Thermodynamics of the electrochemical proton gradient in bovine heart submitochondrial particles.

The electrical and chemical components of the electrochemical proton gradient of submitochondrial particles can be monitored simultaneously by continuously recording optical signals from the probes oxonol-VI and 9-aminoacridine. Either respiration or ATP hydrolysis causes a red shift in the absorption spectrum of oxonol-VI indicative of a membrane potential and a decrease of the fluorescence of 9-aminoacridine indicative of a pH gradient. The magnitude of the membrane potential and pH gradient formed by respiring submitochondrial particles can be modulated by the thermodynamic phosphorylation potential (deltaGp) of the adenine nucleotide system. deltaGp is the Gibbs free energy of ATP synthesis and is defined by the relationship deltaGp = -deltaG'o + RTln([ATP]/[ADP][Pi] where deltaG'o is the standard free energy of ATP hydrolysis. Increasing values of deltaGp cause an increase in the steady state magnitudes of both the membrane potential and pH gradient. Thermodynamic phosphorylation potential titration experiments indicate that the electrochemical proton gradient normally maintained by respiring submitochondrial particles has an energy equivalent to 10.5 to 10.9 kcal/mol.     

Effect of the electrochemical proton gradient and anions on the ATPase activity of soybean submitochondrial particles

Submitochondrial particles from soybean (Glycine max L. cv Jupiter) hypocotyls with an ATPase activity of 0.3 to 1.0 micromole per minute per milligram were prepared by sonication with Mg-ATP. The particles catalyzed ATP synthesis with NADH and succinate; the ratios of ATP/O with these substrates were 1.0 and 0.1, respectively. As monitored by oxonol-VI, the particles built up and maintained a membrane potential that was higher with NADH than with succinate or Mg-ATP. The ATPase activity of the particles increased two to threefold by preincubation with 50 millimolar phosphate at a temperature of 38°C. The increase in ATPase activity became higher (five to sixfold) when particles were preincubated with Mg-ATP plus phosphate. Under the latter conditions, collapse of μH by carbonyl cyanide p-trifluoromethoxyphenylhydrazone prevented the activation. An increase in ATPase activity of the particles was also observed with NADH and succinate, although activation was lower with succinate. With these substrates, phosphate did not increase ATPase activation. When particles were preincubated with Mg-ATP, anions that stimulate ATP hydrolysis (malate, malonate, and bicarbonate) had an activating effect similar to that of phosphate. The data suggest that the soybean mitochondrial ATPase can be activated by μH but that this activation is increased by the binding of certain anions to a conformation of the enzyme that appears during hydrolytic cycles.     

Proton electrochemical gradient and phosphate potential in submitochondrial particles

1. The aerobic uptake of inorganic ions, such as ⁸⁶Rb⁺ or ¹²⁵I^?, by submitochondrial particles, is about one order of magnitude lower than the uptake of organic ions, such as acridines or 8-anilino-1-naphthalene sulphonate. The values of ΔpH, the transmembrane pH differential, and Δψ, the transmembrane membrane potential are between 60 and 100 mV when calculated on the inorganic ions and between 150 and 240 mV when calculated on the organic ions. The discrepancy between the ΔpH and Δψ values from organic and inorganic ions is large at high but not at low ion/protein ratios.2. In the absence of weak bases and strong acids the values of $\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{<mtext>H</mtext>}}$, the proton electrochemical potential difference, are close to 100 mV and the magnitude of ΔpH and Δψ are similar. Weak bases decrease ΔpH and enhance Δψ. Strong acids decrease Δψ and enhance ΔpH. Interchangeability of ΔpH with Δψ occurs at low concentrations of weak bases and strong acids. High concentrations of weak bases and strong acids cause depression of $\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{<mtext>H</mtext>}}$.3. Concentrations of weak bases capable of abolishing ΔpH, do not affect ATP synthesis. Concentrations of strong acids capable of abolishing Δψ affect only slightly ATP synthesis. Concentrations of weak bases and strong acids capable of causing a decline of ΔpH + Δψ inhibit ATP synthesis.4. Depression of $\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{<mtext>H</mtext>}}$ is paralleled by inhibition of ATP synthesis and decline of ΔGp, the phosphate potential. Abolition of ATP synthesis occurs only when $\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{<mtext>H</mtext>}}$ is below 20 mV. The $\text{ΔG}\text{p}\text{/Δ}\text{}\text{?}\text{gm}{}_{\mathrm{<mtext>H</mtext>}}$ ratio increases hyperbolically with the decrease of $\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{<mtext>H</mtext>}}$.     

Proton electrochemical gradient and phosphate potential in submitochondrial particles.

The aerobic uptake of inorganic ions, such as 86Rb+ or 125I-, by submitochondrial particles, is about one order of magnitude lower than the uptake of organic ions, such as acridines or 8-anilino-1-naphthalene sulphonate. The values of deltapH, the transmembrane pH differential, and deltapsi, the transmembrane membrane potential are between 60 and 100 mV when calculated on the inorganic ions and between 150 and 240 mV when calculated on the organic ions. The discrepancy between the deltapH and deltapsi values from organic and inorganic ions is large at high but not at low ion/protein ratios. 2. In the absence of weak bases and strong acids the values of deltamuH, the proton electrochemical potential difference, are close to 100 mV and the magnitude of deltapH and deltapsi are similar. Weak bases decrease deltapH and enhance deltapsi. Strong acids decrease deltapsi and enhance deltapH. Interchangeability of deltapH with deltapsi occurs at low concentrations of weak bases and strong acids. High concentrations of weak bases and strong acids cause depression of deltamuH. 3. Concentrations of weak bases capable of abolishing deltapH, do not affect ATP synthesis. Concentrations of strong acids capable of abolishing deltapsi affect only slightly ATP synthesis. Concentrations of weak bases and strong acids capable of causing a decline of deltapH + deltapsi inhibit ATP synthesis. 4. Depression of deltamuH is paralleled by inhibition of ATP synthesis and decline of deltaGp, the phosphate potential. Abolition of ATP synthesis occurs only when deltamuH is below 20 mV. The deltaGp/deltamuH ratio increases hyperbolically with the decrease of deltamuH.     

Synthesis of adenosine triphosphate by an artificially imposed electrochemical proton gradient in bovine heart submitochondrial particles.

Submitochondrial particles subjected to an artificially imposed electrochemical proton gradient consisting of a pH gradient (acid to base transition) and membrane potential (low to high K-+ transition in the presence of valinomycin) catalyzed the net synthesis of 2.5 nmol of [-32P]ATP per mg of protein from ADP and 32-Pi. Optimal reaction conditions included incubation of submitochondrial particles in malonate at pH 5.0 with valinomycin in the absence of added K-+, followed by a rapid transition to pH 7.5 and 100 mM K-+. ATP synthesis continued for about 6 s and was sensitive to uncouplers or oligomycin but insensitive to inhibitors of electron transport. Lower amounts of ATP were formed by either the pH gradient (25%) of K-+ gradient (15%) alone. These results demonstrate that an electrochemical gradient of protons can drive the synthesis of ATP by reversal of the proton-translocating ATPase independent of electron transport.     

Variable proton conductance of submitochondrial particles.

The relationship between the rate of substrate oxidation and the protonmotive force (electrochemical proton gradient) generated by bovine heart submitochondrial particles has been examined. Unexpectedly, oxidation of succinate generated a higher protonmotive force than the oxidation of NADH, although the rate of proton translocation across the membrane was inferred to be considerably lower with succinate as substrate. The data suggest that the flow of electrons through site 1 of the respiratory chain may increase the conductance of the mitochondrial membrane for protons. Upon reduction of the rate of succinate oxidation by titration with malonate, the protonmotive force remained essentially constant until the extent of inhibition was greater than 75%. The general conclusion from this work is that a constant passive membrane conductance for protons cannot be assumed.     

Energy-dependent accumulation of the uncoupler picrate and proton flux in submitochondrial particles

In the presence of ATP and oxidizable substrate, submitochondrial particles accumulate up to 7 nmol of picrate/mg of protein. Half of this value is reached at 5 μM picrate in the medium, and maximal energy-dependent accumulation occurs at 25 μM picrate. Mitochondrial proton fluxes calculated under such conditions are 0.80 and 1.08 pmol H⁺/cm²·sec at 10 μM and 25 μM picrate, respectively. These values are similar to those reported for state 4, and are therefore not large enough for uncoupling by picrate through proton translocation. The energy-dependent spectral response of oxonol VI is reversed to 50 % by 40 μM picrate, suggesting that abolishment of membrane potential is responsible for uncoupling of submitochondrial particles by picrate.     

The electrochemical proton gradient in Mycoplasma cells

The electrochemical proton gradient, delta mu H+ generated upon glycolysis by Mycoplasma mycoides var. Capri cells has been determined. The components, the transmembrane pH gradient, delta pH, and the membrane potential, delta psi, were measured using several methods. The determination of the delta pH was conducted by measuring the transmembrane distribution of weak acids (acetate and butyrate) and of a weak base (methylamine), using flow dialysis and filtration techniques. The transmembrane electrical potential was determined from the distribution of the lipophilic cation Ph3MeP+ and of Rb+ or K+ in the presence of valinomycin. At extra-cellular pH 7.2, glycolyzing Mycoplasma cells maintain an internal pH more alkaline (0.5 pH unit) than that of the milieu and an electrical potential of - 85 mV, interior negative. The delta mu H+ in M. mycoides var. Capri cells is thus about - 115 mV. When the external pH was altered from 7.7 to 5.7 delta psi decreased from - 90 mV to - 60 mV. On other hand although the internal pH decreased, delta pH was found to increase from 0.2 to 1.0 pH unit. Since the changes in delta psi were largely compensated by the changes in delta pH, delta mu H+ remained practically constant at about - 115 mV throughout the pH range tested. Finally, inhibition of delta pH by N,N'-dicyclohexylcarbodiimide, carbonylcyanide-p-trifluoromethoxyphenylhydrazone or nigericin confirmed that chemiosmotic phenomena contribute to energy transduction across the membranes of M. mycoides var. Capri cells.     

The proton electrochemical gradient in Escherichia coli cells.

The internal pH of Escherichia coli cells was estimated from the distribution of either 5,5-[14C]dimethyl-2,4-oxazolidinedione or [14C]methylamine. EDTA/valinomycin treatment of cells was employed to estimate delta psi from 86Rb+ distribution concomitant with the delta pH for calculation of delta muH. Respiring intact cells maintained an internal pH more alkaline by 0.63-0.75 unit than that of the milieu at extracellular pH 7, both in growth medium and KCl solutions. The delta pH decreased when respiration was inhibited by anaerobiosis or in the presence of KCN. The delta muH, established by EDTA/valinomycin-treated cells, was constant (122-129 mV) over extracellular potassium concentration of 0.01 mM-1 mM. At the lower potassium concentration delta psi (110-120 mV) was the predominant component, and at the higher concentration delta pH increased to 0.7 units (42 mV). At 150 mM potassium delta muH was reduced to 70 mV mostly due to a delta pH component of 0.89 (53 mV). The interchangeability of the delta muH components is consistent with an electronic proton pump and with potassium serving as a counter ion in the presence of valinomycin. Indeed both parameters of delta muH decreased in the presence of carbonylcyanide p-trifluoromethoxyphenylhydrazone. The highest delta pH of 2 units was observed in the intact cells at pH 6; increasing the extracellular pH decreased the delta pH to 0 at pH 7.65 and to -0.51 at pH 9. A similar pattern of dependence of delta pH on extracellular pH was observed in EDTA/valinomycin-treated cells but the delta psi was almost constant over the whole range of extracellular pH values (6-8) implying electroneutral proton movement. Potassium is specifically required for respiration of EDTA-treated E. coli K12 cells since other monovalent or divalent cations could not replace potassium and valinomycin was not required.     

Gradation of the magnitude of the electrochemical proton gradient in Mycoplasma cells

The results presented show that in Mycoplasma mycoides var. Capri, regulation of glucose uptake by its non-metabolizable analogue methyl alpha-D-glucoside, can be used to control intracellular ATP content. This in turn leads to a control of the rate of proton extrusion catalysed by the Mg2+-dependent ATPase (phi (cHxN)2C H+) and the respective amplitudes of the components of delta mu H+. When Mycoplasma cells are incubated with 10 mM methyl alpha-D-glucoside, the amplitude of phi (cHxN)2C H+, of the electrical potential delta psi and of the chemical gradient delta pH become continuous functions of external glucose concentration within the limits of the non-energized and fully energized states. Analysis of the relationships between graduated amplitudes of delta psi, delta pH and phi (cHxN) 2C H+ show that the primary form of energy stored by a delta mu H+ generator is the electrical potential.     

Involvement of the proton electrochemical gradient in genetic transformation in Escherichia coli

Plasmid pIY2 DNA which encodes for ampicillin-resistance was used to study the energetics of Ca⁺⁺-induced transformation in Escherichia coli. When cells are exposed to DNA in the presence of carbonylcyanide-m-chlorophenylhydrazone or 2,4-dinitrophenol, two protonophores that collapse the proton electrochemical gradient across the cell membrane ($\text{Δ}\text{μ}{}_{\mathrm{H}}\text{+}$), transformation to ampicillin-resistance is drastically reduced with little or no effect on viability. Furthermore, when the components of $\text{Δ}\text{μ}{}_{\mathrm{H}}\text{+}$ are altered by varying ambient pH or by performing transformation in the presence of valinomycin or nigericin, the efficiency of transformation is directly correlated with the magnitude of the membrane potential and changes in the pH gradient have no significant effect. It is concluded that $\text{Δ}\text{μ}{}_{\mathrm{H}}\text{+}$, more specifically the membrane potential, plays a critical role in Ca⁺⁺-induced transformation.     

Site-directed alkylation of LacY: effect of the proton electrochemical gradient

Previous N-ethylmaleimide-labeling studies show that ligand binding increases the reactivity of single-Cys mutants located predominantly on the periplasmic side of LacY and decreases reactivity of mutants located for the most part of the cytoplasmic side. Thus, sugar binding appears to induce opening of a periplasmic pathway with closing of the cytoplasmic cavity resulting in alternative access of the sugar-binding site to either side of the membrane. Here we describe the use of a fluorescent alkylating reagent that reproduces the previous observations with respect to sugar binding. We then show that generation of an H⁺ electrochemical gradient (Δ_μ¯H+, interior negative) increases the reactivity of single-Cys mutants on the periplasmic side of the sugar-binding site and in the putative hydrophilic pathway. The results suggest that Δ_μ¯H+, like sugar, acts to increase the probability of opening on the periplasmic side of LacY.     

The electrochemical proton gradient in Escherichia coli membrane vesicles.

Membrane vesicles isolated from Escherichia coli grown under various conditions generate a transmembrane pH gradient (delta pH) of about 2 pH units (interior alkaline) under appropriate conditions when assayed by flow dialysis. Using the distribution of weak acids to measure delta pH and the distribution of the lipophilic cation triphenylmethylphosphonium to measure the electrical potential (delta psi) across the membrane, the vesicles are demonstrated to develop an electrochemical proton gradient (delta-muH+) of almost - 200 mV (interior negative and alkaline) at pH 5.5 in the presence of reduced phenazine methosulfate or D-lactate, the major component of which is a deltapH of about - 120 mV. As external pH is increased, deltapH decreases, reaching 0 at about pH 7.5 and above, while delta psi remains at about - 75 mV and internal pH remains at pH 7.5-7.8. The variations in deltapH correlate with changes in the oxidation of reduced phenazine methosulfate or D-lactate, both of which vary with external pH in a manner similar to that described for deltapH. Finally, deltapH and delta psi can be varied reciprocally in the presence of valinomycin and nigericin with little change in delta-muH+ and no change in respiratory activity. These data and those presented in the following paper (Ramos and Kaback 1976) provide strong support for the role of chemiosmotic phenomena in active transport and extend certain aspects of the chemiosmotic hypothesis.