The deactive form of respiratory complex I from mammalian mitochondria is a Na+/H+ antiporter

Abstract

In mitochondria, complex I (NADH:ubiquinone oxidoreductase) uses the redox potential energy from NADH oxidation by ubiquinone to transport protons across the inner membrane, contributing to the proton-motive force. However, in some prokaryotes, complex I may transport sodium ions instead, and three subunits in the membrane domain of complex I are closely related to subunits from the Mrp family of Na(+)/H(+) antiporters. Here, we define the relationship between complex I from Bos taurus heart mitochondria, a close model for the human enzyme, and sodium ion transport across the mitochondrial inner membrane. In accord with current consensus, we exclude the possibility of redox-coupled Na(+) transport by B. taurus complex I. Instead, we show that the "deactive" form of complex I, which is formed spontaneously when enzyme turnover is precluded by lack of substrates, is a Na(+)/H(+) antiporter. The antiporter activity is abolished upon reactivation by the addition of substrates and by the complex I inhibitor rotenone. It is specific for Na(+) over K(+), and it is not exhibited by complex I from the yeast Yarrowia lipolytica, which thus has a less extensive deactive transition. We propose that the functional connection between the redox and transporter modules of complex I is broken in the deactive state, allowing the transport module to assert its independent properties. The deactive state of complex I is formed during hypoxia, when respiratory chain turnover is slowed, and may contribute to determining the outcome of ischemia-reperfusion injury.

FIGURE 1.

Structural information about complex I, taken from 3M9S.pdb (Thermus thermophilus complex I), 3I9V.pdb (T. thermophilus hydrophilic domain), and 3RKO.pdb (six subunits of the E. coli hydrophobic domain) (2, 57). A, complex I is an L-shaped enzyme, with a large hydrophilic domain containing the flavin mononucleotide, iron-sulfur (FeS) clusters, and the binding site for the ubiquinone headgroup. The membrane domain contains seven subunits; the structure of subunit ND1 has been determined only at low resolution, and two possible positions for helix I of ND3 are shown, from the low resolution structure of the intact enzyme and the E. coli membrane domain. The three helices absent from the mammalian ND2 subunit are shown in black (51), and the ends of the cysteine-containing loop of ND3, which is not resolved in the structure, are highlighted in green (the ends of ND3 helices I and II). The membrane domain is also shown from the top, with the hydrophilic domain removed, and labeled with the names of the B. taurus subunits. The antiporter-like subunits, ND2, ND4, and ND5, are indicated. B, the structure of E. coli NuoM (ND4; the 14 core helices of ND2 and ND5 have the same structure) (2). The transmembrane helices are in light blue; fully conserved residues between all of the antiporter-like subunits of B. taurus and E. coli and MrpA and MrpD from B. subtilis are shown in purple (see supplemental Fig. 1). Contact points with the lateral helix of ND5 are highlighted in green, and the loops in the two broken helices in red. On the right, the same structure is shown from the top.

FIGURE 2.

Mitochondrial complex I is a redox-coupled proton pump. A, quenching of the ACMA fluorescence demonstrates ΔpH formation in BtPLs and BtSMPs. Black: BtPLs (1.3 μg-protein ml⁻¹), 200 μm DQ, and 200 μm NADH; 2 μg ml⁻¹ gramicidin abolishes Δp. Conditions are: 10 mm Tris-SO₄, pH 7.5, 125 mm sucrose, 25 mm KCl, 2 μg ml⁻¹ valinomycin, 20 °C. Gray: BtSMPs (3.8 μg-protein ml⁻¹), and 200 μm NADH; 200 nm rotenone stops proton translocation, and Δp dissipates. Conditions are: 10 mm Tris-SO₄, pH 7.5, 125 mm sucrose, 80 mm KCl, 20 °C. B, the effect of Na⁺, K⁺ and Ch⁺ on the apparent Δp (ΔE/2), generated by ATP hydrolysis by SMPs. The rate of NADH:fumarate oxidoreduction was recorded as a function of ΔE, where ΔE (V) = −0.315 − RT/2F.ln{([NADH][fumarate])/([NAD⁺][succinate])} using 0.1 mm [NADH], 1 mm [NAD⁺], 0.5 mm [succinate], 0.025–40 mm [fumarate]). When the net rate is zero, ΔE/2 = Δp (assuming 4H⁺ per NADH). Conditions are: 10 mm Tris-SO₄, pH 7.5, 32 °C, 0 to 100 mm MCl (M, Na⁺, K⁺, Ch⁺) with variable sucrose concentration (250 to 50 mm) to maintain the osmolarity. Error bars indicate S.D.

FIGURE 3.

Schematic representation of two experiments that reveal the Na⁺/H⁺ antiporter activity of complex I. The diagrams represent a typical complex I PL (see data in Table 1); the complex at the top has been magnified. A, Na⁺-loaded vesicles are placed into a low [Na⁺] buffer. If complex I is a Na⁺/H⁺ antiporter, then Na⁺ efflux drives H⁺ uptake and the pH of the lumen rapidly decreases, forming ΔpH. B, the vesicles have been loaded with protons to create ΔpH. The driving force for H⁺ efflux is high, but uncatalyzed H⁺ efflux is slow; rapid H⁺ efflux is observed if it is coupled to Na⁺ uptake by complex I (the pH of the lumen rapidly increases, and ΔpH collapses).

FIGURE 4.

Sodium ion efflux from PLs drives proton uptake by deactive BtCI (Experiment A). A, BtPLs were prepared in 5 mm Tris-SO₄, 50 mm NaCl, pH 7.4, and then placed into buffer containing 5 mm Tris-SO₄, 5 mm NaCl, 45 mm ChCl, pH 7.4, and 0.25 μm ACMA. Their active/deactive status (A- or D-form) was set beforehand (see “Experimental Procedures”). Each trace is the average from at least three experiments, with the standard deviations indicated by shading. 200 nm rotenone was included in the assay buffer as indicated, and 2 μg ml⁻¹ monensin and 2 μg ml⁻¹ alamethicin were added as indicated. The signal has been normalized to 100% when the BtPLs were added and to 0% in the presence of monensin; ∼70% of the total fluorescence was quenchable. Note that the fluorescence is quenched slightly by the ethanol used for monensin and alamethicin. B, same as A except that the PLs contained 50 mm KCl and were placed into buffer containing 5 mm KCl and 45 mm ChCl. Conditions are: 21 °C, ∼20 μg ml⁻¹ BtCI.

FIGURE 5.

Proton efflux from BtSMPs drives sodium ion uptake by deactive BtCI (Experiment B). A, BtSMPs were placed into buffer containing 10 mm Tris-SO₄, 80 mm ChCl, 250 mm sucrose, pH 7.5, and 0.25 μm ACMA. The active/deactive status of complex I (A- or D-form) was set beforehand (see “Experimental Procedures”). Each trace is the average from at least three experiments, with the standard deviations indicated by shading. 1 mm succinate, 5 mm Na₂SO₄, 0.5 mm KCN, and 2 μg ml⁻¹ monensin were added as indicated. 200 nm rotenone was added along with the Na₂SO₄ and KCN where indicated. The signal has been normalized to 100% at the end of the experiment (to compare the traces during the recovery phase) and to 0% before the addition of M⁺ and cyanide (where M⁺ = Li⁺, Na⁺, K⁺, or Rb⁺); ∼60% of the total fluorescence was quenchable. B, same as A except with 5 mm K₂SO₄ instead of 5 mm Na₂SO₄. Conditions are: 21 °C, ∼30 μg ml⁻¹ protein.

FIGURE 6.

Characteristics of the antiporter reactions in PLs and BtSMPs. A, BtPLs, with deactive CI, were tested using Experiment A in the presence of various inhibitors. A high value for the normalized fluorescence quench (100% fluorescence quench = fluorescence in the presence of monensin) 75 s after the PLs were added indicates high antiporter activity (Fig. 4). Caps.: 50 μm capsaicin; Fenp.: 150 nm fenpyroximate; Aceto.: 30 μm Δlac-acetogenin; MPP⁺, 5 mm 1-methyl-4-phenyl-pyridinium; Pier.: 1 μm piericidin A; Ranol.: 200 μm ranolazine; Rot.: 200 nm rotenone; Stig.: 250 nm stigmatellin. NEM indicates that the BtPLs were preincubated in 1 mm NEM for 25 min and then washed and collected by centrifugation. B, BtPLs and YlPLs were tested using Experiment A following activation (-A) or deactivation (-D). C, BtSMPs, with active or deactive CI, in the presence or absence of 200 nm rotenone (Rot.), were tested for M⁺/H⁺ antiporter activity (M⁺ = Li⁺, Na⁺, K⁺, or Rb⁺). A high 1/t½ (where t½ is the time taken for the fluorescence to recover to half its full value upon the addition of M⁺) indicates significant antiporter activity (Fig. 5).