1. To determine whether controlled (State 4) pyruvate oxidation can support a high energy state, measurements of the redox span NAD–cytochrome c, phosphorylation potential and protonmotive force (the gradient in electrochemical activity of protons across the mitochondrial inner membrane) were made as indices of energy status. For comparison, these three measurements were also made with glycerol 3-phosphate, an alternative substrate. The two substrates gave essentially identical values for the redox span NAD–cytochrome c in State 4, and the phosphorylation potential was of sufficient magnitude to be considered in equilibrium with the redox span over the first two phosphorylation sites. The magnitude of the protonmotive force in State 4 was much less and the implications of this finding are discussed. 2. Measurements made during the controlled (State 4) to active (State 3) transition indicated that with glycerol 3-phosphate as substrate, both the redox span NAD–cytochrome c and the protonmotive force were diminished; the State 4 → State 3 transition with pyruvate as substrate was accompanied by an increase in the redox span but a decrease in protonmotive force. The contrary behaviour of these two energetic parameters in the presence of pyruvate was ascribed to a transient excess in the flux of protons through the adenosine triphosphatase relative to the protonpumping respiratory chain, in spite of the increased dehydrogenase activity. 3. The lower protonmotive force seen in State 3 relative to State 4 with pyruvate as substrate was due to a diminution of both the electrical (ΔΨ) and the chemical (ΔpH) components; with glycerol 3-phosphate, the magnitude of the decrease in protonmotive force during the State 4 → State 3 transition was similar to that seen with pyruvate, but was due to a large decrease in the electrical component (ΔΨ) and a small rise in the chemical component (ΔpH). The reason for the difference seen in the behaviour of the components of the protonmotive force was investigated but not established. 4. In the presence of oligomycin and ADP, oxidation of pyruvate, but not of glycerol 3-phosphate, supported a greater protonmotive force than in State 4, in keeping with the dehydrogenase activation and increased redox span NAD–cytochrome c found under these conditions. 5. Experiments involving the use of uncoupling agent to stimulate respiration are compared with those in which limiting concentrations of ADP were used. Estimates of the proton conductance of the inner membrane indicate a similar non-linear dependence on uncoupler concentration with the two substrates. 6. A model is proposed as an explanation of the high rates of controlled glycerol 3-phosphate oxidation. The model relies on a high permeability of the inner membrane to protons and other ions being induced by glycerol 3-phosphate oxidation in State 4.
The nature of controlled respiration and its relationship to protonmotive force and proton conductance in blowfly flight-muscle mitochondria
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Roger N. Johnson, Richard G. Hansford; The nature of controlled respiration and its relationship to protonmotive force and proton conductance in blowfly flight-muscle mitochondria. Biochem J 15 May 1977; 164 (2): 305–322. doi: https://doi.org/10.1042/bj1640305
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