Iberius

Redox Poise of Complex I



Complex I transfers electrons from the NAD+/NADH pool to the UQ/UQH2 pool pumping two protons per electron. The free energy change (ΔGI) is given by:



Where EhUQ and EhNADH are the redox potentials of the redox potentials of the UQ/UQH2 and NAD+/NADH pools, respectively, np is the proton pumping stoichiometry and ΔP is the proton motive force.

Complex I is an energy transducing enzyme that converts redox energy (the free energy released on transfer of electrons from the NAD+/NADH pool to the UQ/UQH2 pool) into the proton motive force. The thermodynamic efficiency of complex I (ηI) is given by:



Where np is the proton pumping stoichiometry, ΔP is the proton motive force and ΔE is redox energy available which is the difference in redox potentials between the UQ/UQH2 and NAD+/NADH pools.


In order to achieve high thermodynamic efficiency, complex I must work close to equilibrium. At equilibrium (ΔG=0), the flux is zero and so no energy is transduced. Therefore, complex I must achieve high flux close to equilibrium to achieve efficient energy transduction.

Figure 2 shows the force-flux relationship for complex I in untreated Raw 264.7 cells and cells treated with 6nM of piericidin, a specific complex I inhibitor. The flux is plotted as a function of ΔG, which is the force driving the flux. Close to equilibrium (ΔG=0) the flux is linear with respect to the ΔG and, far from equilibrium (ΔG<−80mV), the flux saturates.

The functionality is the gradient of the force-flux relationship close to equilibrium, as shown by the linear regression (see [1]). A high functionality means that complex I can achieve high flux close to equilibrium. Piericidin lowers the functionality of complex I because it inhibits its function.

A good analogy is a person pushing a wheelbarrow. The person applies a force to the wheelbarrow, equivalent to the ΔG, to make the wheel barrow move (the flux). The higher the force (ΔG), the faster the wheel barrow will move (flux). If the bearings on the wheel of the wheelbarrow are well oiled and smooth, then only a small force would be required to move the wheel barrow at pace and it would have a high functionality. If the bearing was rusty, it would require a large force to make the wheel barrow move and it would have a low functionality.

Figure 1 Redox poise of complex I




Figure 2 Force flux relationship for complex I in control RAW 264.7 cells and cells treated with 6nM of Piericidin, a specific complex I inhibitor. Data is -presented as mean±SE (n=6). The lines are a regression to 3 data points closest to equilibrium and extrapolate to meet the x-axis at ΔG=0 with good accuracy, as would be expected.

Measurement of Complex I Functionality



The functionality of complex I is measured by the Iberius by first treating the cells with oligomycin to inhibit the ATP synthase and decrease the electron flux bringing complex I closer to equilibrium (Figure 3).

The cells are then treated with multiple small doses of an uncoupler to increase the electron flux and drive complex I out of equilibrium.

The flux is then plotted as a function of ΔGI and the gradient of the response close to equilibrium calculated from a linear regression.


Figure 3. Measurement of the force flux relationship of complex I and the functionality. Additions: O:oligomycin, U:uncoupler.


References


  1. Rocha, M. and R. Springett, Measuring the functionality of the mitochondrial pumping complexes with multi-wavelength spectroscopy. Biochim Biophys Acta Bioenerg, 2019. 1860(1): p. 89-101.


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