Chemical rescue of H+ delivery in proton transfer mutants of reaction center of photosynthetic bacteria

In the native and most mutant reaction centers of bacterial photosynthesis, the electron transfer is coupled to proton transfer and is rate limiting for the second reduction of Q_B^??→?Q_BH₂. In the presence of divalent metal ions (e.g. Cd²⁺) or in some (“proton transfer”) mutants (L210DN/M17DN or L213DN), the proton delivery to Q_B^? is made rate limiting and the properties of the proton pathway can be directly examined. We found that small weak acids and buffers in large concentrations (up to 1?M) were able to rescue the severely impaired proton transfer capability differently depending on the location of the defects: lesions at the protein surface (proton gate H126H/H128H?+?Cd²⁺), beneath the surface (M17DN?+?Cd²⁺, L210DN/M17DN) or deep inside the protein (L213DN) could be completely, partially or to very small extent recovered, respectively. Small zwitterionic acids (azide/hydrazoic acid) and buffers (tricine) proved to be highly effective rescuers consistent with their enhanced binding affinity and access to any of the proton acceptors (including Q_B^? itself) in the pathway. As a consequence, back titration of the protons at L212Glu could be observed as a pH-dependence of the rate constant of the charge recombination in the presence of azide or formate. Model calculations support the collective influence of the acid cluster on the change of the protonation states upon extension of the cluster with the bound small acid. In proton transfer mutants, the rescuing agents decreased the free energy of activation together with their enthalpic and entropic components. This is in agreement with the hypothesis that they function as protein-penetrating protonophores delivering protons into the chain and select dominating paths out of many alternate routes. We estimate that the proton delivery will be accelerated in one pathway out of 100–200 alternate pathways. The implications for design of the chemical recovery of impaired intra-protein proton transfer pathways in proton transfer mutants are discussed.     

Equilibria in the fibrinogen-fibrin conversion. IX. Effects of calcium ions on the reversible polymerization of fibrin monomer

An investigation was made of the role of calcium ions in the reversible stage of fibrin polymerization, using a direct and relatively simple approach. Purified fibrin monomer in solution (7.5 mg/ml) in 1.0 m NaBr (pH 5.3) was polymerized by raising the pH to 5.7–7.7 by the addition of aliquots of standard NaOH solution and the rate and total extent of proton release during polymerization were measured potentiometrically. In the presence of added CaCl₂ (10⁻⁵-10⁻²m) the rate of proton release was increased and the clotting time was decreased. The profile of equilibrium proton release vs pH of polymerization was also shifted, the maximum being increased and occurring at a lower pH. Sedimentation velocity studies in the intermediate pH range (5.7–6.0) showed that the altered profile of equilibrium proton release was due to a broadening of the pH range of polymerization, and that polymerization remained reversible in the presence of CaCl₂. At pH 5.3, where fibrin is essentially monomeric, addition of CaCl₂ resulted in the release of protons and small increases in sedimentation coefficient and reduced viscosity. Under the same conditions, a similar release of protons was observed from fibrinogen, but there was no effect on its sedimentation coefficient. It was concluded that the proton release at pH 5.3 was due mainly to binding of calcium ions to fibrinogen and fibrin monomer. The effect of CaCl₂ on the sedimentation coefficient of fibrin at pH 5.3 was found to decrease with decreasing protein concentration, indicating that it was the result of a small extent of polymerization, rather than a conformational change. Added MgCl₂ had no effect on fibrin monomer at pH 5.3 and no significant effect on the rate or extent of proton release during polymerization at higher pH, indicating that there are specific binding sites for calcium ions in fibrinogen and fibrin. The observed effects of bound calcium ions on reversible fibrin polymerization are explained most simply in electrostatic terms.     

Dissecting the pattern of proton release from partial process involved in ubihydroquinone oxidation in the Q-cycle

A key feature of the modified Q-cycle of the cytochrome bc₁ and related complexes is a bifurcation of QH₂ oxidation involving electron transfer to two different acceptor chains, each coupled to proton release. We have studied the kinetics of proton release in chromatophore vesicles from Rhodobacter sphaeroides, using the pH-sensitive dye neutral red to follow pH changes inside on activation of the photosynthetic chain, focusing on the bifurcated reaction, in which 4H⁺are released on complete turnover of the Q-cycle (2H⁺/ubiquinol (QH₂) oxidized). We identified different partial processes of the Q_o-site reaction, isolated through use of specific inhibitors, and correlated proton release with electron transfer processes by spectrophotometric measurement of cytochromes or electrochromic response. In the presence of myxothiazol or azoxystrobin, the proton release observed reflected oxidation of the Rieske iron?sulfur protein. In the absence of Q_o-site inhibitors, the pH change measured represented the convolution of this proton release with release of protons on turnover of the Q_o-site, involving formation of the ES-complex and oxidation of the semiquinone intermediate. Turnover also regenerated the reduced iron-sulfur protein, available for further oxidation on a second turnover. Proton release was well-matched with the rate limiting step on oxidation of QH₂ on both turnovers. However, a minor lag in proton release found at pH?7 but not at pH?8 might suggest that a process linked to rapid proton release on oxidation of the intermediate semiquinone involves a group with a pK in that range.     

Modulation of proton efflux from chloroplasts in the light by external pH

The effects of external pH on the efflux of protons from illuminated spinach chloroplasts have been studied by monitoring the rates of proton-pumping electron transport under a variety of steady-state conditions. Phosphorylation-coupled proton efflux through the ATP synthase (CF₀-CF₁), determined from the rates of ATP formation and that portion of the total electron transport attributable to phosphorylation, is strongly dependent upon pH over the range 6–9, with little activity below pH 7 and half-maximal activity at pH ≈ 7.6. Noncoupled proton efflux through the ATP synthase, determined in the absence of ADP and phosphate, was also strongly pH sensitive, with little activity below pH 7.5 and half-maximal activity at pH ～- 7.9. When proton efflux via CF₀ was prevented by triphenyltin, the rate of passive proton leakage across the membrane was very low and practically insensitive to external pH indicating that the major pH-sensitive pathway(s) for proton efflux in the light involves CF₀ · CF₁. Modification of CF₁ sulfhydryls by Ag⁺ resulted in an apparent increase in proton efflux via the normally coupled CF₀ · CF₁ pathway (half-maximal activity = pH 7.6), whereas modification by Hg²⁺ resulted in an apparent increase in proton efflux via the noncoupled CF₀ · CF₁ pathway (half-maximal activity = pH 7.9).     

Effects of nitrofen on chloroplast coupling factor-dependent reactions

Nucleotides induce a conformational change in the proteins of the CF₀-CF₁ complex. They give rise to reduced proton permeability of the thylakoid membranes. This reaction is paralleled by an enhanced yield of the steady-state proton uptake and a reduced nonphosphorylating electron-transport rate. Nitrofen acts as an energy-transfer inhibitor. It inhibits the rate of nucleotide exchange on CF₁ both at ‘loose’ and ‘tight’ binding sites. During illumination the percentage of nucleotide-free CF₀-CF₁ complex seems to be enhanced in the presence of nitrofen. This results in a prevention of the described ADP effects on proton uptake and electron transport. These similar effects of nitrofen on loose and tight nucleotide-binding sites correspond with the idea that both types are different states of identical sites.     

Regulation of proton leakage from broken chloroplasts by CF0.

Measurements of proton translocation in CF₁-depleted, N, N′-dicyclohexylcarbodiimide-resealed broken chloroplasts were made under different light intensities. Kinetic analysis of the data shows that the outward leakage of accumulated protons through CF₀ is still dependent on light intensity with a first-order rate constant equal to mR₀, where R₀ is the initial rate of proton uptake which normally increases with light intensity and m is a characteristic constant which is independent of proton gradient and light intensity. Measurements of proton translocation in these modified chloroplasts cross-linked with glutaraldehyde under illumination and in the dark respectively suggest that the light-dependent proton leakage through CF₀ is regulated by conformation change in the membrane. It is proposed that the ovserved regulation of proton leakage through the CF₁.CF₀ complex in native chloroplasts is for optimizing the steady state synthesis of ATP under different light intensities.     

Proton transfer in catalysis and the role of proton shuttles in carbonic anhydrase

The undisputed role of His64 in proton transfer during catalysis by carbonic anhydrases in the α class has raised questions concerning the details of its mechanism. The highly conserved residues Tyr7, Asn62, and Asn67 in the active-site cavity function to fine tune the properties of proton transfer by human carbonic anhydrase II (HCA II). For example, hydrophobic residues at these positions favor an inward orientation of His64 and a low pK_a for its imidazole side chain. It appears that the predominant manner in which this fine tuning is achieved in rate constants for proton transfer is through the difference in pK_a between His64 and the zinc-bound solvent molecule. Other properties of the active-site cavity, such as inward and outward conformers of His64, appear associated with the change in ΔpK_a; however, there is no strong evidence to date that the inward and outward orientations of His64 are in themselves requirements for facile proton transfer in carbonic anhydrase.     

Relevance of Divalent Cations to ATP-Driven Proton Pumping in Beef Heart Mitochondrial F0F1-ATPase

The ATP hydrolysis rate and the ATP hydrolysis-linked proton translocation by the F₀F₁-ATPase of beef heart submitochondrial particles were examined in the presence of several divalent metal cations. All Me–ATP complexes tested sustained ATP hydrolysis, although to a different extent. However, only Mg- and Mn-ATP-dependent hydrolysis could sustain a high level of proton pumping activity, as determined by acridine fluorescence quenching. Moreover, the K _m of the Me-ATP hydrolysis-induced proton pumping activity was very similar to the K _m value of Me-ATP hydrolysis. Both oligomycin and DCCD caused the full recovery of the fluorescence, providing clear evidence for the association of Mg-ATP hydrolysis with proton translocation through the F₀F₁-ATPase complex. In contrast, with other Me-ATP complexes, including Ca-ATP as substrate, the proton pumping activity was undetectable, implicating an uncoupling nature for these substrates. Attempts to demonstrate the involvement of the subunit of the enzyme in the coupling mechanism failed, suggesting that the participation of at least the N-terminal segment of the subunit in the coupling mechanism of the mitochondrial enzyme is unlikely.     

The influence of energy-transfer inhibitors on proton permeability and photophosphorylation in normal and preilluminated Rhodospirillum rubrum chromatophores

(1) Chromatophores were preilluminated in the presence of phenazine methosulphate or diaminodurene, and without phosphorylation substrates; next they were transferred to fresh medium and assayed for light-induced proton uptake, light-induced 9-aminoacridin fluorescence quenching, and photophosphorylation.(2) Preillumination in the presence of phenazine methosulphate or diaminodurene causes an inhibition of the photophosphorylation rate. The presence of ADP + MgCl₂ + phosphate, or ADP + MgCl₂ + arsenate during preillumination provides full protection against this effect.(3) Preilluminated chromatophores are leaky for protons. The leak is expressed as an accelerated dark decay, and a diminished extent of succinate-supported, light-induced proton uptake. The extent of light-induced 9-aminoacridin fluorescence quenching is also diminished.(4) The proton leak can be closed by oligomycin and by dicyclohexyl carbodiimide (at concentrations similar to those used to inhibit photophosphorylation), but not by aurovertin. Closure of the proton leak results in partial restoration of the photophosphorylation rate.(5) The inhibition of phosphorylation by oligomycin or dicyclohexyl carbodiimide is time-dependent. In untreated chromatophores, the time-dependence is determined by the extent of membrane energization. In preilluminated chromatophores, the time-dependence is determined in addition by the extent to which the proton leaks have been closed. The reasons for this are briefly discussed.     

Conformational and kinetic features of the morphine-Mn(II) interaction as probed by nuclear paramagnetic resonance investigations

The nature of binding between manganese ions and morphine was studied using Fourier transform proton nuclear magnetic resonance techniques. Proton relaxation times in the presence of Mn(II) ions were determined together with their temperature dependence. Slow exchange conditions were observed for the NCH₃ group, while fast exchange conditions applied for all the other protons. The rotational correlation time of the complex was approximated by that of the free morphine molecule, as measured by selective and nonselective proton relaxation rate measurements. The distances between the metal ion and proton nuclei of morphine were evaluated on the basis of an association constant, measured from water proton spin-lattice relaxation rate binding studies. The results indicate that the metal binds directly to the two oxydryls with K_ass = 9.7 × 10^?3.The rate constant for the interaction of Mn(II) with the opiate is 2.25 × 10⁴ sec^?1 at 27°C, as determined from the temperature dependence of longitudinal relaxation rate of the NCH₃ group.     

Effects of Carbonic Anhydrase Inhibitors on Proton Exchange and Photosynthesis in Pea Protoplasts

At concentrations of 100–200 M, ethoxyzolamide, a lipophilic inhibitor of carbonic anhydrase, considerably (by 60%) inhibited light-induced CO₂-dependent oxygen evolution in pea protoplasts at the optimum concentration of inorganic carbon (100 M CO₂) in the medium. At the same concentrations of the inhibitor, electron transport in isolated pea thylakoids was inhibited only by 6–9%. Acetazolamide, a water-soluble inhibitor of carbonic anhydrase, affected neither the rate of CO₂-dependent O₂evolution in protoplasts nor electron transport in thylakoid membranes. A light-dependent proton uptake by protoplasts was demonstrated. At pH 7.2, the induction kinetics and the rate of proton uptake were similar to those for CO₂-dependent O₂evolution. The rate of proton uptake was decreased twofold by 1 mM acetazolamide. This fact agrees with the notion that a membrane-bound carbonic anhydrase is operative in the plasma membrane of higher plant cells. A mechanism of its functioning is suggested. Possible functions of carbonic anhydrases in the cells of C₃-plants are discussed.     

Mechanistic Insight into the H<Subscript>2</Subscript>O/D<Subscript>2</Subscript>O Isotope Effect in the Proton Transport of the Influenza Virus M2 Protein

The M2 proton channel is essential for the replication of the flu virus and is a known drug target. The functional mechanism of channel activation and conductance is key to both the basic biology of viral replication and the design of drugs that can withstand mutations. A quantitative model was previously developed for calculating the rate of proton transport through the M2 channel. The permeant proton was assumed to diffuse to the pore, obligatorily bind to the His37 tetrad, and then dissociate and be released to either side of the tetrad. Here the model is used to calculate the effect of a change in solvent from H₂O to D₂O on the rate of proton transport. The solvent substitution affects two parameters in the model: the proton diffusion constant and the pK _a for proton binding to the His37 tetrad. When the known effects on these two parameters are included, the deuterium isotope effect calculated from the model is in quantitatively agreement with experimental results. This strict test of the theoretical model provides strong support for the hypothesis that the permeant proton obligatorily binds to and then unbinds from the His37 tetrad. This putatively essential role of the His37 tetrad in the functional mechanism of the M2 channel makes it a promising target for designing mutation-tolerant drugs.     

Observation of the phosphatidyl ethanolamine amino proton magnetic resonance in phospholipid vesicles: inside/outside ratios and proton transport.

The amino proton resonance of phosphatidyl ethanolamine in sonicated mixed phospholipid vesicles is observed 3.3 ppm downfield from H₂O. Above pH ～ 5 it is broadened beyond detectability as a result of exchange with H₂O protons. In low salt, resonances of amino protons inside the vesicles appear to persist as the pH is raised, while those on the outside disappear. Solvent catalized proton conduction along the surface is proposed, with an effective -NH₂ to -NH₃ transfer rate of about 8 × 10⁵ sec^?1 at 25°C.     

How chloride functions to enable proton conduction in photosynthetic water oxidation: Time-resolved kinetics of intermediates (S-states) in vivo and bromide substitution

Chloride (Cl⁻) is essential for O₂ evolution during photosynthetic water oxidation. Two chlorides near the water-oxidizing complex (WOC) in Photosystem II (PSII) structures from Thermosynechococcus elongatus (and T. vulcanus) have been postulated to transfer protons generated from water oxidation. We monitored four criteria: primary charge separation flash yield (P* → P⁺Q_A⁻), rates of water oxidation steps (S-states), rate of proton evolution, and flash O₂ yield oscillations by measuring chlorophyll variable fluorescence (P* quenching), pH-sensitive dye changes, and oximetry. Br-substitution slows and destabilizes cellular growth, resulting from lower light-saturated O₂ evolution rate (−20 %) and proton release (−36 % ΔpH gradient). The latter implies less ATP production. In Br- cultures, protonogenic S-state transitions (S₂ → S₃ → S₀’) slow with increasing light intensity and during O₂/water exchange (S₀’ → S₀ → S₁), while the non-protonogenic S₁ → S₂ transition is kinetically unaffected. As flash rate increases in Cl⁻ cultures, both rate and extent of acidification of the lumen increase, while charge recombination is suppressed relative to Br⁻. The Cl⁻ advantage in rapid proton escape from the WOC to lumen is attributed to correlated ion-pair movement of H₃O⁺Cl⁻ in dry water channels vs. separated Br⁻ and H⁺ ion movement through different regions (>200-fold difference in Bronsted acidities). By contrast, at low flash rates a previously unreported reversal occurs that favors Br⁻ cultures for both proton evolution and less PSII charge recombination. In Br⁻ cultures, slower proton transfer rate is attributed to stronger ion-pairing of Br⁻ with AA residues lining the water channels. Both anions charge-neutralize protons and shepherd them to the lumen using dry aqueous channels.     

Kinetic analysis of proton transport by the vanadate-sensitive ATPase from maize root microsomes

Proton transport by the nitrate-insensitive, vanadate-sensitive ATPase in Kl-washed microsomes and reconstituted vesicles from maize (Zea mays L.) roots was followed by changes in acridine orange absorbance in the presence of either KNO₃ or KCl. Data from such studies obeyed a kinetic model in which net proton transport by the pump is the difference between the rate of proton transport by the action of the ATPase and the leak of protons from the vesicles in the direction opposite from the pump. After establishing the steady state proton gradient, the rate of return of transported protons was found to obey first-order kinetics when the activity of the ATPase was completely and rapidly stopped. The rate of return of these protons varied with the quencher used. When the substrate Mg:ATP was depleted by the addition of either EDTA or hexokinase, the rate at which the proton gradient collapsed was faster than when vanadate was used as the quencher. These trends were independent of the anion accompanying the K and the transport assay used.     

Cluster-root formation, carboxylate exudation and proton release of Lupinus pilosus Murr. as affected by medium pH and P deficiency

The study examined the interactive effect of pH and P supply on cluster-root formation, carboxylate exudation and proton release by an alkaline-tolerant lupin species (Lupinus pilosus Murr.) in nutrient solution. The plants were exposed to 1 (P₁, deficient) and 50 μM P (P₅₀, adequate) for 34 days in nutrient solution at either pH 5.6 or 7.8. Plant biomass was not influenced by pH at P₁, but at P₅₀ shoot and root dry weights were 23 and 18% higher, respectively, at pH 7.8 than at pH 5.6. There was no significant difference in plant biomass between two P treatments regardless of medium pH. Phosphorus deficiency increased significantly the number of the second-order lateral roots compared with the P₅₀ treatment. Both total root length and specific root length of plants grown at pH 5.6 were higher than those at pH 7.8 regardless of P supply. Cluster roots were formed at P₁, but cluster-root number was 2-fold higher at pH 7.8 than pH 5.6. Roots released 16 and 31% more protons at pH 5.6 and 7.8, respectively, in P₁ than in P₅₀ treatments, and the rate of proton release followed the similar pattern. At pH 5.6, citrate exudation rate was 0.39 μmol g⁻¹ root DW h⁻¹ at P₁, but was under the detection limit at P₅₀; at pH 7.8, it was 2.4-fold higher in P₁ than in P₅₀ plants. High pH significantly increased citrate exudation rate in comparison to pH 5.6. The uptake of anions P and S was inhibited at P₁ and high pH increased cations Na, Mg and Ca uptake. The results suggested that enhanced cluster-root formation, proton release and citrate exudation may account for the mechanism of efficient P acquisition by alkaline-tolerant L. pilosus well adapted to calcareous soils. Cluster-root formation and citrate exudation in L. pilosus can be altered by medium pH and P deficiency. Phosphorus deficiency-induced proton release may be associated with the reduced anion uptake, but high pH-induced proton release may be partly attributed to increased cation uptake.     

A reevaluation of the events leading to the electrogenic reaction and proton translocation in the ubiquinol-cytochrome c oxidoreductase of Rhodopseudomonas sphaeroides

Chromatophore membranes from Rhodopseudomonas sphaeroides activated by light display a carotenoid band shift (phase III) that occurs in response to the electrogenic event (charge separation) in the ubiquinol-cytochrome c oxidoreductase. The rate of formation of this electrogenic event has previously been shown to be strongly dependent on the initial redox state of a bound ubiquinone species (designated Q_z) associated with the oxidoreductase. When Q_z is reduced (quinol form; Q_zH₂) the electrogenic event takes place in less than 5 ms. When Q_z is oxidized (quinone form; Q_z) it is much slower; under these conditions the fact that it occurs has been ignored. In this report, we address this issue and describe events that lead to the generation of carotenoid band shift phase III when the total population of Q_z of the chromatophore is oxidized before flash activation. The following characteristics are apparent: (1) When oxidized Q_z is present before activation, the half-time of formation of carotenoid band-shift phase III is 10–20-times slower than when Q_zH₂ is present before activation. (2) When oxidized Q_z is present, the measured full extent of phase III generated by a single-turnover flash is diminished by about one-half of that observed when Q_zH₂ is present before activation. (3) The rate of formation of the carotenoid band shift phase III when Q_z is initially oxidized corresponds closely to the rate of completion of the flash-activated electron-transfer cycle. This can be seen under two different conditions: (a) as the partial reduction of cytochrome c₁ + c₂ (at redox potentials of 200–300 mV) or (b) as the partial reduction of flash-oxidized bacteriochlorophyll dimer, (BChl)₂⁺ (at redox potentials above 300 mV). (4) At the higher redox potentials (above 300 mV), antimycin-sensitive proton binding shares a common, rate-limiting step with the carotenoid band shift phase III and (BChl)₂⁺ reduction. (5) However, proton binding at redox potentials above 300 mV is not observed at all unless valinomycin (K⁺) is present. Thus, proton binding occurs only when the carotenoid band shift is collapsed in milliseconds, whereas, conversely, the carotenoid band shift is stably generated when proton binding is not observed. These and other observations are the basis of a reevaluation of our current views on the coupling of electron transfer and proton translocation in photosynthetic bacteria.     

Functionally-distinct proton-binding in HERG suggests the presence of two binding sites

HERG (Human ether-à-go-go-related gene) potassium channels are crucial for cardiac action potential repolarization. HERG channels are also found in neuronal and tumor cells. The effect of pH₀ on HERG is of clinical significance because of changes in pH during myocardial ischemia, inflammation, and respiratory alkalosis. We present evidence for the presence of multiple proton binding sites in HERG. Extracellular protons bind rapidly and reversibly to affect both activation and deactivation. However, these effects occur in two distinct pH₀ ranges. The deactivation rate has a pK_a of 6.76±0.02 compared to pK_a of 5.50±0.02 for changes in current suppression, which suggests the presence of at least two proton binding sites on HERG with functionally distinct properties.     

Acclimation to low temperature by microsomal membranes from tomato cell cultures

Sealed vesicles were prepared from microsomal membranes from cell suspension cultures of tomato (Lycopersicon esculentum Mill cv VF36). ATP-dependent proton transport activity by the vesicles was measured as quenching of fluorescence of acridine orange. Measurements of proton transport were correlated with the activity of a nitrate-inhibitable ATPase. The initial rate of proton influx into the vesicles was strongly temperature dependent with a Q₁₀ of 2 and a maximum rate near 35°C. The data suggest that passive permeability did not increase at chilling temperatures but did increase rapidly with temperatures above 30°C. A comparison was made between membranes from cell cultures grown at 28°C and 9°C. The temperature optimum for proton transport broadened and shifted to a lower temperature range in membranes from cells maintained at 9°C.     

Continuous ECS-indicated recording of the proton-motive charge flux in leaves

Technical features and examples of application of a special emitter–detector module for highly sensitive measurements of the electrochromic pigment absorbance shift (ECS) via dual-wavelength (550–520 nm) transmittance changes (P515) are described. This device, which has been introduced as an accessory of the standard, commercially available Dual-PAM-100 measuring system, not only allows steady-state assessment of the proton motive force (pmf) and its partitioning into ΔpH and ΔΨ components, but also continuous recording of the overall charge flux driven by photosynthetic light reactions. The new approach employs a double-modulation technique to derive a continuous signal from the light/dark modulation amplitude of the P515 signal. This new, continuously measured signal primarily reflects the rate of proton efflux via the ATP synthase, which under quasi-stationary conditions corresponds to the overall rate of proton influx driven by coupled electron transport. Simultaneous measurements of charge flux and CO₂ uptake as a function of light intensity indicated a close to linear relationship in the light-limited range. A linear relationship between these two signals was also found for different internal CO₂ concentrations, except for very low CO₂, where the rate of charge flux distinctly exceeded the rate of CO₂ uptake. Parallel oscillations in CO₂ uptake and charge flux were induced by high CO₂ and O₂. The new device may contribute to the elucidation of complex regulatory mechanisms in intact leaves.