Respiration rates and coupling efficiency of oxidative phosphorylation using succinate as substrate in mitochondria isolated from cybrids. Closed bars, haplogroup H; open bars, haplogroup T. (A) State 3, state 4 and uncoupled respiration rates. (B) Respiratory control ratio (state 3 respiration rate/state 4 respiration rate). (C) Coupling efficiency at selected Δψ values between state 3 and state 4. Data are means ± SEM (n = 15 cell clones- five different cybrid clones for each donor; three donors for each haplogroup). Values were calculated from the results shown in Fig. 1. There were no significant differences between haplogroups H and T for any of the data in Fig. 2.

Cellular and mitochondrial respiration rates in intact cybrids. Closed symbols and bars, haplogroup H; open symbols and bars, haplogroup T. (A) Oxygen consumption rates of cells grown in 24-well plates measured using a Seahorse XF24 Extracellular Flux Analyzer; the time after the first measurement is indicated. Rates were expressed as a percentage of the basal rate in each well measured at about 20 min, just before oligomycin addition. At the times indicated, final concentrations of 1 µg/ml oligomycin, 3 µM FCCP and 1 µM rotenone plus 2 µM myxothiazol were injected automatically. (B) Mitochondrial respiration rates in intact cybrids calculated from (A). Non-mitochondrial oxygen consumption was calculated as the mean of the last three points in (A), and subtracted from the mean of the first four points in (A). The difference was scaled to give 100% of basal mitochondrial respiration. Respiration driving ATP synthesis was calculated as the mitochondrial rate sensitive to oligomycin, and respiration driving proton leak as the mitochondrial rate insensitive to oligomycin, both calculated from the mean of the three points after addition of oligomycin in (A). Uncoupled mitochondrial respiration was calculated arbitrarily as the mean of the first three points after addition of FCCP. Data are means ± SEM (n = 6 cell clones, two different cybrid clones for each donor and three donors for each haplogroup). There were no significant differences between haplogroups H and T for any of the data in Fig. 4.

Modular kinetic analysis of oxidative phosphorylation in mitochondria isolated from cybrids. Modular kinetic analysis, using 4 mM succinate as substrate, of the kinetic responses to membrane potential, Δψ, of respiration driving (A) substrate oxidation (Δψ titrated with uncoupler, FCCP), (B) proton leak (Δψ titrated with malonate, starting in state 4) and (C) the phosphorylating system, calculated by subtracting respiration driving proton leak from respiration driving the Δψ-consumers (Δψ titrated with malonate starting in state 3; not shown) at each Δψ. (D) Kinetic response to Δψ of respiration driving substrate oxidation using 3.2 mM 2-oxoglutarate + 0.8 mM malate as substrate, with rotenone omitted. Closed symbols, haplogroup H; open symbols, haplogroup T. Results are means ± SEM (n = 15 cell clones — five different cybrid clones for each donor; three donors for each haplogroup). There were no significant differences between haplogroups H and T for any of the data in Fig. 1.

Respiration rates and coupling efficiency of oxidative phosphorylation using 2-oxoglutarate + malate as substrate in mitochondria isolated from cybrids. Closed bars, haplogroup H; open bars, haplogroup T. (A) State 3, state 4 and uncoupled respiration rates, all in the absence of nigericin. (B) Respiratory control ratio (state 3 respiration rate/state 4 respiration rate) in the absence of nigericin. (C) Coupling efficiency at selected Δψ values between state 3 and state 4 in the presence of nigericin. Data are means ± SEM (n = 15 cell clones — five different cybrid clones for each donor; three donors for each haplogroup). Values in (C) were calculated from the results shown in Fig. 1. H⁺/O ratios were taken to be 10 and 6 for respiration on 2-oxoglutarate + malate and succinate, respectively (Brand, 2005); the curves for proton leak with succinate as substrate (Fig. 1B) were scaled by 6/10 and used to calculate the coupling efficiencies with oxoglutarate + malate as substrate using the data in Fig. 1D. ^⁎P < 0.05.