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.

Acute Mitochondrial Inhibition by Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase (MEK) 1/2 Inhibitors Regulates Proliferation

“…Our previous work with the protonophore CCCP has shown the Cytc oxidation state changes very little at low concentrations, where VO 2 increases, but becomes highly oxidized at higher concentrations where VO 2 decreases (35). PD198306 produced exactly this pattern of oxidation and electron flux changes consistent with it acting as a protonophore.…”

Mammalian Complex I Pumps 4 Protons per 2 Electrons at High and Physiological Proton Motive Force in Living Cells*

“…Spectroscopy and Spectral Analysis-The spectroscopy system has been described previously (13). In brief, heme attenuation spectra and NADH fluorescence spectra were measured with two separate CCD-spectrograph systems working in timemultiplexed mode at 50 Hz, and contiguous spectra were averaged to give a temporal resolution of 0.5 s. A warm white light emitting diode (LED) was used for the attenuation spectra illumination, and a 365 nm UV LED was used for NADH fluorescence excitation.…”

“…Recently we have developed a methodology to calculate the membrane potential (⌬⌿) and pH gradient (⌬pH) components of ⌬P as well as the redox potential of the UQH 2 /UQ pool from the redox poise of the bc 1 complex measured using multiwavelength cell spectroscopy in living cells (13). Combined with NADH fluorescence spectroscopy to measure the oxidation state of mitochondrial NADH, it provides all the necessary parameters to examine the thermodynamic poise of complex I in mitochondria respiring in the authentic environment of a living cell.…”

Mitochondrial energetic metabolism—some general principles

“…This has been experimentally shown: Kim et al (12) measured all the thermodynamical parameters in whole cells and found at state 4 in the presence of oligomycin with a Dp % 200 mV associated with E h UQ ¼ 70 mV and E h Cytc ¼ þ280 mV, which gives 210 mV as redox span for complex III and places E h NADH at at least À330 mV, that is, 68% of…”