into proteoliposomes and monitored the generation of ΔpH and Δψ. We tested several detergents to solubilize the asolectin used for liposome formation. Tightly coupled proteoliposomes containing highly active complex I were obtained by detergent removal with BioBeads after total solubilization of the phospholipids with n-octyl-β-d-glucopyranoside. We have used dyes to monitor the formation of the two components of the proton motive force,ΔpH and Δψ, across the liposomal membrane, and analyzed the effects of inhibitors, uncouplers and ionophores on this process. We show that electron transfer of complex I of the lower eukaryote Y. lipolytica is clearly linked to proton translocation. While this study was not specifically designed to demonstrate possible additional sodium translocating properties of complex I, we did not find indications for primary or secondary Na⁺ translocation by Y. lipolytica complex I.

Keywords: Mitochondria; Complex I; Reconstitution; H⁺-pumping; Yarrowia lipolytica

Abbreviations: ACMA, 9-amino-6-chloro-2-methoxyacridine; DBQ, n-decylubiquinone; LM, n-lauryl-β-d-maltoside; ETH 157, N,N′-dibenzyl-N,N′-diphenyl-1,2-phenylenedioxydiacetamide; FCCP, carbonyl-cyanide-p-trifluoro-methoxy-phenylhydrazone; HAR, hexaammineruthenium(III)-chloride; OG, n-octyl-β-d-glucopyranoside; Q-1, 2,3-dimethoxy-5-methyl-6-(3-methyl-2-butenyl)-1,4-benzoquinone; TX-100, Triton X-100

Article Outline

1. Introduction

2. Material and methods

2.1. Materials

2.2. Analytical methods

2.3. Yeast growth and preparation of mitochondrial membranes

2.4. Purification of complex I

2.5. Complex I proteoliposomes

2.6. Measurement of catalytic activity

2.7. Measurement of proton gradient

2.8. Measurement of the membrane potential

3. Results

3.1. Properties of complex I proteoliposomes

3.2. Proton pumping by reconstituted complex I

3.3. Generation of Δψ by reconstituted complex I

4. Discussion

Acknowledgements

References

(28K)
Fig. 1. Specific activities of complex I after reconstitution with different detergents. Proteoliposomes were prepared using ‘onset’ (open columns) or ‘total’ conditions (grey columns) and specific NADH:ubiquinone oxidoreductase activities of FCCP-uncoupled proteoliposomes were measured in 20 mM K⁺-Mops, 50 mM KCl, pH 7.4 as detailed in Materials and methods. For the calculation of the specific activities, the complex I recovery and the percentage of inside-out oriented pumps were taken into account.

(35K)
Fig. 2. Effect of lipid-to-protein ratio used for reconstitution on the magnitude of ACMA-quenching. Complex I was reconstituted using OG-total solubilized phospholipids at protein-to-lipid-ratios of 1:33, 1: 67, 1:133 and 1:267 (w/w). The specific activities were 2.9, 4.9, 3.1 and 2.6 μmol·min⁻¹·mg⁻¹ and the coupling factors were 5.1, 9.2, 7.4 and 8.3 for these batches of proteoliposomes. Proteoliposomes were added at approximately the same amount of phospholipids in each measurement and the ACMA quenching was monitored as detailed under Materials and methods. 1 μM of DQA was added to inhibit complex I.

(37K)
Fig. 3. Proton pumping of reconstituted complex I. Purified complex I (NADH:HAR activity 39.1 μmol·min⁻¹·mg⁻¹) was reconstituted as detailed in Materials and Methods using OG-total solubilized asolectin at protein-to-lipid-ratios of 1:50. Uncoupled NADH:DBQ activity was 4.1 μmol·min⁻¹·mg⁻¹ and uncoupled NADH:Q₁ activity was 4.8 μmol·min⁻¹·mg⁻¹. Respiratory control ratios were 3.0 and 3.2, respectively. All measurements were done in presence of 50 mM KCl and 5 μM valinomycin with 9.8 μg of reconstituted complex I. 0.5 μM ACMA, 60 μM DBQ or Q₁ and 100 μM NADH, 1 μM DQA, 12.5 μM nigericin, 1 μM rotenone, 1 μM FCCP were added as indicated. In panel F, 1 μM DQA was added before starting the measurement.

(34K)
Fig. 4. Effect of K⁺/valinomycin and Na⁺/ETH157 on proton pumping of reconstituted complex I. Purified complex I (NADH:HAR activity 65,5 μmol·min⁻¹·mg⁻¹) was reconstituted either in 1 mM Na⁺/Mops, 50 mM NaCl, pH 7.4 or 1 mM K⁺/Mops, 50 mM KCl, pH 7.4 using OG-total solubilized asolectin at protein-to-lipid-ratios of 1:50. Uncoupled NADH:DBQ activity was 17.0 μmol·min⁻¹·mg⁻¹ in the K⁺-buffer and 12.5 μmol·min⁻¹·mg⁻¹ in Na⁺-buffer. Respiratory control ratios were 4.7 and 3.9, respectively. Measurements were done in the respective buffers (traces A and B: K⁺-buffer; traces C and D: Na⁺-buffer). Additions: 0.5 μM ACMA, 60 μM DBQ, 100 μM NADH, 0,5 μM valinomycin, 1 μM nigericin, 1 μM monensin, 1 μM FCCP, 1 μM (trace C) or 5 μM (trace D, to ensure a fast collapse of ΔΨ, comparable to valinomycin) ETH 157.

(52K)
Fig. 5. Generation of Δψ by reconstituted complex I in the presence of potassium ions. Absorbance changes (623–604 nm) of the potential-sensitive dye Oxonol VI and the NADH oxidation (340–400 nm) were recorded simultaneously with a Shimadzu MultiSpec diode array spectrophotometer as detailed under Materials and methods. Proteoliposomes were reconstituted in 20 mM K⁺/Mops, 50 mM KCl with OG-total solubilized asolectin. For each measurement, proteoliposomes (approx. 5 μg protein) were diluted in 1 ml of the same buffer. Substrates/effectors were added as indicated at the following concentrations: 2 μM Oxonol VI, 100 μM NADH, 100 μM DBQ, 5 μM valinomycin (Val), 1 μM DQA. Note, that the respiratory control ratios that can be deduced from the traces are somewhat lower than the values given in Table 1. This can be explained by the partial uncoupling effect of the dye oxonol VI.

(50K)
Fig. 6. Generation of Δψ by reconstituted complex I in the presence of sodium ions. The general conditions were as indicated in the legend of Fig. 5. For these experiments, complex I was reconstituted in 20 mM Na⁺/Mops, 50 mM NaCl with OG-total solubilized asolectin. Measurements were performed in the identical Na⁺/Mops buffer. Substrates/effectors were added as indicated at the following concentrations: 2 μM Oxonol VI, 100 μM NADH, 100 μM DBQ, 1 μM FCCP, 1 μM monensin (Mon).

Table 1.
Effect of solubilization method on proteoliposome formation

	Complex I recoverya (%)	Inside-outa %	RCRa,b
Onset	46–57	70–100	1.7–2.2
Total	49–60	40–65	3.1–5.8c

^a The range reflects the values obtained with different detergents (octylglucoside, Triton X-100, C₁₂E₈, laurylmaltoside) that were used to solubilize asolectin. The protein to phospholipids ratio was 1:50 (w/w) throughout.
^b No respiratory control ratios (RCR) were obtained for C₁₂E₈. See text for details.
^c These values were determined with different batches of proteoliposomes made with one representative complex I preparation. In some measurements with another preparation, even higher RCRs were observed (see Fig. 2).

References

[1] U. Brandt, S. Kerscher, S. Dröse, K. Zwicker and V. Zickermann, Proton pumping by NADH:ubiquinone oxidoreductase. A redox driven conformational change mechanism?, FEBS Lett. 545 (2003), pp. 9–17.

[2] J. Hirst, J. Carroll, I.M. Fearnley, R.J. Shannon and J.E. Walker, The nuclear encoded subunits of complex I from bovine heart mitochondria, Biochim. Biophys. Acta 1604 (2003), pp. 135–150.

[3] A. Abdrakhmanova, V. Zickermann, M. Bostina, M. Radermacher, H. Schägger, S. Kerscher and U. Brandt, Subunit composition of mitochondrial complex I from the yeast Yarrowia lipolytica, Biochim. Biophys. Acta 1658 (2004), pp. 148–156.

[4] U. Brandt, Proton-translocation by membrane-bound NADH:Ubiquinone-oxidoreductase (complex I) through redox-gated ligand conduction, Biochim. Biophys. Acta 1318 (1997), pp. 79–91.

[5] U. Brandt, Proton translocation in the respiratory chain involving ubiquinone—A hypothetical semiquinone switch mechanism for complex I, BioFactors 9 (1999), pp. 95–101.

[6] S. Magnitsky, L. Toulokhonova, T. Yano, V.D. Sled, C. Hagerhall, V.G. Grivennikova, D.S. Burbaev, A.D. Vinogradov and T. Ohnishi, EPR characterization of ubisemiquinones and iron–sulfur cluster N2, central components of the energy coupling in the NADH- ubiquinone oxidoreductase (complex I) in situ, J. Bioenerg. Biomembr. 34 (2002), pp. 193–208.

[7] T. Ohnishi, S. Magnitsky, L. Toulokhonova, T. Yano, T. Yagi, D.S. Burbaev and A.D. Vinogradov, EPR studies of the possible binding sites of the cluster N2, semiquinones, and specific inhibitors of the NADH:quinone oxidoreductase (complex I), Biochem. Soc. Trans. 27 (1999), pp. 586–591.

[8] P.L. Dutton, C.C. Moser, V.D. Sled, F. Daldal and T. Ohnishi, A reductant-induced oxidation mechanism for Complex I, Biochim. Biophys. Acta 1364 (1998), pp. 245–257.

[9] U. Brandt and B.L. Trumpower, The protonmotive Q cycle in mitochondria and bacteria, CRC Crit. Rev. Biochem. 29 (1994), pp. 165–197.

[10] V. Zickermann, M. Bostina, C. Hunte, T. Ruiz, M. Radermacher and U. Brandt, Functional implications from an unexpected position of the 49 kDa subunit of complex I, J. Biol. Chem. 278 (2003), pp. 29072–29078.

[11] A.A. Mamedova, P.J. Holt, J. Carroll and L.A. Sazanov, Substrate-induced conformational change in bacterial complex I, J. Biol. Chem. 279 (2004), pp. 23830–23836.

[12] P. Hellwig, S. Stolpe and T. Friedrich, Fourier transform infrared spectroscopic study on the conformational reorganization in Escherichia coli complex I due to redox-driven proton translocation, Biopolymers 74 (2004), pp. 69–72.

[13] M. Yamaguchi, G. Belogrudov and Y. Hatefi, Mitochondrial NADH-ubiquinone oxidoreductase (complex I). Effect of substrates on the fragmentation of subunits by trypsin, J. Biol. Chem. 273 (1998), pp. 8094–8098.

[14] M.K.F. Wikström, Two protons are pumped from the mitochondrial matrix per electron transferred between NADH and ubiquinone, FEBS Lett. 169 (1984), pp. 300–304.

[15] A.S. Galkin, V.G. Grivennikova and A.D. Vinogradov, H⁺/2e⁻ stoichiometry of the NADH:ubiquinone reductase reaction catalyzed by submitochondrial particles, Biochemistry (Mosc.) 66 (2001), pp. 435–443.

[16] H.G. Lawford, J.C. Cox, P.B. Garland and B.A. Haddock, Electron transport in aerobically grown Paracoccus denitrificans: kinetic characterization of the membrane-bound cytochromes and the stoichiometry of respiration-driven proton translocation, FEBS Lett. 64 (1976), pp. 369–374.

[17] A.V. Bogachev, R.A. Murtazina and V.P. Skulachev, H⁺/e⁻ stoichiometry for NADH dehydrogenase I and dimethyl sulfoxide reductase in anaerobically grown Escherichia coli cells, J. Bacteriol. 178 (1996), pp. 6233–6237.

[18] A. Baccarini Melandri, D. Zannoni and B.A. Melandri, Energy transduction in photosynthetic bacteria: VI. Respiratory sites of energy conservation in membranes from dark-grown cells of Rhodopseudomonas capsulata, Biochim. Biophys. Acta 314 (1973), pp. 298–311.

[19] S. Stolpe and T. Friedrich, The Escherichia coli NADH:ubiquinone oxidoreductase (complex I) is a primary proton pump but may be capable of secondary sodium antiport, J. Biol. Chem. 279 (2004), pp. 18377–18383.

[20] A.C. Gemperli, P. Dimroth and J. Steuber, Sodium ion cycling mediates energy coupling between complex I and ATP synthase, Proc. Natl. Acad. Sci. U. S. A. 100 (2003), pp. 839–844.

[21] A.C. Gemperli, P. Dimroth and J. Steuber, The respiratory complex I (NDH I) from Klebsiella pneumoniae, a sodium pump, J. Biol. Chem. 277 (2002), pp. 33811–33817.

[22] Y.V. Bertsova and A.V. Bogachev, The origin of the sodium-dependent NADH oxidation by the respiratory chain of Klebsiella pneumoniae, FEBS Lett. 563 (2004), pp. 207–212.

[23] P. Ahlers, K. Zwicker, S. Kerscher and U. Brandt, Function of conserved acidic residues in the PSST-homologue of complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica, J. Biol. Chem. 275 (2000), pp. 23577–23582.

[24] N. Kashani-Poor, K. Zwicker, S. Kerscher and U. Brandt, A central functional role for the 49-kDa subunit within the catalytic core of mitochondrial complex I, J. Biol. Chem. 276 (2001), pp. 24082–24087.

[25] A. Garofano, K. Zwicker, S. Kerscher, P. Okun and U. Brandt, Two aspartic acid residues in the PSST-homologous NUKM subunit of complex I from Yarrowia lipolytica are essential for catalytic activity, J. Biol. Chem. 278 (2003), pp. 42435–42440.

[26] L. Grgic, K. Zwicker, N. Kashani-Poor, S. Kerscher and U. Brandt, Functional significance of conserved histidines and arginines in the 49 kDa subunit of mitochondrial complex I, J. Biol. Chem. 279 (2004), pp. 21193–21199.

[27] S. Dröse, K. Zwicker and U. Brandt, Full recovery of the NADH:ubiquinone activity of complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica by the addition of phospholipids, Biochim. Biophys. Acta-Bioenerg. 1556 (2002), pp. 65–72.

[28] N. Kashani-Poor, S. Kerscher, V. Zickermann and U. Brandt, Efficient large scale purification of his-tagged proton translocating NADH:ubiquinone oxidoreductase (complex I) from the strictly aerobic yeast Yarrowia lipolytica, Biochim. Biophys. Acta 1504 (2001), pp. 363–370.

[29] O.H. Lowry, N.R. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193 (1951), pp. 265–275.

[30] S. Kerscher, S. Dröse, K. Zwicker, V. Zickermann and U. Brandt, Yarrowia lipolytica, a yeast genetic system to study mitochondrial complex I, Biochim. Biophys. Acta-Bioenerg. 1555 (2002), pp. 83–91.

[31] S. Kerscher, J.G. Okun and U. Brandt, A single external enzyme confers alternative NADH:ubiquinone oxidoreductase activity in Yarrowia lipolytica, J. Cell Sci. 112 (1999), pp. 2347–2354.

[32] J.-L. Rigaud, B. Pitard and R.M. Levy, Reconstitution of membrane proteins into liposomes: application to energy-transducing membrane proteins, Biochim. Biophys. Acta 1231 (1995), pp. 223–246.

[33] P.W. Holloway, Anal. Biochem. 53 (1973), pp. 304–307.

[34] R. Djafarzadeh, S. Kerscher, K. Zwicker, M. Radermacher, M. Lindahl, H. Schägger and U. Brandt, Biophysical and structural characterization of proton-translocating NADH-dehydrogenase (complex I) from the strictly aerobic yeast Yarrowia lipolytica, Biochim. Biophys. Acta 1459 (2000), pp. 230–238.

[35] J.G. Okun, V. Zickermann and U. Brandt, Properties of the common inhibitor binding domain in mitochondrial NADH-dehydrogenase (complex I), Biochem. Soc. Trans. 27 (1999), pp. 596–601.

[36] J.G. Okun, V. Zickermann, K. Zwicker, H. Schägger and U. Brandt, Binding of detergents and inhibitors to bovine complex I—A novel purification procedure of bovine complex I retaining full inhibitor sensitivity, Biochim. Biophys. Acta-Bioenerg. 1459 (2000), pp. 77–87.

[37] C.S. Huang, J. Kopecky and C.P. Lee, Mechanistic differences in the energy-linked fluorescence decreases of 9-aminoacridine dyes associated with bovine heart submitochondrial membranes, Biochim. Biophys. Acta 722 (1983), pp. 107–115.

[38] C.L. Bashford and W.S. Thayer, Thermodynamics of the electrochemical proton gradient in bovine heart submitochondrial particles, J. Biol. Chem. 252 (1977), pp. 8459–8463.

[39] C.I. Ragan and P.C. Hinkle, Ion transport and respiratory control in vesicles formed from reduced nicotinamid adenine dinucleotide coenzyme Q reductase and phospholipids, J. Biol. Chem. 250 (1975), pp. 8472–8476.

[40] J. Steuber, C. Schmid, M. Rufibach and P. Dimroth, Na⁺ translocation by complex I (NADH:quinone oxidoreductase) of Escherichia coli, Mol. Microbiol. 35 (2000), pp. 428–434.

[41] J. Steuber, The Na⁺-translocating NADH:quinone oxidoreductase (NDH I) from Klebsiella pneumoniae and Escherichia coli: implications for the mechanism of redox-driven cation translocation by complex I, J. Bioenerg. Biomembr. 33 (2001), pp. 186–197.

[42] W. Krebs, J. Steuber, A.C. Gemperli and P. Dimroth, Na⁺ translocation by theNADH:ubiquinone oxidoreductase (complex I) from Klebsiella pneumoniae, Mol. Microbiol. 33 (1999), pp. 590–598.

Corresponding author. Fax: +49 69 6301 6970.