An ancient pathway combining carbon dioxide fixation with the generation and utilization of a sodium ion gradient for ATP synthesis

Synthesis of acetate from carbon dioxide and molecular hydrogen is considered to be the first carbon assimilation pathway on earth. It combines carbon dioxide fixation into acetyl-CoA with the production of ATP via an energized cell membrane. How the pathway is coupled with the net synthesis of ATP has been an enigma. The anaerobic, acetogenic bacterium Acetobacterium woodii uses an ancient version of this pathway without cytochromes and quinones. It generates a sodium ion potential across the cell membrane by the sodium-motive ferredoxin:NAD oxidoreductase (Rnf). The genome sequence of A. woodii solves the enigma: it uncovers Rnf as the only ion-motive enzyme coupled to the pathway and unravels a metabolism designed to produce reduced ferredoxin and overcome energetic barriers by virtue of electron-bifurcating, soluble enzymes.     

ATP formation coupled to caffeate reduction by H2 in Acetobacterium woodii NZva16

We have addressed the question, whether the reduction of caffeate in Acetobacterium woodii strain NZva16 is coupled to ATP synthesis by electron transport phosphorylation. The following results were obtained: 1. Cultures of A. woodii with H₂ and CO₂, grew to greater cell densities, when caffeate was also present. Caffeate was reduced to give hydrocaffeate and less acetate was formed. The cell yield based on the amount of caffeate reduced was approximately 1 g dry cells/mol. 2. Non-growing bacterial suspensions catalyzed the reduction of caffeate by H₂. The specific activity (0.2–1.0 mol · min^–1 · mg^–1 bacterial protein) was as high as expected for a catabolic reaction. 3. The ATP content of bacteria incubated, with H₂ increased from < 1 to about 7 mol per g cellular protein on the addition of caffeate. The ATP yield was calculated as 0.06 mol ATP · mol^–1 caffeate from the initial velocity of ATP formation and the activity of caffeate reduction. Valinomycin together with nigericin inhibited ATP formation and caused a 2–3-fold increase of the activity of caffeate reduction. Protonophores were without, effect. 4. Caffeate in the presence of H₂ caused the uptake of tetraphenylphosphonium cation by the bacteria. The uptake was abolished by valinomycin plus nigericin, and was considerably enhanced by monensin. Protonophores were without effect, even in the presence of monensin. It is concluded that caffeate reduction by H₂ is coupled to ATP formation by electron transport phosphorylation. However, the failure of protonophores to prevent phosphorylation and TPP uptake cannot be explained.Abbreviations Caffeate 3,4-Dihydroxycinnamate - Hydrocaffeate 3,4-dihydroxyphenylpropionate - TPP⁺ tetraphenylphosphonium cation - FCCP carbonylcyanide-4-trifluoromethoxyphenylhydrazone - TTGB 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazol - TCS 3,5,3,4-tetrachlorosalicylanilide     

Platelet free calcium concentrations measured with fura-2 are influenced by the transmembrane sodium gradient

The influence of the transmembrane Na+ gradient on the intracellular free calcium concentration, [Ca2+]i, was studied in Sepharose gel-filtered platelets from healthy human subjects, using the Ca-sensitive fluorescent dye, fura-2. Raising the internal Na+ concentration, [Na+]i, by Na+ pump inhibition with 0.05 mM ouabain, without changing external Na+ did not cause a significant increase in [Ca2+]i. Substitution of extracellular Na+ by iso-osmolar sucrose induced a rapid (half-time about 2 min) and significant rise in [Ca2+]i; this effect was amplified in Na-loaded platelets. Partial restitution of external Na+ in these cells with increased [Ca2+]i promoted a significant and rapid Na+ concentration-dependent fall in [Ca2+]i; little decline in [Ca2+]i was observed if K+ was used instead of Na+. These observations represent in vitro evidence for the existence of a Na/Ca exchange mechanism in human platelets that may, in vivo, participate in the control of [Ca2+]i.     

Kinetic equivalence of transmembrane pH and electrical potential differences in ATP synthesis

ATP synthase is the key player of Mitchell's chemiosmotic theory, converting the energy of transmembrane proton flow into the high energy bond between ADP and phosphate. The proton motive force that drives this reaction consists of two components, the pH difference (ΔpH) across the membrane and transmembrane electrical potential (Δψ). The two are considered thermodynamically equivalent, but kinetic equivalence in the actual ATP synthesis is not warranted, and previous experimental results vary. Here, we show that with the thermophilic Bacillus PS3 ATP synthase that lacks an inhibitory domain of the ε subunit, ΔpH imposed by acid-base transition and Δψ produced by valinomycin-mediated K(+) diffusion potential contribute equally to the rate of ATP synthesis within the experimental range examined (ΔpH -0.3 to 2.2, Δψ -30 to 140 mV, pH around the catalytic domain 8.0). Either ΔpH or Δψ alone can drive synthesis, even when the other slightly opposes. Δψ was estimated from the Nernst equation, which appeared valid down to 1 mm K(+) inside the proteoliposomes, due to careful removal of K(+) from the lipid.     

Evidence from ATP synthesis driven by a proton gradient in Methanosarcina barkeri.

Hepatoma Tissue Culture (HTC) cell nuclei were isolated from untreated cells and from cells treated with sodium butyrate to increase the levels of acetylated histone. Nuclei from sodium butyrate treated cells exhibited a dramatic increased rate of digestion with DNase I as compared to control cell nuclei. Micrococcal nuclease showed no preference for chromatin containing hyperacetylated histones.     

Conductivity noise in transmembrane ion channels due to ion concentration fluctuations via diffusion.

A Green's function approach is developed from first principles to evaluate the power spectral density of conductance fluctuations caused by ion concentration fluctuations via diffusion in an electrolyte system. This is applied to simple geometric models of transmembrane ion channels to obtain an estimate of the magnitude of ion concentration fluctuation noise in the channel current. Pure polypeptide alamethicin forms stable ion channels with multiple conductance states in artificial phospholipid bilayers isolated onto tips of micropipettes with gigaohm seals. In the single-channel current recorded by voltage-clamp techniques, excess noise was found after the background instrumental noise and the intrinsic Johnson and shot noises were removed. The noise que to ion concentration fluctuations via diffusion was isolated by the dependence of the excess current noise on buffer ion concentration. The magnitude of the concentration fluctuation noise derived from experimental data lies within limits estimated using our simple geometric channel models. Variation of the noise magnitude for alamethicin channels in various conductance states agrees with theoretical prediction.     

ATP synthesis in chromatophores driven by artificially induced ion gradients

An electrochemical potential difference for protons (delta mu H+) across the membrane of bacterial chromatophores was induced by an artificially generated pH difference (delta pH) and a K+/valinomycin diffusion potential, delta phi. The initial rate of ATP synthesis was measured with a rapid-mixing quenched-flow apparatus in the time range between 70 ms and 30 s after the acid-base transition. The rate of ATP synthesis depends exponentially on delta pH. Increasing diffusion potentials shift the delta pH dependency to lower delta pH values. Diffusion potentials were calculated from the Goldman equation. Using estimated permeability coefficients, the rate of ATP synthesis depends only on the electrochemical potential difference of protons irrespective of the relative contribution of delta pH and delta phi.     

Light-driven primary sodium ion transport in halobacterium halobium membranes

Light-induced sodium extrusion from H halobium cell envelope vesicles proceeds largely through an uncoupler-sensitive pathway involving bacteriorhodopsin and a proton/sodium antiporter. Vesicles from bacteriorhodopsin-negative strains also extrude sodium ions during illumination, but this transport is not sensitive to uncouplers and has been proposed to involve a light-energized primary sodium pump. Proton uptake in such vesicles is passive, and under steady-state illumination the large electrical potential (negative inside) is just balanced by a pH difference (acid inside), so that the protonmotive force is near zero. Action spectra indicated that this effect of illumination is attributable to a pigment absorbing near 585 nm (of 568 for bacteriorhodopsin). Bleaching of the vesicles by prolonged illumination with hydroxylamine results in inactivation of the transport; retinal addition causes partial return of the activity. Retinal addition also causes the appearance of an absorption peak at 588 nm, while the absorption of free retinal decreases. The 588 nm pigment is present in very small quantities (0.13 nmole/mg protein), and behaves differently from bacteriorhodopsin in a number of respects. Vesicles can be prepared from bacteriorhodopsin-containing H halobium strains in which primary transport for both protons and sodium can be observed. Both pumps appear to cause the outward transport of the cations. The observations indicate the existence of a second retinal protein, in addition to bacteriorhodopsin, in H halobium, which is associated with primary sodium translocation. The initial proton uptake normally observed during illumination of whole H halobium cells may therefore be a passive flux in response to the primary sodium extrusion.     

Hydrolysis of urea by Ureaplasma urealyticum generates a transmembrane potential with resultant ATP synthesis.

When urea is added to Ureaplasma urealyticum, it is hydrolysed internally by a cytosolic urease. Under our measuring conditions, and at an external pH of 6.0, urea hydrolysis caused an ammonia chemical potential equivalent to almost 80 mV and, simultaneously, an increase in proton electrochemical potential (delta p) of about 24 mV with resultant de novo ATP synthesis. Inhibition of the urease with the potent inhibitor flurofamide abolished both the chemical potential and the increase of delta p such that ATP synthesis was reduced to approximately 5% of normally obtained levels. Uncouplers of electrochemical gradients had little or no effect on these systems. The electrochemical parameters and ATP synthesis were measured similarly at three other external pH values. Any change in delta p was primarily via membrane potential (delta psi), and the level of de novo ATP synthesis was related to the increase in delta p generated upon addition of urea and more closely to the ammonia chemical potential. Although the organisms lack an effective mechanism for internal pH homeostasis, they maintained a constant delta pH. The data reported are consistent with, and give evidence for, the direct involvement of a chemiosmotic mechanism in the generation of around 95% of the ATP by this organism. Furthermore, the data suggest that the ATP-generating system is coupled to urea hydrolysis by the cytosolic urease via an ammonia chemical potential.     

Characterization of the ATP synthase of Propionigenium modestum as a primary sodium pump

The ATP synthase (F1F0) of Propionigenium modestum has been purified to a specific ATPase activity of 5.5 units/mg of protein, which is about 6 times higher than that of the bacterial membranes. Analysis by SDS gel electrophoresis indicated that in addition to the five subunits of the F1 ATPase, subunits of Mr 26,000 (a), 23,000 (b), and 7500 (c) have been purified. The ATPase activity of F1F0 was specifically activated about 10-fold by Na+ions. The enzyme was strongly inhibited by dicyclohexylcarbodiimide, venturicidin, tributyltin chloride, and azide. After incubation with [14C]dicyclohexylcarbodiimide, about 3-4 mol of the inhibitor was bound per 500,000 g of the enzyme. The radioactive label was specifically bound to submit c. These subunits form stable aggregates which resist dissociation by SDS at 100 degrees C. The monomer is formed upon heating with SDS to 121 degrees C or by extraction of the membranes with chloroform/methanol. The ATP synthase was incorporated into liposomes by a freeze-thaw-sonication procedure. The reconstituted proteoliposomes catalyzed the transport of Na+ions upon ATP hydrolysis. The transport was completely abolished by dicyclohexylcarbodiimide. Whereas monensin prevented the accumulation of Na+ions, the uptake rate was stimulated 4-5-fold in the presence of valinomycin or carbonyl cyanide m=chlorophenylhydrazone. These results indicate an electrogenic Na+ transport and also that it is a primary event and not accomplished by a H+-translocating ATP synthase in combination with a Na+/H+ antiporter.     

The role of transmembrane sodium gradient in rabbit ileal smooth muscle: Utilization of harmaline

Decreasing extracellular sodium concentration was found to produce a contractile response of rabbit ileal smooth muscle. As the concentration decreases, the amplitude of contraction increases, thus producing a dose-dependent curve. Harmaline, a competitor for sodium, was found to inhibit the sodium gradient-dependent contractions in a dose-dependent manner. The results are interpreted as harmaline inhibiting a Na–Ca exchange mechanism present in ileal smooth muscle.     

Mathematical modeling of electron and protein transport, coupled with ATP synthesis in chloroplasts

A mathematical model of electron and proton transport in chloroplasts of higher plants was developed, which takes into account the lateral heterogeneity of the lamellar system. Based on the results of numerical experiments, lateral profiles of pH in the thylakoid lumen and in the narrow gap between grana thylakoids under different metabolic conditions (in the state of photosynthetic control and under photophosphorylation conditions) were simulated. Lateral profiles of pH in the thylakoid lumen and in the intrathylakoid gap were simulated for different values of the proton diffusion coefficient and stroma pH. The model demonstrated that there might be two mechanisms of regulation of electron and proton transport in chloroplasts: (1) the slowing down of noncyclic electron transport due to a decrease in the intrathylakoid pH, and (2) the retardation of plastoquinone reduction due to slow diffusion of protons inside the narrow gap between the thylakoids of grana.     

ATP synthesis driven by a potassium diffusion potential in Methanobacterium thermoautotrophicum is stimulated by sodium

Abstract ATP synthesis driven by a potassium diffusion potential was studied in cell suspensions of Methanobacterium thermoautotrophicum (Marburg). This transient increase in the intracellular ATP content was stimulated five-fold by the addition of sodium ions, from about 2 nmol ATP/min × mg cells (dry weight) at 0.07 mM Na⁺ to about 10 nmol ATP/min × mg cells at 25 mM Na⁺.     

Proton transport coupled ATP synthesis by the purified yeast H-ATP synthase in proteoliposomes

The H⁺/ATP synthase from yeast mitochondria, MF₀F₁, was purified and reconstituted into liposomes prepared from phosphatidylcholine and phosphatidic acid. Analysis by mass spectrometry revealed the presence of all subunits of the yeast enzyme with the exception of the K-subunit. The MF₀F₁ liposomes were energized by acid-base transitions (ΔpH) and a K⁺/valinomycin diffusion potential (Δφ). ATP synthesis was completely abolished by the addition of uncouplers as well as by the inhibitor oligomycin. The rate of ATP synthesis was optimized as a function of various parameters and reached a maximum value (turnover number) of 120 s^− 1 at a transmembrane pH difference of 3.2 units (at pH_in = 4.8 and pH_out = 8.0) and a Δφ of 133 mV (Nernst potential). Functional studies showed that the monomeric MF₀F₁ was fully active in ATP synthesis. The turnover increased in a sigmoidal way with increasing internal and decreasing external proton concentration. The dependence of the turnover on the phosphate concentration and the dependence of K_M on pH_out indicated that the substrate for ATP synthesis is the monoanionic phosphate species H₂PO₄⁻.     

An analysis of proton fluxes coupled to electron transport and ATP synthesis in chloroplast thylakoids

Electron transport, phosphorylation and internal proton concentration were measured in illuminated spinach chloroplast thylakoid membranes under a number of conditions. Regardless of the procedure used to vary these parameters, the data fit a simple chemiosmotic model. Protons from Photosystem II did not appear to be utilized differently from those derived from Photosystem I. The maximal phosphorylation efficiency ($\text{P}\text{e}{}_2$) for photophosphorylation in washed thylakoids under oxidizing conditions is likely to be $\text{4}\text{3}$. This value is consistent with a proton-to-electron-pair ratio of 4 for electron flow through both photosystems and a proton-to-ATP ratio of 3 for the chloroplast proton-ATPase.     

Generation of a transmembrane gradient of Na+ in Methanosarcina barkeri

A transmembrane Na+ gradient was generated by Methanosarcina barkeri during methanogenesis. The intracellular Na+ concentration amounted to approximately one fifth of the extracellular one. A secondary Na+/H+ antiport system was shown to be responsible for Na+ extrusion. This system could be inhibited by amiloride. In the presence of amiloride the delta pH across the cytoplasmic membrane increased and a transmembrane Na+ gradient could neither be generated nor maintained. The possible role of Na+ in the oxidation of methanol to the level of formaldehyde is discussed.     

Extracellular ATP perturbs transmembrane ion fluxes, elevates cytosolic [Ca2+], and inhibits phagocytosis in mouse macrophages

In addition to its important role in intracellular metabolic pathways, ATP appears to function as a neurotransmitter in mammalian neurones. The extracellular effects of ATP are not restricted to neurones. We describe the effects of ATP on transmembrane fluxes of monovalent and divalent cations and on phagocytosis in the J774 mouse macrophage cell line and in mouse macrophages elicited by intraperitoneal injection of thioglycollate broth. Of all nucleotides tested, only ATP is capable of depolarizing the macrophage plasma membrane potential, promoting Na+ influx and K+ efflux, effecting an increase in intracellular free Ca2+, and inhibiting phagocytosis. Nonhydrolyzable ATP analogs had no effect on membrane permeability or phagocytosis. The effect mediated by ATP is not accompanied by an increase in membrane permeability to nucleotides, indicating that the action of ATP is restricted to the external surface of macrophages.     

On the relationship between rate of ATP synthesis and H+ electrochemical gradient in rat-liver mitochondria

The relationship between rate of ATP synthesis, JATP, and value of the proton electrochemical gradient, delta mu H, has been analyzed in intact mitochondria. Onset of phosphorylation causes a depression of delta mu H of 1.5 kJ/mol. There is a close parallelism between inhibition of JATP and restoration of delta mu H to its state-4 value during titrations with oligomycin or atractyloside. Titrations with ionophores display the following features: (a) delta mu H can be depressed by 3-4 kJ/mol by valinomycin + K+ without affecting the rate of ATP synthesis; (b) uncouplers abolish JATP completely while depressing delta mu H by 3 kJ/mol; (c) complete abolition of ATP synthesis by inhibitors of electron transport is accompanied by a depression of delta mu H of only 1 kJ/mol. The results indicate that: (a) there is a close functional relationship between redox and ATPase H+ pumps, whereby inhibition of electron transfer is accompanied by simultaneous inhibition of the ATPase H+ pumps; and (b) uncoupling of oxidative phosphorylation is not due to depression of delta mu H per se. The consistence of the present data with either a chemiosmotic model where delta mu H is the sole and obligatory intermediate for energy coupling, or models where there is a direct transfer of energy between the two pumps is discussed.     

Plastid and nuclear DNA synthesis are not coupled in suspension cells ofNicotiana tabacum

The relationship between nuclear and plastid DNA synthesis in cultured tobacco cells was measured by following³H-thymidine incorporation into total cellular DNA in the absence or presence of specific inhibitors. Plastid DNA synthesis was determined by hybridization of total radiolabeled cellular DNA to cloned chloroplast DNA. Cycloheximide, an inhibitor of nuclear encoded cytoplasmic protein synthesis, caused a rapid and severe inhibition of nuclear DNA synthesis and a delayed inhibition of plastid DNA synthesis. By contrast, chloramphenicol which only inhibits plastid and mitochondrial protein production, shows little inhibition of either nuclear or plastid DNA synthesis even after 24 h of exposure to the cells. The inhibition of nuclear DNA synthesis by aphidicolin, which specifically blocks the nuclear DNA polymeraseα, has no significant effect on plastid DNA formation. Conversely, the restraint of plastid DNA synthesis exerted by low levels of ethidium bromide has no effect on nuclear DNA synthesis. These results show that the synthesis of plastid and nuclear DNA are not coupled to one another. However, both genomes require the formation of cytoplasmic proteins for their replication, though our data suggest that different proteins regulate the biosynthesis of nuclear and plastid DNA.