Respiratory Complex I in Bos taurus and Paracoccus denitrificans Pumps Four Protons across the Membrane for Every NADH Oxidized

Abstract

Respiratory complex I couples electron transfer between NADH and ubiquinone to proton translocation across an energy-transducing membrane to support the proton-motive force that drives ATP synthesis. The proton-pumping stoichiometry of complex I (i.e. the number of protons pumped for each two electrons transferred) underpins all mechanistic proposals. However, it remains controversial and has not been determined for any of the bacterial enzymes that are exploited as model systems for the mammalian enzyme. Here, we describe a simple method for determining the proton-pumping stoichiometry of complex I in inverted membrane vesicles under steady-state ADP-phosphorylating conditions. Our method exploits the rate of ATP synthesis, driven by oxidation of NADH or succinate with different sections of the respiratory chain engaged in catalysis as a proxy for the rate of proton translocation and determines the stoichiometry of complex I by reference to the known stoichiometries of complexes III and IV. Using vesicles prepared from mammalian mitochondria (from Bos taurus) and from the bacterium Paracoccus denitrificans, we show that four protons are pumped for every two electrons transferred in both cases. By confirming the four-proton stoichiometry for mammalian complex I and, for the first time, demonstrating the same value for a bacterial complex, we establish the utility of P. denitrificans complex I as a model system for the mammalian enzyme. P. denitrificans is the first system described in which mutagenesis in any complex I core subunit may be combined with quantitative proton-pumping measurements for mechanistic studies.

Keywords: complex I; electron transfer complex; mitochondria; mitochondrial respiratory chain complex; oxidative phosphorylation; proton motive force.

FIGURE 1.

Schematic representation of ATP synthesis in the SMP and SBP systems. A, the NADH:O₂ reaction drives proton translocation by complexes I, III, and IV ((n + 6) H⁺ per NADH). B, the NADH:Q₁ reaction drives proton translocation by complex I (n H⁺ per NADH); complexes III and IV are inhibited. C, the succinate:O₂ reaction drives proton translocation by complexes II, III, and IV (6 H⁺ per succinate). The number of protons required to synthesize 1 ATP is 2.7 in B. taurus and 4 in P. denitrificans.

FIGURE 2.

ATP synthesis driven by the NADH:O₂ reaction in SMPs is sensitive to dissipation of Δp and inhibition of complex I. NADH oxidation (A) and ATP synthesis (B) were initiated with 200 μm NADH and monitored simultaneously (see “Experimental Procedures”). The data from the standard reaction (red) are compared with data recorded in the presence of 4 μm FCCP to dissipate Δp (orange) and in the presence of 5 μm piericidin A to inhibit complex I catalysis (magenta). Rates of NADH oxidation and ATP synthesis are marked in μmol min⁻¹ mg⁻¹.

FIGURE 3.

Data from experiments to determine the stoichiometry of B. taurus complex I in SMPs and P. denitrificans complex I in SBPs. A and B show data from SMPs and C and D data from SBPs. The rates of substrate consumption (A and C) and ATP synthesis (B and D) were monitored simultaneously (see “Experimental Procedures”). Rates of ATP synthesis have been matched using inhibitors (see text). The NADH:O₂ reaction (red) was initiated by the addition of 200 μm NADH in the presence of 13 mm (SMPs) or 5 mm (SBPs) ADP-ribose. The succinate:O₂ reaction (green) was initiated by 5 mm succinate in the presence of 5 nm atpenin (SMPs). The NADH:Q₁ reaction (blue) was initiated by 200 μm NADH in the presence of 150 μm ubiquinone-1 and 500 nm (SMPs) or 1.25 μm (SBPs) myxothiazole. Rates of substrate oxidation and ATP synthesis are marked in μmol min⁻¹ mg⁻¹; the inhibitor-insensitive rates of the NADH:Q₁ reaction (0.091 ± 0.011 in SMPs and 0.101 ± 0.009 μmol min⁻¹ mg⁻¹ in SBPs) have been subtracted.

FIGURE 4.

The strategy taken to delete the two hydrogenase genes in P. denitrificans. Horizontal arrows in flanking region 2 show the Pden_3101 ORF and promoter region.