The relationship between the rate of respiration and the protonmotive force. The role of proton conductivity.
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ABSTRACT:
SUBMITTER: O'Shea PS
PROVIDER: S-EPMC1153494 | biostudies-other | 1984 Apr
REPOSITORIES: biostudies-other
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Project description:The rate of respiration in mitochondria is not a unique function of the protonmotive force, depending on whether the protonmotive force is varied by addition of ADP or uncouplers. This result has been generally considered to contradict the chemiosmotic theory. Recently, O'Shea & Chappell [Biochem. J. (1984) 219, 401-404] claimed that this observation can be reconciled with the chemiosmotic theory, provided only that the proton conductance of the membrane is different in the presence of ADP or uncouplers. This hypothesis is shown here to be necessary but not sufficient to account for the experimental data and the reason for the contradiction between this recent interpretation and earlier interpretations is pointed out.
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Project description: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 (DeltaPsi) and the chemical (DeltapH) 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 (DeltaPsi) and a small rise in the chemical component (DeltapH). 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.
| S-EPMC1164795 | biostudies-other
Project description:The TonB system of Escherichia coli (TonB/ExbB/ExbD) transduces the protonmotive force (pmf) of the cytoplasmic membrane to drive active transport by high-affinity outer membrane transporters. In this study, chromosomally encoded ExbD formed formaldehyde-linked complexes with TonB, ExbB and itself (homodimers) in vivo. Pmf was required for detectable cross-linking between TonB-ExbD periplasmic domains. Consistent with that observation, the presence of inactivating transmembrane domain mutations ExbD(D25N) or TonB(H20A) also prevented efficient formaldehyde cross-linking between ExbD and TonB. A specific site of periplasmic interaction occurred between ExbD(A92C) and TonB(A150C) and required functional transmembrane domains in both proteins. Conversely, neither TonB, ExbB nor pmf were required for ExbD dimer formation. These data suggest two possible models where either dynamic complex formation occurred through transmembrane domains or the transmembrane domains of ExbD and TonB configure their respective periplasmic domains. Analysis of T7-tagged ExbD with anti-ExbD antibodies revealed that a T7 tag was responsible both for our previous failure to detect T7-ExbD-ExbB and T7-ExbD-TonB formaldehyde-linked complexes and for the concomitant artefactual appearance of T7-ExbD trimers.
| S-EPMC2729267 | biostudies-literature
Project description:The rate of phosphorylation of ADP by isolated mitochondria respiring on succinate was set by addition of ATP, ADP or ADP plus malonate. We measured the rates of phosphorylation and respiration and the protonmotive force under each of these conditions. We measured the oxygen consumption required to drive the proton leak at the protonmotive force reached under each condition and subtracted it from the respiration rate during phosphorylation to determine the oxygen consumption driving phosphorylation. By dividing the rate of phosphorylation by the rate of respiration driving phosphorylation we calculated the mechanistic P/O ratio (number of molecules of ADP phosphorylated per oxygen atom reduced). This ratio was the same at high, intermediate and low values of protonmotive force, indicating that the relative stoichiometries of the mitochondrial protonmotive-force-producing and protonmotive-force-consuming pumps (i.e. H+/O:H+/ATP) are independent of the protonmotive force. This greatly weakens the case for a decrease in stoichiometry, or 'slip', in the mitochondrial proton pumps at high protonmotive force.
| S-EPMC1150015 | biostudies-other
Project description:Although mitochondrial respiration is believed to explain a substantial part of the variation in resting metabolic rate (RMR), few studies have empirically studied the relationship between organismal and cellular metabolism. We therefore investigated the relationship between RMR and mitochondrial respiration of permeabilized blood cells in wild great tits (Parus major L.). We also studied the correlation between mitochondrial respiration traits and blood cell count, as normalizing mitochondrial respiration by the cell count is a method commonly used to study blood metabolism. In contrast to previous studies, our results show that there was no relationship between RMR and mitochondrial respiration in intact blood cells (i.e. with the ROUTINE respiration). However, when cells were permeabilized and interrelation re-assessed under saturating substrate availability, we found that RMR was positively related to phosphorylating respiration rates through complexes I and II (i.e. OXPHOS respiration) and to the mitochondrial efficiency to produce energy (i.e. net phosphorylation efficiency), though variation explained by the models was low (i.e. linear model: R2=0.14 to 0.21). However, unlike studies in mammals, LEAK respiration without [i.e. L(n)] and with [i.e. L(Omy)] adenylates was not significantly related to RMR. These results suggest that phosphorylating respiration in blood cells can potentially be used to predict RMR in wild birds, but that this relationship may have to be addressed in standardized conditions (permeabilized cells) and that the prediction risks being imprecise. We also showed that, in our conditions, there was no relationship between any mitochondrial respiration trait and blood cell count. Hence, we caution against normalising respiration rates using this parameter as is sometimes done. Future work should address the functional explanations for the observed relationships, and determine why these appear labile across space, time, taxon, and physiological state.
| S-EPMC10958200 | biostudies-literature
Project description:Mitochondrial respiration generates an electrochemical proton gradient across the mitochondrial inner membrane called protonmotive force (PMF) to drive diverse functions and synthesize ATP. Current techniques to manipulate the PMF are limited to its dissipation; yet, there is no precise and reversible method to increase the PMF. To address this issue, we aimed to use an optogenetic approach and engineered a mitochondria-targeted light-activated proton pump that we name mitochondria-ON (mtON) to selectively increase the PMF in Caenorhabditis elegans. Here we show that mtON photoactivation increases the PMF in a dose-dependent manner, supports ATP synthesis, increases resistance to mitochondrial toxins, and modulates energy-sensing behavior. Moreover, transient mtON activation during hypoxic preconditioning prevents the well-characterized adaptive response of hypoxia resistance. Our results show that optogenetic manipulation of the PMF is a powerful tool to modulate metabolism and cell signaling.
| S-EPMC7132214 | biostudies-literature
Project description:It is well known that loss of aerobic respiration in Gram-negative bacteria can diminish the efficacy of a variety of bactericidal antibiotics, which has lead to subsequent demonstrations that the formation of reactive oxygen species (ROS) and the proton motive force (PMF) can both play a role in antibiotic toxicity. The susceptibility of Gram-negative bacteria to aminoglycoside antibiotics, particularly gentamicin, has previously been linked to both the production of ROS and the rate of antibiotic uptake that is mediated by the PMF, although the relative contributions of ROS and PMF to aminoglycoside toxicity has remained poorly understood. Herein, gentamicin was shown to elicit a very modest increase in ROS levels in an aerobically grown Escherichia coli clinical isolate. The well-characterised uncoupler 2,4-dinitrophenol (DNP) was used to disrupt the PMF, which resulted in a significant decrease in gentamicin lethality towards E. coli. DNP did not significantly alter respiratory oxygen consumption, supporting the hypothesis that this uncoupler does not increase ROS production via elevated respiratory oxidase activity. These observations support the hypothesis that maintenance of PMF rather than induction of ROS production underpins the mechanism for how the respiratory chain potentiates the toxicity of aminoglycosides. This was further supported by the demonstration that the uncoupler DNP elicits a dramatic decrease in gentamicin lethality under anaerobic conditions. Together, these data strongly suggest that maintenance of the PMF is the dominant mechanism for the respiratory chain in potentiating the toxic effects of aminoglycosides.
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Project description:Proton conductivity is important in many natural phenomena including oxidative phosphorylation in mitochondria and archaea, uncoupling membrane potentials by the antibiotic Gramicidin, and proton actuated bioluminescence in dinoflagellate. In all of these phenomena, the conduction of protons occurs along chains of hydrogen bonds between water and hydrophilic residues. These chains of hydrogen bonds are also present in many hydrated biopolymers and macromolecule including collagen, keratin, chitosan, and various proteins such as reflectin. All of these materials are also proton conductors. Recently, our group has discovered that the jelly found in the Ampullae of Lorenzini- shark's electro-sensing organs- is the highest naturally occurring proton conducting substance. The jelly has a complex composition, but we proposed that the conductivity is due to the glycosaminoglycan keratan sulfate (KS). Here we measure the proton conductivity of hydrated keratan sulfate purified from Bovine Cornea. PdHx contacts at 0.50 ± 0.11 mS cm -1, which is consistent to that of Ampullae of Lorenzini jelly at 2 ± 1 mS cm -1. Proton conductivity, albeit with lower values, is also shared by other glycosaminoglycans with similar chemical structures including dermatan sulfate, chondroitin sulfate A, heparan sulfate, and hyaluronic acid. This observation supports the relationship between proton conductivity and the chemical structure of biopolymers.
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Project description:1. Oxidation of ferrocytochrome c by cytochrome c oxidase incorporated into proteoliposomes induces a transient acidification of the external medium. This change is dependent on the presence of valinomycin and can be abolished by carbonyl cyanide p-trifluoromethoxyphenylhydrazone or by nigericin. The H+/e- ratio for the initial acidification varies with the internal buffering capacity of the vesicles, and under suitable conditions approaches + 1, the pulse slowly decaying to give a net alkalinity change with H+/e- value approaching -1. 2. Inhibition of cytochrome c oxidase turnover by ferricytochrome c or by azide addition results in ferrocytochrome c-dependent H+ pulses with decreasing H+/e- ratios. The rate of the initial H+ production remains higher than the rate of equilibration of the pH gradient, indicating an intrinsic dependence of the H+/e- ratio on enzyme turnover. The final net alkalinity changes are relatively unaffected by turnover inhibition.
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