Analysis of relationships between the protonmotive force and rates and extents of ATP synthesis

1. The inference, implicit in the chemiosmotic hypothesis, that protons move into the bulk phase during ATP synthesis was investigated. 2. Incubation of rat liver mitochondria in the presence of the cation exchanger CM-Sephadex C-50 caused alkalinization in the medium, though total ATP synthesis remained unchanged. The addition of N-ethylmaleimide prevented the alkalinization, but there was still no indication of protons passing into the medium. The expected proton movement [Mitchell & Moyle (1967) Biochem. J. 105, 1147–1162] was readily detected when as an equivalent acid pulse. 3. Analysis of delta H+ decay curves after O2 pulses (3 micrograms-atoms of O/g of protein) indicated the presence of fast and slow components of decay, with first-order rate constants (k) of 0.24s-1 and 0.032s-1. The fast decay was finite and was eliminated in the presence of N-ethylmaleimide. 4. These observations are interpreted as evidence for the development of unmasking of fixed charges on the outer surface of the mitochondrial inner membrane during energization and for the existence of proton-retentive electrical fields (rho-zones) on this surface. The charge concentration is calculated as about 1 charge/10nm2. 5. A cycle of changes in a single fixed-charge molecule is proposed which mediates both Ca2+ uptake and the first step in the utilization of the rho-zone protonmotive force, delta p rho.

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Neuronal AMPK coordinates mitochondrial energy sensing and hypoxia resistance

10.1101/2020.05.01.073007 ◽

2020 ◽

Author(s):

Brandon J. Berry ◽

Aksana Baldzizhar ◽

Andrew P. Wojtovich

Keyword(s):

Protein Kinase ◽

Mitochondrial Function ◽

Stress Resistance ◽

Atp Synthesis ◽

Protonmotive Force ◽

Electrochemical Gradient ◽

Energy Sensing ◽

Amp Activated Protein Kinase ◽

Spatiotemporal Control

ABSTRACTOrganisms adapt to their environment through coordinated changes in mitochondrial function and metabolism. The mitochondrial protonmotive force (PMF) is an electrochemical gradient that powers ATP synthesis and adjusts metabolism to energetic demands via cellular signaling. It is unknown how or where transient PMF changes are sensed and signaled due to lack of precise spatiotemporal control in vivo. We addressed this by expressing a light-activated proton pump in mitochondria to spatiotemporally “turn off” mitochondrial function through PMF dissipation in tissues with light. We applied our construct – mitochondria-OFF (mtOFF) – to understand how metabolic status impacts hypoxia resistance, a response that relies on mitochondrial function. mtOFF activation induced starvation-like behavior mediated by AMP-activated protein kinase (AMPK). We found prophylactic mtOFF activation increased survival following hypoxia, and that protection relied on neuronal AMPK. Our study links spatiotemporal control of mitochondrial PMF to cellular metabolic changes that mediate behavior and stress resistance.

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Photosensitive phosphoproteins in Halobacteria: regulatory coupling of transmembrane proton flux and protein dephosphorylation.

The Journal of Cell Biology ◽

10.1083/jcb.91.3.895 ◽

1981 ◽

Vol 91 (3) ◽

pp. 895-900 ◽

Cited By ~ 11

Author(s):

E N Spudich ◽

J L Spudich

Keyword(s):

Protein Phosphorylation ◽

Action Spectrum ◽

Atp Synthesis ◽

Energy Coupling ◽

Proton Flux ◽

Protonmotive Force ◽

Proton Efflux ◽

Ph Gradients ◽

Protein Dephosphorylation ◽

Reversible Protein Phosphorylation

A photoregulated reversible protein phosphorylation system controlled by the halobacterial rhodopsins was recently reported. The results presented in this paper identify the initial steps in the pathway from the absorption of light to the photoregulated protein phosphorylation and dephosphorylation reactions. Action spectrum, biochemical, and genetic analyses show that the proton pump bacteriorhodopsin mediates light-induced dephosphorylation of three photoregulated phosphoproteins. Light absorbed by bacteriorhodopsin is used to establish a proton efflux from the cells. The increase in the inwardly directed protonmotive force (pmf) from this efflux induces dephosphorylation of the three phosphoproteins, as demonstrated by the effects of the protonophore CCCP and of artificially imposed transmembrane pH gradients. Upon darkening the cells, cessation of the proton efflux through bacteriorhodopsin causes a decrease in pmf, which induces rephosphorylation of the proteins. Pmf appears to function as a regulator rather than a driving force in this system. Measurements of pmf-driven ATP synthesis in our conditions indicate the regulation of protein phosphorylation by pmf is probably not a consequence of proton flux through the H+ ATPase, a known energy coupling structure in these cells. The properties of this system may indicate the existence of a pmf detector which regulates kinase or phosphatase activity; i.e., a regulatory coupling device.

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Mitochondrial transmembrane potential and pH gradient during anoxia

AJP Cell Physiology ◽

10.1152/ajpcell.1987.252.4.c349 ◽

1987 ◽

Vol 252 (4) ◽

pp. C349-C355 ◽

Cited By ~ 139

Author(s):

B. S. Andersson ◽

T. Y. Aw ◽

D. P. Jones

Keyword(s):

Transmembrane Potential ◽

Adenine Nucleotide ◽

Electron Flow ◽

Atp Synthesis ◽

Isolated Hepatocytes ◽

Mitochondrial Transmembrane Potential ◽

Ph Gradient ◽

Protonmotive Force ◽

Ion Distribution ◽

Cytosolic Ph

The effect of anoxia on the mitochondrial transmembrane potential and pH gradient was studied in a preparation of isolated hepatocytes. Transmembrane potential (delta psi) was calculated from the distribution of triphenylmethylphosphonium between the mitochondrial, cytosolic, and extracellular compartments, which were separated by digitonin fractionation and centrifugation. Mitochondrial and cytosolic pH values were calculated from the distribution of the weak acid, dimethadione, which was determined similarly. After 30 min anoxia, the magnitude of mitochondrial delta psi was decreased from -163 to -133 mV and the delta pH (mitochondria vs. cytoplasm) was essentially unchanged (aerobic, 0.78 +/- 0.08; anaerobic, 0.76 +/- 0.11). Thus the protonmotive force (delta p = delta psi-Z delta pH), is largely retained even in the absence of electron flow and ATP synthesis. Inhibitors of the ATP synthase (oligomycin), mitochondrial adenine nucleotide carrier (atractyloside), and glycolytic pathway (2-deoxy-D-glucose) do not affect the ability of the cell to maintain delta psi during anoxia. Therefore, the results indicate that retention of the protonmotive force is not due to utilization of ATP produced by glycolysis and suggest that mechanisms exist to preserve ion distribution during anoxia.

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The rate of ATP synthesis by submitochondrial particles can be independent of the magnitude of the protonmotive force

Biochemical Journal ◽

10.1042/bj1880945 ◽

1980 ◽

Vol 188 (3) ◽

pp. 945-948 ◽

Cited By ~ 40

Author(s):

M C Sorgato ◽

D Branca ◽

S J Ferguson

Keyword(s):

Atp Synthesis ◽

Substrate Oxidation ◽

Protonmotive Force ◽

Submitochondrial Particles

The problem of whether the rate of ATP synthesis is proportional to the magnitude of the protonmotive force has been studied in submitochondrial particles. It was found that the rate of ATP synthesis can decrease at constant protonmotive force and is more closely related to the rate of substrate oxidation.

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ATP synthesis driven by a protonmotive force inStreptococcus lactis

The Journal of Membrane Biology ◽

10.1007/bf01868580 ◽

1975 ◽

Vol 25 (1) ◽

pp. 285-310 ◽

Cited By ~ 42

Author(s):

Peter C. Maloney ◽

T. Hastings Wilson

Keyword(s):

Atp Synthesis ◽

Protonmotive Force

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The protonmotive force and ATP synthesis/membrane-fixed-charge relationships

Biochemical Society Transactions ◽

10.1042/bst0080453 ◽

1980 ◽

Vol 8 (4) ◽

pp. 453-454 ◽

Cited By ~ 2

Author(s):

G. P. R. ARCHBOLD ◽

C. L. FARRINGTON ◽

SYLVIA A. LAPPIN ◽

A. M. McKAY ◽

F. H. MALPRESS

Keyword(s):

Atp Synthesis ◽

Fixed Charge ◽

Protonmotive Force

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A Protonmotive Force Drives ATP Synthesis in Bacteria

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.71.10.3896 ◽

1974 ◽

Vol 71 (10) ◽

pp. 3896-3900 ◽

Cited By ~ 70

Author(s):

P. C. Maloney ◽

E. R. Kashket ◽

T. H. Wilson

Keyword(s):

Atp Synthesis ◽

Protonmotive Force

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The protonmotive force in bovine heart submitochondrial particles. Magnitude, sites of generation and comparison with the phosphorylation potential

Biochemical Journal ◽

10.1042/bj1740237 ◽

1978 ◽

Vol 174 (1) ◽

pp. 237-256 ◽

Cited By ~ 59

Author(s):

M C Sorgato ◽

S J Ferguson ◽

D B Kell ◽

P John

Keyword(s):

Membrane Potential ◽

Reaction Medium ◽

Atp Synthesis ◽

Ph Gradient ◽

Protonmotive Force ◽

Submitochondrial Particles ◽

Electron Mediator ◽

Phosphorylation Potential ◽

Bovine Heart ◽

Gradient Component

1. The magnitude of the protonmotive force in respiring bovine heart submitochondrial particles was estimated. The membrane-potential component was determined from the uptake of S14CN-ions, and the pH-gradient component from the uptake of [14C]methylamine. In each case a flow-dialysis technique was used to monitor uptake. 2. With NADH as substrate the membrane potential was approx. 145mV and the pH gradient was between 0 and 0.5 unit when the particles were suspended in a Pi/Tris reaction medium. The addition of the permeant NO3-ion decreased the membrane potential with a corresponding increase in the pH gradient. In a medium containing 200mM-sucrose, 50mM-KCl and Hepes as buffer, the total protonmotive force was 185mV, comprising a membrane potential of 90mV and a pH gradient of 1.6 units. Thus the protonmotive force was slightly larger in the high-osmolarity medium. 3. The phosphorylation potential (= deltaG0′ + RT ln[ATP]/[ADP][Pi]) was approx. 43.1 kJ/mol (10.3kcal/mol) in all the reaction media tested. Comparison of this value with the protonmotive force indicates that more than 2 and up to 3 protons must be moved across the membrane for each molecule of ATP synthesized by a chemiosmotic mechanism. 4. Succinate generated both a protonmotive force and a phosphorylation potential that were of similar magnitude to those observed with NADH as substrate. 5. Although oxidation of NADH supports a rate of ATP synthesis that is approximately twice that observed with succinate, respiration with either of these substrates generated a very similar protonmotive force. Thus there seemed to be no strict relation between the size of the protonmotive force and the phosphorylation rate. 6. In the presence of antimycin and/or 2-n-heptyl-4-hydroxyquinoline N-oxide, ascorbate oxidation with either NNN'N′-tetramethyl-p-phenylenediamine or 2,3,5,6-tetramethyl-p-phenylenediamine as electron mediator generated a membrane potential of approx. 90mV, but no pH gradient was detected, even in the presence of NO3-. These data are discussed with reference to the proposal that cytochrome oxidase contains a proton pump.

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The Non-Ohmic Proton Leak—25 Years On

Bioscience Reports ◽

10.1023/a:1027376426860 ◽

1997 ◽

Vol 17 (3) ◽

pp. 251-257 ◽

Cited By ~ 28

Author(s):

David G. Nicholls

Keyword(s):

Ion Transport ◽

Respiratory Chain ◽

Atp Synthesis ◽

Proton Leak ◽

Protonmotive Force ◽

Proton Conductance ◽

Inner Membrane ◽

Ohm’S Law ◽

Mitochondrial Inner Membrane ◽

Proton Current

The proton conductance of the mitochondrial inner membrane can be quantified by applying Ohm's law to the experimentally determined protonmotive force and the proton current flowing around the proton circuit in the absence of ATP synthesis or ion transport. This last parameter is derived from the rate of State 4 respiration multiplied by the H+/O stoichiometry for the substrate. When the activity of the dehydrogenase supplying electrons to the respiratory chain is progressively increased the proton conductance increases rapidly when the protonmotive force is greater than 220 mV. The consequences of this non-ohmic relationship are discussed.

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