Measurement of the mitochondrial membrane potential and pH gradient from the redox poise of the hemes of the bc1 complex

Abstract

The redox potentials of the hemes of the mitochondrial bc(1) complex are dependent on the proton-motive force due to the energy transduction. This allows the membrane potential and pH gradient components to be calculated from the oxidation state of the hemes measured with multi-wavelength cell spectroscopy. Oxidation states were measured in living RAW 264.7 cells under varying electron flux and membrane potential obtained by a combination of oligomycin and titration with a proton ionophore. A stochastic model of bc(1) turnover was used to confirm that the membrane potential and redox potential of the ubiquinone pool could be measured from the redox poise of the b-hemes under physiological conditions assuming the redox couples are in equilibrium. The pH gradient was then calculated from the difference in redox potentials of cytochrome c and ubiquinone pool using the stochastic model to evaluate the ΔG of the bc(1) complex. The technique allows absolute quantification of the membrane potential, pH gradient, and proton-motive force without the need for genetic manipulation or exogenous compounds.

Figure 1

Cartoon of the bc₁ complex showing the relative position in the membrane of the redox centers (b_L, b_H, c₁, Rieske), the ubiquinone binding centers (Q_o, Q_i), the bound Cyt, and the high and low potential chain electron pathways.

Figure 2

Oxidation changes in b_H, b_L, c₁, and Cytc and ΔΨ during the experimental protocol <50 μM. (Arrows) Additions to the chamber. (O) 5 μg/mL oligomycin, (C) 75 nM CCCP, (N) 1 mM 3-NPA, and (R) 1 μM Rotenone. The hemes were assumed to be fully oxidized after rotenone.

Figure 3

Mitochondrial oxygen consumption and oxidation state of b_H, b_L, c₁, and Cytc under baseline conditions (B), after oligomycin (O) and subsequent addition of CCCP.

Figure 4

The bc₁ turnover, redox potentials of Cytc, c₁, and UQ, bc₁ redox span, and ΔΨ under baseline conditions (B), after oligomycin (O) and subsequent addition of CCCP.

Figure 5

Stochastic model estimations of bc₁ turnover (upper panels), oxidation states of b_H and b_L (middle panels), and the difference between ΔΨ and $E_h^{UQ}$ used the model and calculated from the oxidation state of the hemes assuming equilibrium (lower panels) as a function of $E_{h,7}^{UQ}$ for ΔΨ = 120 mV (left panels), 150 mV (center panels), and 180 mV (right panels). The values $E_h^{Cytc}$ and ΔH⁺ were set to 285 and 20 mV, respectively.

Figure 6

Difference between the measured and simulated bc₁ turnover as a function of ΔG using values of bc₁ turnover, $E_h^{Cytc}$, $E_h^{UQ}$, and ΔΨ from oligomycin-inhibited cells (O) and after addition of 150, 300, 450, and 600 nM of CCCP. The value ΔG was varied in the simulation by varying ΔH⁺.

Figure 7

Disequilibrium of the bc₁ complex calculated from the stochastic simulations compared to the difference between the redox span of the bc₁ complex and ΔΨ, which is equal to 2ΔH⁺+ΔG under baseline conditions (B), after oligomycin (O) and subsequent addition of CCCP.

Figure 8

Values ΔP, ΔΨ, and ΔH⁺ under baseline conditions (B), after oligomycin (O) and subsequent addition of CCCP.