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Originally published In Press as doi:10.1074/jbc.M311242200 on February 17, 2004

J. Biol. Chem., Vol. 279, Issue 18, 18377-18383, April 30, 2004
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The Escherichia coli NADH:Ubiquinone Oxidoreductase (Complex I) Is a Primary Proton Pump but May Be Capable of Secondary Sodium Antiport*

Stefan Stolpe and Thorsten Friedrich{ddagger}

From the Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität Freiburg, Albertstrasse 21, D-79104 Freiburg, Germany

Received for publication, October 13, 2003 , and in revised form, February 9, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The NADH:ubiquinone oxidoreductase (complex I) couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Recently, it was demonstrated that complex I from Klebsiella pneumoniae translocates sodium ions instead of protons. Experimental evidence suggested that complex I from the close relative Escherichia coli works as a primary sodium pump as well. However, data obtained with whole cells showed the presence of an NADH-induced electrochemical proton gradient. In addition, Fourier transform IR spectroscopy demonstrated that the redox reaction of the E. coli complex I is coupled to a protonation of amino acids. To resolve this contradiction we measured the properties of isolated E. coli complex I reconstituted in phospholipids. We found that the NADH:ubiquinone oxidoreductase activity did not depend on the sodium concentration. The redox reaction of the complex in proteoliposomes caused a membrane potential due to an electrochemical proton gradient as measured with fluorescent probes. The signals were sensitive to the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), the inhibitors piericidin A, dicyclohexylcarbodi-imide (DCCD), and amiloride derivatives, but were insensitive to the sodium ionophore ETH-157. Furthermore, monensin acting as a Na+/H+ exchanger prevented the generation of a proton gradient. Thus, our data demonstrated that the E. coli complex I is a primary electrogenic proton pump. However, the magnitude of the pH gradient depended on the sodium concentration. The capability of complex I for secondary Na+/H+ antiport is discussed.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The NADH:ubiquinone oxidoreductase, also known as respiratory complex I,1 couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane (1–3). The bacterial complex consists, in general, of 14 different subunits named NuoA to NuoN. Seven peripheral subunits bear all known redox groups of the complex, namely one FMN and up to nine iron-sulfur (Fe/S) clusters. The remaining seven subunits are mostly hydrophobic proteins predicted to fold into 54 {alpha}-helices across the membrane (4). These hydrophobic subunits do not contain a conserved sequence motif for the binding of a cofactor, nor has a cofactor been identified in these subunits as yet. However, because of their location within the membrane, they should be involved in proton translocation. The 14 subunits present in bacteria are called the minimal subunits because their homologues are found in the complex I of all species investigated as yet (2, 4, 5). They comprise the minimal structural and functional framework for redox-driven proton translocation in complex I. In addition to the homologues of the 14 minimal subunits the mitochondrial complex I of eukaryotes contains at least 32 extra proteins but no additional redox groups (6). It has been shown that both the bacterial and the mitochondrial complex I have a proton to electron stoichiometry of 4H+/2e– (7–11).

Until recently it was believed that complex I works exclusively as a proton pump. It has been elegantly demonstrated that the Klebsiella pneumoniae complex I acts as a Na+ pump, with a stoichiometry of 2Na+/2e– (12–14). It was also shown that the K. pneumoniae complex I is not capable of proton translocation (14). Recent experimental evidence disclosed that complex I from Escherichia coli, a close relative of K. pneumoniae, acts as a sodium pump as well (15, 16). This proposal is based on experiments with the E. coli strain EP432 lacking the antiporter genes nhaA and nhaB, which leads to an inability to grow under elevated Na+ concentrations (17). However, growth on glycerol and fumarate enabled growth of strain EP432 at about 450 mM NaCl (15). Under these conditions, the expression of the nuo genes coding for the subunits of complex I is 2-fold increased (18, 19). Inverted membrane vesicles catalyzed NADH-driven Na+ uptake, which was not cancelled by addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP, Sigma) but by the addition of rotenone, a specific complex I inhibitor (15). It was also shown that the overproduced subunit NuoL of the E. coli complex I mediates Na+ uptake when reconstituted into proteoliposomes (20). This transport was inhibited by the addition of EIPA, an inhibitor of Na+/H+ antiporters (21).

However, data obtained with E. coli wild-type cells and membrane vesicles indicated the presence of an NADH-induced proton gradient (10, 22, 23). In addition, FT-IR spectroscopy has shown that the redox reaction of the E. coli complex I is associated with the protonation of tyrosines and acidic amino acids (24, 25). As activity measured in whole cells and membrane vesicles are sometimes hard to assign, we reconstituted the isolated E. coli complex I in proteoliposomes, measured the effect of Na+ ions on enzyme activity, and determined the type of cation responsible for the electrochemical gradient generated by the redox reaction of complex I. Our data showed that the E. coli complex I is a primary proton pump but may be capable of secondary H+/Na+ antiport.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of Complex I—E. coli complex I was isolated from the overproducing strain ANN003/pAR1219 similar to the procedure described (26) but treatment of the membranes with NaBr was omitted. All steps were carried out at 4 °C. 55 g of cells were resuspended in the 5-fold volume 50 mM MES/NaOH, 0.1 mM phenylmethylsulfonyl fluoride, pH 6.0, with 10 µg/ml DNase I and 50 µg/ml lysozyme and disrupted by a single pass through a French Pressure cell (SLM Aminco) at 110 mega Pascal units. Cell debris was removed by centrifugation at 36,000 x g, and cytoplasmic membranes were obtained by centrifugation at 250,000 x g for 1 h. The membranes were resuspended in 50 mM MES/NaOH, 50 mM NaCl, 5 mM MgCl2, pH 6.0 at a concentration of 80 mg/ml. Dodecyl maltoside (Glycon) was added to a final concentration of 3%, and the solution was gently homogenized and centrifuged for 20 min at 250,000 x g. The supernatant was applied to an 80-ml Source 15Q (Amersham Biosciences) column equilibrated in 50 mM MES/NaOH, 50 mM NaCl and 0.1% dodecyl maltoside, pH 6.0. The column was eluted with a 700-ml linear gradient of 50–350 mM NaCl in 50 mM MES/NaOH, 0.1% dodecyl maltoside, pH 6.0 at a flow rate of 5 ml/min. Fractions containing NADH/ferricyanide reductase activity were combined, concentrated by precipitation with 9% (w/v; final concentration) polyethylene glycol 4,000, and dissolved in 1 ml of 50 mM MES/NaOH, 50 mM NaCl, 5 mM MgCl2, and 0.1% dodecyl maltoside, pH 6.0. The proteins were subjected to size-exclusion chromatography on a 450-ml Sephacryl S-300HR (Amersham Biosciences) column in 50 mM MES/NaOH, 50 mM NaCl, 5 mM MgCl2, and 0.1% dodecyl maltoside, pH 6.0, at a flow rate of 20 ml/h. Peak fractions of NADH/ferricyanide reductase activity were pooled and stored at –80 °C.

Reconstitution of Complex I—To determine the most suitable lipid for reconstitution experiments we examined the NADH:decyl-ubiquinone reductase activity of complex I in the presence of soybean L-{alpha}-phosphatidylcholine (Sigma), egg yolk L-{alpha}-phosphatidylcholine (Lipoid), E. coli polar lipid extract (Avanti), and total lipid extract (Avanti). Lipids (30 mg/ml) were dissolved in 50 mM MES/NaOH, 50 mM NaCl, 0.1% (w/v) dodecyl maltoside, pH 6.0 by sonication in five intervals of 20 s at 80% output level with a microtip (Branson sonifier). The suspensions were diluted to 3 mg/ml in the same buffer and either directly added to the assay or mixed in a 1:1 (v/v) ratio with complex I (1 mg/ml) and incubated for 20 min on ice before addition to the assay.

Complex I was reconstituted in proteoliposomes by the BioBeads (Bio-Rad) method (27). 80 mg of E. coli polar lipid extract (Avanti) were suspended in 4 ml of 50 mM MES/NaOH, 50 mM NaCl, pH 6.0, and 800 µl of 20% (w/v) dodecyl maltoside were added. The lipids were dissolved by sonication of the suspension in five intervals of 20 s at 80% output level with a microtip (Branson sonifier). Complex I (2 mg/ml) and the lipid solution were mixed in a 1:4 (w/w) ratio. BioBeads with a capacity of absorbing 105 mg of dodecyl maltoside per g of beads (28) were added at an 8-fold excess. The mixture was stirred for 3 h at 4 °C and centrifuged for 30 min at 150,000 x g (Beckmann Airfuge) after separation of the beads. The sediment was resuspended in 50 mM MES/NaOH, 100 mM NaCl, pH 6.0. Various Na+ concentrations in the assay were obtained by dialyzing complex I overnight against the desired buffer at 4 °C. In these cases the lipids and the proteoliposomes were resuspended in the corresponding buffer.

Determination of Complex I Activity—The NADH:decyl-ubiquinone oxidoreductase activity of complex I was measured with a Perkin Elmer 156 dual-wavelength spectrophotometer at 340 and 400 nm using an {epsilon} of 6.3 mM–1 cm–1 in a final volume of 1 ml (29). To obtain various Na+ concentrations in the assay, the E. coli polar lipid extract (30 mg/ml) was suspended in the desired buffer containing 0.1% (w/v) dodecyl maltoside. The enzyme (20 mg/ml) was dialyzed for 16 h against 50 mM MES/LiOH, 100 mM LiCl, pH 6.0 at 4 °C, mixed in 1:1 (v/v) ratio with the lipid and incubated for 20 min on ice. 3 µl of this mixture were incubated for 5 min at room temperature in the assay buffer (50 mM MES/LiOH, 100 mM LiCl, pH 6.0). The Na+ concentration of the buffer was varied from 25 µM to 100 mM by adding the corresponding amount of 50 mM MES/LiOH, 100 mM NaCl, pH 6.0. 50 µM decyl-ubiquinone was added to the assay, and the reaction was started by addition of 50 µM NADH (potassium salt, Sigma). The sodium content of the assay buffer was determined by Christine Höher, Institut für Mineralogie, Petrologie und Geochemie, Albert-Ludwigs-Universität Freiburg by atomic absorption spectroscopy.

For inhibitor titrations of the isolated and reconstituted E. coli complex I an ethanolic solution of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and an aqueous solution of benzamil (both from Sigma) was added to the assay. The samples were preincubated for 5 min at room temperature with inhibitors in the enzyme assay. Measurements were performed either in 50 mM MES/NaOH, 100 mM NaCl, pH 6.0 or 50 mM MES/KOH, 100 mM KCl, pH 6.0. The final ethanol concentration was kept constant in each enzyme assay and did not exceed 2%.

Determination of a Membrane Potential ({Delta}{psi})—The generation of {Delta}{psi} by complex I was monitored with 8-anilino-1-naphthalene-sulfonic acid (ANS, Sigma; Ref. 30) at various NaCl concentrations. The ionic strength of the assay buffer was kept constant by adding the corresponding amount of tetramethylammonium chloride (TMA). The respective ion concentrations are given in the figure legends. 3 µl of proteoliposomes, 1 µM ANS, and 50 µM decyl-ubiquinone were added to the assay buffer (5 mM MES/KOH, 80–x mM NaCl, x mM TMA, pH 6.0) and preincubated for 5 min at 25 °C. The reaction was started by addition of 50 µM NADH (Gerbu) in a final volume of 1 ml. The increase of ANS fluorescence caused by the generation of {Delta}{psi} was followed with an SFM 25 spectrofluorometer (Kontron), using an excitation wavelength of 380 nm and an emission wavelength of 480 nm. When desired, the proton ionophore CCCP, the Na+-specific ionophore N,N'-dibenzyl-N,N'-diphenyl-1,2-phenylene diacetamide (ETH-157, Fluka; Ref. 31), and the specific complex I inhibitor piericidin A were added to a final concentration of 10 µM, respectively.

Determination of a Proton Gradient ({Delta}pH)—The generation of a proton gradient caused by the redox reaction of complex I was determined by quenching of the fluorescence of 9-amino-6-chloro-2-methoxyacridine (ACMA, Sigma; Ref. 32). 3 µl of proteoliposomes, 1 µM ACMA, and 50 µM decyl-ubiquinone were incubated for 5 min at 25 °C in the assay buffer (5 mM MES/KOH, 80–x mM NaCl, x mM TMA, pH 6.0) in 1 ml final volume. The respective ion concentrations are given in the figure legends. The fluorescence was detected with a SFM 25 spectrofluorometer (Kontron), using an excitation wavelength of 410 nm and an emission wavelength of 480 nm. The reaction was started by addition of 50 µM NADH (Gerbu). When desired CCCP, piericidin A, and the Na+/H+ exchanger monensin (Sigma, Ref. 33) were added at a final concentration of 10 µM, respectively. The proton specificity of ACMA was determined using liposomes without enzyme in 5 mM MES/NaOH, 80 mM NaCl, pH 6.0. 1 µM ACMA was added to the liposomes, and the fluorescence change was detected after addition of either 20 mM NaCl or 20 mM HCl.

Effect of Dicyclohexylcarbodi-imide (DCCD)—The effect of DCCD on the NADH:decyl-ubiquinone oxidoreductase activity of complex I and its ability to generate a membrane potential and a proton gradient was measured as described above. Complex I reconstituted in phospholipids was incubated for 60 min at 4 °C with various amounts from a 0.1 M DCCD (Sigma) stock solution in ethanol. The final ethanol concentration was kept constant and did not exceed 1% (v/v). After incubation the enzyme was immediately used for NADH:decyl-ubiquinone oxidoreductase activity measurement in 50 mM MES/NaOH, 100 mM NaCl, pH 6.0. Addition of 10 µM CCCP had no effect on the measurement. Complex I reconstituted into proteoliposomes was incubated for 10 min with 0.3 mM DCCD final concentration at room temperature and immediately used for measurements in 5 mM MES/KOH, 80 mM NaCl, pH 6.0. Prolongation of the incubation time had no effect.

Other Analytical Assays—NADH/ferricyanide reductase activity was measured at room temperature with an Ultrospec 1000 (Amersham Biosciences) spectrophotometer at 410 nm in 50 mM MES/NaOH, 50 mM NaCl, pH 6.0 using an {epsilon} of 1 mM–1 cm–1 (34). The reaction was started with 0.1 mM NADH. Protein concentrations were determined according to the biuret method.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Purification of Complex I—Our standard purification method was modified to circumvent the washing of the membranes with NaBr, which led to an incubation of complex I with 1.4 M Na+ (26). Complex I was extracted from the membranes with 3% dodecyl maltoside and eluted by anion exchange chromatography on Source 15Q at 280 mM NaCl. The final size-exclusion chromatography on Sephacryl S-300 showed a homogenous peak coeluting with the complex I activity (Fig. 1). Residual traces of ATP synthase were present in the late fractions of the peak. These fractions were discarded. The molecular mass of the preparation was estimated by comparison of the elution volume with standards of known molecular mass to 600,000 Da. Upon SDS/PAGE the preparation was resolved into 12 subunits corresponding to the proteins encoded by the nuo operon (Fig. 1; Refs. 26, 35, and 36). The subunits NuoE and J were not separated by SDS-PAGE as reported previously (26, 35). Approximately 25 mg of complex I were obtained from 55 g of cells (Table I).



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  		FIG. 1.
Isolation of E. coli complex I from strain ANN003/pAR1219. A, chromatography on Source 15Q; B, chromatography on Sephacryl S-300; absorbance at 280 nm (–); NADH/ferricyanide reductase activity (); NaCl gradient (—). C, SDS/PAGE of the indicated fractions from size exclusion chromatography.

 


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  		TABLE I
Isolation of E. coli complex I from 55 g of wet cells of strain ANN003/pAR1219

 
Effect of Phospholipids on Enzymatic Activity—To choose a convenient lipid for the reconstitution of the E. coli complex I, the effect of soybean L-{alpha}-phosphatidylcholine, egg yolk L-{alpha}-phosphatidylcholine, and E. coli polar and total lipid extracts on the NADH:decyl-ubiquinone reductase activity was examined (Table II). Addition of soybean L-{alpha}-phosphatidylcholine and E. coli polar lipid extract to the enzyme assay doubled the enzymatic activity, whereas addition of the other lipids had only a minor effect. However, the NADH:decyl-ubiquinone oxidoreductase activity was increased 7–8-fold after a 20-min incubation of the enzyme with the lipids (Table II). Again, soybean L-{alpha}-phosphatidylcholine and E. coli polar lipid extract showed the strongest activation of the complex. Further experiments were therefore performed with E. coli polar lipid extract, and activity of the isolated complex was measured after 20 min of incubation with lipids.


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  		TABLE II
Specific NADH:decyl-ubiquinone reductase activity of complex I in the presence of various phospholipids

The lipids were either added just prior to the start of the reaction or the enzyme was incubated with phospholipids for 20 min on ice. The activity was measured as decrease of the NADH concentration at 340–400 nm in the presence of 50 µM decyl-ubiquinone and 50 µM NADH (enzyme:lipid = 1:3 (w/w)).

 
Effect of the Sodium Concentration on the Enzymatic Activity—The electron transfer activity of the Na+-translocating complex I from K. pneumoniae and the Na+-translocating NQR from Vibrio cholerae strongly depend on the Na+ concentration (13, 37). Therefore, we measured the influence of Na+ concentration on the NADH:decyl-ubiquinone oxidoreductase activity of the E. coli complex I. The activity was measured with complex I reconstituted in E. coli polar lipids in 50 mM MES/LiOH, 100 mM LiCl, pH 6.0 as described above. The Na+ concentration of the buffer was varied from 25 µM to 100 mM by adding the corresponding amount of 50 mM MES/LiOH, 100 mM NaCl, pH 6.0. Under the chosen conditions, the average activity of ~5.4 µmol of NADH/min·mg was determined. The highest activity was measured around 80 µM Na+ (Fig. 2). Unlike Na+-dependent enzymes, the average activity of complex I did not increase by raising the Na+ concentration from 100 µM to 100 mM. The NADH:decyl-ubiquinone reductase activity was 95% inhibited by 10 µM piericidin A, showing that the physiological activity of the complex was measured.



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  		FIG. 2.
Steady state activity of E. coli complex I reconstituted in E. coli polar lipids. The dependence of the NADH oxidation coupled with decyl-ubiquinone reduction on the Na+ concentration was followed by the decrease of the NADH concentration at 340–400 nm. The assay contained 30 µg of purified complex I in 50 mM MES/NaOH, 100 mM NaCl, pH 6.0. Variation of the Na+ concentration was achieved by mixing the buffer with the corresponding amount of 50 mM MES/LiOH, 100 mM NaCl, pH 6.0. 50 µM decyl-ubiquinone was added to the assay, and the reaction was started by addition of 50 µM NADH (potassium salt). Average values of three independent assays are shown.

 
Reconstitution of Complex I in Proteoliposomes—Isolated complex I was reconstituted in proteoliposomes by the BioBeads method (27). The best results were achieved with E. coli polar lipid extract in a 4:1 (w/w) ratio with complex I. BioBeads were added at an 8-fold excess, and the mixture was incubated at 4 °C for 3 h. The vesicles were sedimented by ultracentrifugation and carefully resuspended in 50 mM MES/NaOH, 100 mM NaCl, pH 6.0. The tightness of the proteoliposomes was confirmed by application of a potassium/valinomycin diffusion potential (data not shown). The orientation of complex I in the liposomes was determined by measuring the NADH/ferricyanide reductase activity before and after addition of 0.05% (w/v, final concentration) dodecyl maltoside. Addition of detergent led to a 50% increase of the NADH/ferricyanide reductase activity indicating that two-thirds of the complex was incorporated in the proteoliposomes so that the NADH binding site was accessible. Approximately 80–90% of complex I was found to be incorporated judging by the total NADH/ferricyanide reductase activity. The complex reconstituted in vesicles showed a specific NADH:decyl-ubiquinone oxidoreductase activity of 5.5 µmol/min·mg in the presence of 50 µM NADH, 50 µM decyl-ubiquinone, and 10 µM CCCP.

Determination of {Delta}{psi}—The membrane potential arising from the NADH:decyl-ubiquinone oxidoreductase activity of complex I reconstituted in proteoliposomes was monitored with ANS in 5 mM MES/NaOH, pH 6.0, and 80 mM NaCl. Addition of NADH to the proteoliposomes incubated with decyl-ubiquinone and ANS showed a 90% enhanced fluorescence signal (Fig. 3). Thus, complex I reconstituted in proteoliposomes generated a membrane potential. This membrane potential was due to the redox reaction of complex I as the signal was completely cancelled by addition of piericidin A, a specific complex I inhibitor (Fig. 3). Addition of an equal volume of ethanol had no effect on the signal. The ANS fluorescence signal was completely sensitive to the protonophore CCCP indicating that the membrane potential is established as an electrochemical proton gradient (Fig. 3). In contrast, the Na+-specific ionophore ETH-157 had no effect on the fluorescence signal, indicating that there was no contribution to the membrane potential from a primary NADH-induced Na+ gradient (Fig. 3).



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  		FIG. 3.
Membrane potential generated by the redox reaction of E. coli complex I reconstituted in proteoliposomes monitored by ANS fluorescence. The assay contained 20 µg of purified complex I in 5 mM MES/KOH, 80 mM NaCl, 50 µM decyl-ubiquinone, pH 6.0. The reaction was started by addition of 50 µM NADH indicated by an arrow. The ANS fluorescence was followed at an excitation wavelength of 380 nm and an emission wavelength of 480 nm. A, no further additions. Signal after additions of piericidin A (B), CCCP (C), and ETH-157 (D), each at 10 µM are shown. The signal offset in B caused by the presence of piericidin A in the assay is corrected.

 
Determination of {Delta}pH—The sensitivity of the membrane potential generated by the E. coli complex I to the protonophore CCCP indicated the presence of a proton gradient. We determined the presence of a pH gradient directly by using the fluorophore ACMA. The specificity of ACMA to protons was determined by addition of either HCl or NaCl to liposomes without enzyme. While HCl caused a 70% rise in fluorescence, NaCl had no effect on the fluorescent signal (data not shown). Addition of NADH to the proteoliposomes incubated with decylubiquinone and ACMA showed a 40% quenched fluorescence signal (Fig. 4). The ACMA fluorescence signal was completely sensitive both to the preincubation and the addition of the protonophore CCCP demonstrating that it was generated by a proton gradient (Fig. 4). The ACMA signal was also sensitive to the Na+/H+ exchanger monensin again showing the presence of a proton gradient. The ACMA signal was completely cancelled by adding piericidin A. Addition of an equal volume ethanol had no effect on the signal. This demonstrated that the E. coli complex I is a primary proton pump.



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  		FIG. 4.
Generation of a pH gradient by the redox reaction of E. coli complex I reconstituted in proteoliposomes monitored by ACMA fluorescence. The assay contained 20 µg of purified complex I in 5 mM MES/KOH, 80 mM NaCl, 50 µM decyl-ubiquinone, pH 6.0. The reaction was started by addition of 50 µM NADH indicated by an arrow. The ACMA fluorescence was detected at an excitation wavelength of 410 nm and an emission wavelength of 480 nm. A, no further additions. Signal after additions of 10 µM piericidin A (B), 20 µM CCCP (C), 10 µM monensin (D), and 20 µM CCCP (E) added during enzyme turnover are shown.

 
Effect of DCCD on E. coli Complex I—It is well known that DCCD simultaneously inhibits electron transfer and proton translocation of the mitochondrial complex I (38, 39). To examine whether the same holds true for the bacterial complex I, we titrated the NADH:decyl-ubiquinone reductase activity of the isolated complex I with DCCD (Fig. 5). DCCD inhibited 90% of the enzyme acitivity with an apparent IC50 of 0.25 mM, which was comparable to the value reported for the mitochondrial enzyme (38, 39). Incubation of complex I proteoliposomes with DCCD completely prohibited the generation of a membrane potential and a pH gradient (Fig. 5).



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  		FIG. 5.
Effect of DCCD on the isolated E. coli complex I reconstituted in E. coli polar lipids. A, inhibition of the NADH:decyl-ubiquinone reductase activity monitored as the decrease of the NADH concentration at 340–400 nm. The assay contained 30 µg of purified complex I in 50 mM MES/NaOH, 100 mM NaCl, pH 6.0, 50 µM decyl-ubiquinone, and 50 µM NADH. B, effect on the membrane potential generated by complex I monitored as ANS fluorescence at 380 nm (excitation) and 480 nm (emission). The assay contained 20 µg of purified complex I in 5 mM MES/KOH, 80 mM NaCl, 50 µM decyl-ubiquinone, pH 6.0. C, effect on the pH gradient generated by complex I monitored as ACMA fluorescence at 410 nm (excitation) and 480 nm (emission). The assay contained 20 µg of purified complex I in 5 mM MES/KOH, 80 mM NaCl, 50 µM decyl-ubiquinone, pH 6.0. The reaction in B and C was started by addition of 50 µM NADH indicated by an arrow. The upper curves in B and C were control experiments without DCCD, and the lower curves were obtained in the presence of 100 µM DCCD.

 
Effect of Sodium Concentration on the {Delta}pH—It has been reported that the overproduced and isolated subunit NuoL facilitates Na+ transport across the membrane (20, 40).2 Devoid of the residual complex I subunits, NuoL is capable of mediating a passive Na+ uptake along a concentration gradient, which coincides with an increased proton permeability of the vesicles. This is interpreted in terms of an H+/Na+ antiport (20). We therefore addressed the question, whether this is an intrinsic property of NuoL assembled in the complex I as well. Complex I was reconstituted in proteoliposomes in 80 mM NaCl and was added to an ACMA assay in 5 mM MES/KOH, 1 mM NaCl, 79 mM TMA, pH 6.0 without addition of decyl-ubiquinone and NADH. If NuoL assembled in complex I was capable of a Na+ gradient-driven Na+/H+ antiport, a quench of an ACMA fluorescence signal would be expected. However, no such signal was detected (Fig. 6). The addition of proteoliposomes containing 1 mM NaCl and 79 mM TMA to a buffer of 5 mM MES/KOH, 80 mM NaCl, pH 6.0 did not lead to a rise of the ACMA signal as well. In both cases, the ACMA signal was quenched by subsequent addition of 50 µM decyl-ubiquinone and 50 µM NADH showing that the complex translocates protons across the membrane under the applied conditions (Fig. 6). Thus, we found no evidence for a passive Na+/H+ antiport by complex I.



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  		FIG. 6.
Influence of the Na+ concentration on the proton gradient. Complex I in proteoliposomes containing 5 mM MES/KOH and 80–x mM NaCl, x mM TMA, pH 6.0 were added to an assay buffer of 5 mM MES/KOH, 80–x mM NaCl, x mM TMA, pH 6.0, and 1 µM ACMA. A, proteoliposomes containing 80 mM NaCl were added to an assay buffer of 1 mM NaCl. B, proteoliposomes containing 1 mM NaCl were added to an assay buffer of 80 mM NaCl. At the time indicated by arrows 3 µl of proteoliposomes (PL), 50 µM decyl-ubiquinone (DQ), and 50 µM NADH were added, respectively. C, proteoliposomes containing 80 mM NaCl were added to an assay containing 80 mM NaCl, D, proteoliposomes containing 80 mM NaCl were added to an assay containing 1 mM NaCl. Reactions in C and D were started by addition of 50 µM NADH. Addition of 10 µM ETH-157 to C prior to NADH led to a signal comparable with D.

 
To examine the possibility that a Na+/H+ antiport is coupled to the redox reaction of complex I, we determined the amplitude of the pH gradient generated by complex I in the presence of an Na+ gradient. Proteoliposomes containing 80 mM NaCl were added to an ACMA assay in 5 mM MES/KOH, 1 mM NaCl, 79 mM TMA, and in 5 mM MES/KOH, 80 mM NaCl, respectively. The reaction was started by addition of NADH (Fig. 6). The amplitude of the ACMA signal was ~20% larger in the buffer containing 1 mM Na+ than in the buffer containing 80 mM Na+ (Fig. 6). The amplitude of the ACMA signal was independent of the applied Na+ gradient when the proteoliposomes were preincubated with 10 µM ETH-157 prior to the addition of NADH (Fig. 6). Thus, our data are in line with the idea that complex I is capable of secondary Na+/H+ antiport coupled to its redox reaction.

Effect of Amiloride Inhibitors on E. coli Complex I—It has been shown that the electron transfer of mitochondrial and bacterial complex I is inhibited by amiloride derivatives, inhibitors of Na+/H+ antiporters (41, 42). In addition, these substances prevent labeling of the homologue of NuoL in bovine heart complex I with a photoaffinity analogue of fenpyroximate, an inhibitor binding to the ubiquinone site of complex I (41). The Na+ transport mediated by the overproduced and isolated subunit NuoL was shown to be sensitive to EIPA, an amiloride inhibitor (20). To test the effect of these inhibitors on the isolated E. coli complex I we monitored the NADH:decylubiquinone reductase activity at various EIPA and benzamil concentrations (Fig. 7). The inhibitor concentrations needed for half-maximal inhibition were 100 and 70 µM for EIPA and benzamil, respectively. The residual activity was less than 5% at 500 µM EIPA and 300 µM benzamil. These values are comparable to those obtained for the enzyme in the bacterial membrane of >100 µM and 45 µM, respectively (42). The IC50 did not depend on the Na+ concentration (Fig. 7). Comparable values were obtained in the presence of 25 µM and 100 mM NaCl, respectively. Thus, these inhibitors did not act at a hypothetical Na+ binding site in complex I. Beside their effect on electron transfer we also measured the influence of EIPA and benzamil on proton translocation by complex I. At concentrations needed for 95% inhibition of the electron transfer activity, the NADH-induced ACMA signal of complex I proteoliposomes was completely gone (Fig. 7). This showed that amiloride inhibitors effect both electron transfer and proton translocation of complex I.



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  		FIG. 7.
Effect of amiloride derivates on the isolated E. coli complex I reconstituted in E. coli polar lipids. Inhibition of the NADH:decyl-ubiquinone reductase activity with EIPA (A) and benzamil (B) monitored as the decrease of the NADH concentration at 340–400 nm. The assay contained 15 µg of reconstituted complex I in either 50 mM MES/NaOH, 100 mM NaCl, pH 6.0 (closed squares) or 50 mM MES/KOH, 100 mM KCl, pH 6.0 (closed circles), and 50 µM decylubiquinone. C, effect on the pH gradient generated by complex I monitored as ACMA fluorescence at 410 nm (excitation) and 480 nm (emission). The assay contained 20 µg of purified complex I in 5 mM MES/KOH, 80 mM NaCl, 50 µM decyl-ubiquinone, pH 6.0. The reaction was started by addition of 50 µM NADH as indicated by arrows. Left side, control reaction without any additions; right side, signal in the presence of either 500 µM EIPA or 300 µM benzamil.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Because of its enormous complexity in combination with the lack of structural data, knowledge about the mechanism of complex I is rather limited. The mitochondrial complex I is made up of at least 46 different subunits (6) seven of which are encoded on the mitochondrial genome (1, 2). Although the bacterial complex shows a simpler composition of 14 different subunits, this number is still quite large. In addition, its exact cofactor composition is still under debate (43–46). While it is generally accepted that the mitochondrial complex I translocates four H+ across the membrane for every NADH oxidized (7–11), it has recently been shown that complex I from K. pneumoniae pumps two Na+ per NADH oxidized but is not capable of translocating protons (14). The different type of ions translocated and the different stoichiometries have general consequences for models of the enzyme mechanism (47). It needs to be established whether Na+ translocation by the K. pneumoniae complex I is a unique exception to the rule or whether it represents a general, so far overlooked feature of complex I.

As a substantiation of the latter, it was reported that the E. coli complex I is capable of pumping Na+ as well (15). This proposal was derived from data obtained with inverted membrane vesicles. Because measurements with membrane vesicles may contain enzyme activity obscuring the activity in question we set out to advance measurements with the isolated complex I reconstituted in artificial phospholipid vesicles. Therefore, a suitable mixture of phospholipids and a suitable method for reconstitution had to be established. The best results in terms of enzyme activity and amount of enzymes reconstituted into liposomes were obtained with E. coli polar lipid extract reconstituted by the BioBeads method (Ref. 27 and Table 2). It is a well known property of E. coli membrane proteins that they show the highest activity when reconstituted into E. coli phospholipids (48).

As isolated in detergent, the E. coli complex I has a very low activity of 0.3 µmol of NADH/(min·mg). This low activity can be stimulated by addition of endogenous phospholipids up to 8-fold (Table 2) as described in the literature (34, 36, 49, 50). As pointed out for the Yarrowia lipolytica enzyme, complex I has to be preincubated with phospholipids to achieve sufficient interaction, which is limited in the diluted assay buffer (50). Reconstitution in proteoliposomes led to an enzymatic activity of about 5.5 µmol of NADH/(min·mg) in the presence of CCCP, which is close to the estimated turnover of the enzyme in the bacterial membrane of 6 µmol of NADH/(min·mg) (29). Titration of the NADH:decyl-ubiquinone oxidoreductase activity with E. coli polar lipid extract revealed that the full activity of the E. coli complex I is recovered when incubated with the 4-fold amount (w/w) of phospholipids (data not shown). This was in accordance with previously reported values (36).

In the absence of Na+ ions, primary sodium pumps show apparently no turnover (51). The activity of the Na+-pumping complex I from K. pneumoniae drops to 50% of the Vmax at 332 µM Na+ (13). The enzymatic activity of the Na+-translocating NQR from V. cholerae is lowered by 50% at ~10 mM Na+ (37). However, the NADH:decyl-ubiquinone reductase activity of the isolated E. coli complex I did not depend on the Na+ concentration (Fig. 2), indicating that this enzyme is not involved in primary Na+ translocation.

This was confirmed by measurements of the membrane potential using ANS as a fluorescent probe. An NADH-induced membrane potential was detected using complex I reconstituted into liposomes (Fig. 3). No signal was detected when either NADH or decyl-ubiquinone was omitted from the assay or when complex I was preincubated with the specific inhibitor piericidin A (Fig. 3). Thus, the membrane potential is due to the redox reaction of complex I. This potential was completely sensitive to the protonophore CCCP but not to the Na+ ionophore ETH-157 (Fig. 3) indicating no contribution from a Na+ gradient to the membrane potential. This was confirmed by the direct measurement of a pH gradient using ACMA as a fluorescent probe (Fig. 4). The ACMA signal was sensitive to piericidin A, CCCP, and monensin demonstrating that the ion gradient raised by the E. coli complex I is a proton gradient. Treatment of the isolated complex with DCCD led to a nearly complete inhibition of the electron transport and proton translocation activity (Fig. 5). It is well known, that DCCD inhibits electron transfer activity of the mitochondrial complex I parallel with its proton translocation activity (38, 39, 52). Here, we have shown that this holds true for the bacterial complex I as well. Taking it all together, the data obtained with the isolated E. coli complex demonstrated that it is a primary proton pump.

Our measurements agree with those obtained from whole E. coli cells, where it has been reported that complex I is a proton pump with a stoichiometry of at least 1.5 H+/e– (10). However, this contradicts data claiming that the E. coli complex I is a primary sodium pump (15, 16). It is more than unlikely that a discrepancy exists between data derived from different materials used for different studies. Proton pumping has been reported for the complex in whole cells as mentioned above, and the intactness of our preparation was demonstrated by the complete sensitivity of the NADH:decyl-ubiquinone reductase activity to piericidin A and by its capability to generate membrane potential. However, by using inverted membrane vesicles, it is possible that electrons from NADH entering the respiratory chain via complex I are distributed to other energy-converting enzymes, with one of these probably acting as primary sodium pump. This would also explain the rotenone sensitivity of the sodium gradient because rotenone prevents electrons from entering the respiratory chain. The same would hold true for the measurements performed with the E. coli strain deficient in complex I. The electron entry to the respiratory chain would be missing, and rotenone does not have any effect on this mutant.

Sequence comparisons have shown that the complex I subunits NuoL, -M, and -N are related to cation/proton antiporter (5, 53–57). Experiments with individually overproduced NuoL from E. coli and Rhodobacter capsulatus have shown that these subunits are capable of passive transport of sodium ions along a concentration gradient (20, 40). The sodium transport of the overproduced E. coli NuoL reconstituted into proteoliposomes is accompanied by an increase in the proton permeability of the membrane. This was interpreted as a Na+/H+ antiport (20). Overproduced NuoL and -M from R. capsulatus rescued Bacillus subtilis mutants lacking the Na+/H+ antiporter subunits MrpA and -D at elevated Na+ concentrations (40).2 Our data on complex I reconstituted into proteoliposomes showed that these subunits assembled into the entire complex do not function as a passive Na+/H+ antiporter (Fig. 6). A Na+ gradient across the membrane did not induce a proton translocation (Fig. 6). However, the magnitude of the pH gradient generated by the redox activity of complex I varied in dependence on Na+ concentration (Fig. 6). If the Na+ concentration in the proteoliposomes was higher than in the assay buffer, the amplitude of the ACMA signal was larger than that obtained with equal Na+ concentration on both sides of the membrane (Fig. 6). This points to the possibility that the E. coli complex I may be capable of secondary Na+/H+ antiport being coupled to the redox reaction of the enzyme. The efflux of Na+ would be coupled to a further proton uptake enlarging the pH gradient. It is possible that this antiport is caused by the operation of subunits NuoL and/or NuoM, as proposed (20, 40).

Amiloride derivatives, which are specific inhibitors of Na+/H+ antiporters, inhibit complex I from different sources although with different affinities (39, 40). The IC50 value we measured for the isolated and reconstituted E. coli complex I is comparable to the values reported for the complex in the membrane (42). The inhibition was independent from the Na+ concentration in the measured range, which is further evidence for the finding that the E. coli complex I is not involved in primary Na+ translocation and that the amiloride type inhibitors do not act at a Na+ binding site. Here, we showed that in addition to their effect on electron transfer, EIPA and benzamil also prevent proton translocation (Fig. 7). Thus, their inhibitory action is comparable to that of DCCD. As inhibition of complex I by amiloride derivatives is not restricted to the bacterial complex (20, 40) but was also detected for the mitochondrial complex (41–42), it is possible that complex I in general may be capable of redox-driven secondary H+/cation antiport.


   FOOTNOTES
 
* This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 49-(0)761-203-6060; Fax: 49-(0)761-203-6096; E-mail: tfriedri{at}uni-freiburg.de.

1 The abbreviations used are: complex I, proton-pumping NADH: ubiquinone oxidoreductase; NQR, Na+-translocating NADH:quinone oxidoreductase; MES, 2-(N-morpholino)-ethanesulfonic acid; decylubiquinone, 2,3-dimethoxy-5-methyl-6-decylbenzoquinone; ACMA, 9-amino-6-chloro-2-methoxyacridin; ANS, 8-anilino-1-naphthaline-sulfonic acid; CCCP, carbonylcyanide-m-chlorophenylhydrazone; DCCD, dicyclohexylcarbodi-imide; EIPA, 5-(N-ethyl-N-isopropyl)-amiloride; TMA, tetramethylammonium chloride. Back

2 C. Hägerhall, personal communication. Back


   ACKNOWLEDGMENTS
 
We thank Heinrich Jung, Osnabrück, and Peter Gräber, Freiburg, for advice in the preparation of liposomes and to Uli Brandt, Frankfurt, for his advice in measuring pH gradients. We thank Dr. Peter Heiss and Joachim Grams (Roche Applied Science, Penzberg) for the donation of the Kontron spectrofluorometer.



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