Intrinsic uncoupling of mitochondrial proton pumps. 2. Modeling studies

Pietrobon, Daniela; Zoratti, Mario; Gf, Azzone; Caplan,

doi:10.1021/bi00352a005

Cited by 58 publications

(26 citation statements)

References 29 publications

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“…We performed crucial validation tests of the upgraded model by combining experimental and theoretical data as follows: 1), simulation of the respiratory (VO 2 ) and ATP synthesis (V ATPsynthase ) fluxes as related to their driving forces (i.e., Dp, redox, and phosphorylation potentials) in agreement with data reported in the literature (Fig. 2) (16,31,38,39); 2), simulation of experimental profiles of DJ m and NADH during the transition between states 2/4 and 3, hypoxia (mimicked by CN À addition), and uncoupling by DNP (Fig. 4); 3), simulation of the increase in VO 2 and VATP synthase upon addition of ADP, followed by their decrease under hypoxia and reversal of the ATP synthase during uncoupling (Fig.…”

Section: Discussionmentioning

confidence: 88%

Mitochondrial Energetics, pH Regulation, and Ion Dynamics: A Computational-Experimental Approach

Wei

Aon

O’Rourke

et al. 2011

Biophysical Journal

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We developed a computational model of mitochondrial energetics that includes Ca(2+), proton, Na(+), and phosphate dynamics. The model accounts for distinct respiratory fluxes from substrates of complex I and complex II, pH effects on equilibrium constants and enzyme kinetics, and the acid-base equilibrium distributions of energy intermediaries. We experimentally determined NADH and ΔΨ(m) in guinea pig mitochondria during transitions from de-energized to energized, or during state 2/4 to state 3 respiration, or into hypoxia and uncoupling, and compared the results with those obtained in model simulations. The model quantitatively reproduces the experimentally observed magnitude of ΔΨ(m), the range of NADH levels, respiratory fluxes, and respiratory control ratio upon transitions elicited by sequential additions of substrate and ADP. Simulation results are also able to mimic the change in ΔΨ(m) upon addition of phosphate to state 4 mitochondria, leading to matrix acidification and ΔΨ(m) polarization. The steady-state behavior of the integrated mitochondrial model qualitatively simulates the dependence of respiration on the proton motive force, and the expected flux-force relationships existing between respiratory and ATP synthesis fluxes versus redox and phosphorylation potentials. This upgraded mitochondrial model provides what we believe are new opportunities for simulating mitochondrial physiological behavior during dysfunctional states involving changes in pH and ion dynamics.

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Section: Discussionmentioning

confidence: 88%

Mitochondrial Energetics, pH Regulation, and Ion Dynamics: A Computational-Experimental Approach

Wei

Aon

O’Rourke

et al. 2011

Biophysical Journal

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“…The ICm of the DCCD block to this extra proton uptake was about 4.5 ,uM. The extent of the flash-induced electron transport and ofproton pumping was not influenced by DCCD, unless the concentration was raised to 20 ,M [at 10 Fig. 3 shows transients of the lumenal pH (B) and of the transmembrane voltage (A).…”

mentioning

confidence: 98%

“…4 and 8). Unambiguous conclusions were not reached, as clearly stated in articles from laboratories that had supported localized concepts (9,10). In none ofthese studies was proton flow by way of the ATP synthase kinetically and spatially resolved.…”

mentioning

confidence: 98%

Complete tracking of transient proton flow through active chloroplast ATP synthase

Junge

1987

Proc. Natl. Acad. Sci. U.S.A.

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Proton pumping in thylakoid membranes and backflow of protons through the active ATP synthase CFO-CF1 (where CFO is the proton channel and CF1 is the catalytic portion) were investigated by flash spectrophotometry. A steady pH difference across the membrane was generated by continuous measuring light, supplemented by voltage transients that were generated by flashing light. In the presence of Pi and ADP, the electric potential transients elicited transients of proton flow via CFO-CF1, typically 1.3 H+ per CF1 and flash group. Proton flow was blocked by N,N'-dicyclohexylcarbodiimide, acting on the channel component CFO, and tentoxin, acting on the catalytic component CF1.The half-rise time was 40 ms in '1120 and 78 ms in 2H20. ATP synthesis under conditions of flashing light and transient proton flow was characterized by a Km(Pi) of only 14 ,uM, contrasting with a Km of several hundred micromolar for continuous AtP synthesis at high rate. This might reflect a resistance to Pi dhffusion. The degree of proton delocalization in the chemiosmotic coupling between redox reactions and ATP synthesis is under debate. In thylakoids, it has been proposed that intramembrane proton buffering domains act as ducts for protons between pumps and ATP synthases. In this work, transient proton flow by way of CFO-CF1 was completely tracked from the lumen, across the membrane, and into the suspending medium. Proton uptake from the lumen and charge flow across the membrane occurred synchronously and in stoichiometric proportion. The uptake of protons from the lumen by CFO-CF1, half completed in 40 ms, was preceded by release of protons from water oxidation into the lumen, half completed in <1 ms. Hence, pumps and ATP synthases were coupled through the lumen without involvement of intramembrane domains.The chemiosmotic theory of oxidative and of photophosphorylation places proton pumps and proton translocating ATP synthases in a membrane and couples them by lateral proton flow through two aqueous bulk phases at different electrochemical potentials of the proton (1, 2). Though supported by a vast body of evidence, the delocalized coupling concept has been challenged from its origin (3). In one class of alternative models, proton pumps and ATP synthases are thought to be coupled in pairs rather than sharing common bulk phases (localized coupling or microchemiosmotic concepts, e.g., ref. 4). In another class, proton flow is thought to be contained in the membrane (e.g., ref. 5) or confined to the membrane/ water interface (e.g., refs. 6 and 7), and the protons in these localized domains are supposed not to be equilibrated with adjacent aqueous phases. The bulk of the information interpreted to favor localized coupling concepts was obtained using mitochondria. It resulted from an evaluation of the energetics of phosphorylation or from a comparison of the rates of stationary electron flow and of ATP synthesis in the presence of inhibitors to the former and the latter (reviewed in refs. 4 and 8). Unambiguous conclusions were not reac...

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“…This is effectively to allow a degree of slip into the enzyme. Evidence has been presented that slip in some proton pumps might be variable, depending on the value of Ap [23]. However, this is not crucial to the considerations raised here.…”

Section: Implications For the Mechanism Of H+-thasementioning

confidence: 86%

The ratio of protons translocated/hydride ion equivalent transferred by nicotinamide nucleotide transhydrogenase in chromatophores from Rhodospirillum rubrum

Bizouarn

Jackson

1993

European Journal of Biochemistry

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The reduction of acetylpyridine adenine dinucleotide (AcPdAD', an NAD' analogue) by NADPH, in chromatophores treated with valinomycin, was accompanied by alkalinisation of the external medium, as measured by the absorbance change of added cresol red, a simple, non-binding pH indicator. Experiments with a stopped-flow spectrophotometer showed that initial (linear) rates of alkalinisation persisted for 1-2s. From the results of experiments in which H' uptake was driven by a series of short flashes of light, the dependence of the outward proton leak on the extent of H ' uptake was established. Thus, the proton leak was subtracted from the initial rate of alkalinisation during transhydrogenation to give the true proton-uptake rate. The correction factor was usually about 10%. The ratio of protons translocated/H-transferred from NADPH to AcPdAD' (the H' / H-ratio) was 0.60 2 0.06.The transhydrogenation reaction between NAD ' and NADPH was measured in the presence of a regeneration system for NAD' (pyruvate and lactate dehydrogenase). In addition to the accompanying proton-translocation reaction, scalar H' consumption linked to the regeneration system was observed and permitted internal checks on the calibration of the cresol red absorbance changes. After correction for the proton leak and scalar proton uptake, an H + / K ratio of 0.60 2 0.30 was calculated from the initial rates.The water-soluble polypeptide of transhydrogenase (Th,) was washed from a sample of chroinatophores to inhibit transhydrogenation activity and the accompanying H + uptake. Re-addition of purified Th, to depleted chromatophores led to recovery of transhydrogenation activity and of H i uptake. In this reconstituted system the H'/H-was similar to that in the native membranes. These results make it unlikely that the H'/H-ratio is artefactually low because chromatophores have a population of transhydrogenase which is not coupled to proton translocation. Further evidence that the mechanistic H'/H-ratio of chromatophore transhydrogenase is less than 1 was provided by an analysis of the kinetics of alkalinisation of the medium during reduction of AcPdAD' by NADPH. It was shown that the progress of the transhydrogenation-induced alkalinisation was fitted by the sum of H' uptake (the rate of transhydrogenation multiplied by the H '/Hratio) plus the H' leak, when the ratio was 0.6 but not when it was 1.0. rubrum ; CTparticles, chromatophore membranes depleted of the soluble transhydrogenase component by repeated washing; dp, proton electrochemical potential gradient; ApH, transmembrane pH gradient; d y , transmembrane electrical potential.

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Intrinsic uncoupling of mitochondrial proton pumps. 2. Modeling studies

Cited by 58 publications

References 29 publications

Mitochondrial Energetics, pH Regulation, and Ion Dynamics: A Computational-Experimental Approach

Mitochondrial Energetics, pH Regulation, and Ion Dynamics: A Computational-Experimental Approach

Complete tracking of transient proton flow through active chloroplast ATP synthase

The ratio of protons translocated/hydride ion equivalent transferred by nicotinamide nucleotide transhydrogenase in chromatophores from Rhodospirillum rubrum

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