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.