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).     

Activation of the CF0-CF1, ATP synthase from spinach chloroplasts by chloroplast lipids

The interactions of CF₀-CF₁ with different lipids were studied by following the stimulation of Mg-ATPase and of P_i-ATP exchange activities of reconstituted CF₀-CF₁ proteoliposomes. The following results were obtained: (1) Both P_i-ATP exchange and Mg-ATPase activities are stimulated by lipids. Furthermore, the inhibition of Mg-ATPase by N,N′-dicyclohexylcarbodiimide is dependent on the interactions of CF₀-CF₁ with lipids. (2) A polar lipid extract of thylakoid membranes stimulates Mg-ATPase activity of CF₀-CF₁ more efficiently than phospholipids. The relative effectiveness of Mg-ATPase stimulation is: chloroplast lipids > soybean phospholipids > phosphatidylcholine/phosphatidylserine (4: 1) > phosphatidylcholine. The rate of P_i-ATP exchange in chloroplast lipids CF₀-CF₁ proteoliposomes is, however, lower than in soybean lipids CF₀-CF₁ proteoliposomes, due to their higher permeability to protons. Addition of 10% phosphatidylserine to chloroplast lipids reduces their permeability to protons and stimulates P_i-ATP exchange. (3) The kinetic mechanism of ATPase stimulation by chloroplast lipids is by decreasing the K_m (ATP) and by increasing V_max in comparison to soybean lipid proteoliposomes. This may explain the low affinity for ATP and the slow turnover rate of the purified enzyme in artificial lipids in comparison to the native enzyme in chloroplast thylakoids. (4) Chloroplast lipids lacking monogalactosyldiacylglycerols only poorly activate CF₀-CF₁. A large stimulation of P_i-ATP exchange is obtained by a mixture of 60% monogalactosyldiacylglycerol and 40% of the rest of the chloroplast lipids, but not by mixtures of monogalactosyldiacylglycerol with phospholipids. Hydrogenation of the unsaturated fatty acids of monogalactosyldiacylglycerol inhibits the activation of CF₀-CF₁. (5) The results suggest that: (a) interactions of specific chloroplast lipids with CF₀-CF₁ activates the enzyme by increasing its turnover and its affinity for ATP; (b) specific requirements for CF₀-CF₁ activation are the presence of monogalactosyldiacylglycerols together with another chloroplast lipid component and of highly unsaturated fatty acids.     

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.     

The role of different thylakoid glycolipids in the function of reconstituted chloroplast ATP synthase

ATPase activity of CF₀CF₁ from spinach chloroplasts is specifically stimulated by chloroplast lipids (Pick, U., Gounaris, K., Admon, A. and Barber, J. (1984) Biochim. Biophys. Acta 765, 12–20). The association of CF₀-CF₁ with isolated lipids and their mixtures has been examined by analyzing the stimulation of ATPase and ATP-P_i exchange activities, by binding studies and by measurement of proton conductance of reconstituted proteoliposomes. Monogalactosyldiacylglycerol is the only chloroplast lipid which by itself activates ATP hydrolysis. A mild saturation of the fatty acids of the lipid partially inhibits the activation. CF₀-CF₁ has a higher binding capacity for monogalactosyldiacylglycerol (1.5 mg/mg protein) than for other thylakoid glycolipids. However, ATPase activation is not correlated with the amount of bound lipid but rather with its type. For the same amount of bound lipid, monogalactosyldiacylglycerol best activates ATP hydrolysis, while the acidic lipids phosphatidylglycerol and sulphoquinovosyldiacylglycerol inhibit ATPase activity. Optimal activation of ATP-P_i exchange requires, in addition to monogalactosyldiacylglycerol, digalactosyldiacylglycerol and sulphoquinovosyldiacylglycerol at a ratio of 6:3:1, respectively. Correlations between proton conductance, ATP-P_i exchange and uncoupler stimulation of ATPase activity indicate that sulphoquinovosyldiacylglycerol reduces the permeability of the proteoliposomes to protons. The results suggest that: (a) association of CF₀-CF₁ with polyunsaturated monogalactosyldiacylglycerol greatly stimulates ATPase activity; (b) reconstitution of coupled CF₀-CF₁ proteoliposomes requires a careful balance of the natural glycolipids of thylakoid membranes in similar proportions to their occurrence in chloroplasts, and (c) sulphoquinovosyldiacylglycerol may control the permeability of chloroplast membranes to protons.     

Inhibition of the transthylakoid gradient of electrochemical proton potential by the local anesthetic dibucaine

The effects of the local anesthetic dibucaine on coupling between electron transport and ATP synthesis-hydrolysis by the coupling-factor complex (CF₀CF₁ ATPase) were investigated in thylakoid membranes from Spinacia oleracea L. cv. Monatol. Evidence is presented that inhibition of ATP synthesis was produced by a specific uncoupling mechanism which was based on dibucaine-membrane surface interactions rather than on the interaction of dibucaine with the ATPase complex. Dibucaine reduced the osmotic space of thylakoid vesicles. At low pH of the medium it stimulated ATP hydrolysis beyond the rates obtained with optimum concentrations of ‘classical’ uncouplers. After addition of dibucaine, there was displacement of membrane-bound Mg²⁺ and strong thylakoid stacking in the presence of only low Mg²⁺ concentrations. Inhibition of ATP synthesis and transmembrane pH gradient increased with medium pH. Hydrolysis of ATP by isolated CF₁ and the CF₀CF₁ complex was only slightly affected by dibucaine. The data are discussed assuming the involvement of localized proton channels on the membrane surface in protonic coupling of electron transport and ATP synthesis. A hypothesis for the mechanisms of action of local anesthetics at the thylakoid membrane is presented.     

Structural analysis of the isolated chloroplast coupling factor and the N,N'-dicyclohexylcarbodiimide binding proteolipid

Negative staining of purified spinach dicyclohexylcarbodiimide (DCCD) sensitive ATPase revealed a population of 110 Å subunits attached by stalks to short string-like aggregates. The interpretation of these data is that 110 Å CF₁ are attached by stalks to an aggregate of CF₀.The CF₁-CF₀ complex was incorporated into phospholipid vesicles; freezefracture analysis of this preparation revealed a homogeneous population of particles spanning the lipid bilayer; these averaged 96 Å in diameter. The DCCD binding proteolipid (apparent molecular weight 7500), an integral component of CF₀, was isolated from membranes by butanol extraction and was incorporated rated into phospholipid vesicles. Freeze-fracture analysis of the DCCD-binding proteolipid/vesicle preparation revealed a population of particles averaging 83 Å in diameter suggesting that the DCCD-binding proteolipid self-associates in lipid to form a stable complex. This complex may be required for proton transport across chloroplast membranes in vivo. The size difference between CF₀ and DCCD-proteolipid freeze-fracture particles may be related to differences in polypeptide composition of the two complexes.     

Purification and Characterization of a Glycerol-Resistant CF(0)-CF(1) and CF(1)-ATPase from the Halotolerant Alga Dunaliella bardawil

The isolation of the chloroplast ATP synthase complex (CF₀-CF₁) and of CF₁ from Dunaliella bardawil is described. The subunit structure of the D. bardawil ATPase differs from that of the spinach in that the D. bardawil α subunit migrates ahead of the β subunit and ε-migrates ahead of subunit II of CF₀ when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The CF₁ isolated from D. bardawil resembles the CF₁ isolated from Chladmydomonas reinhardi in that a reversible, Mg²⁺-dependent ATPase is induced by selected organic solvents. Glycerol stimulates cyclic photophosphorylation catalyzed by D. bardawil thylakoid membranes but inhibits photophosphorylation catalyzed by spinach thylakoid membranes. Glycerol (20%) also stimulates the rate of ATP-P_i exchange catalyzed by D. bardawil CF₀-CF₁ proteoliposomes but inhibits the activity with the spinach enzyme. The ethanol-activated, Mg²⁺-ATPase of the D. bardawil CF₁ is more resistant to glycerol inhibition than the octylglucoside-activated, Mg²⁺-ATPase of spinach CF₁ or the ethanol-activated, Mg²⁺-dependent ATPase of the C. reinhardi CF₁. Both cyclic photophosphorylation and ATP-P_i exchange catalyzed by D. bardawil CF₀-CF₁ are more sensitive to high concentrations of NaCl than is the spinach complex.     

Tightly bound sulpholipids in chloroplast CF0-CF1

Highly purified preparations of CF₀-CF₁ from chloroplasts contain a small amount of tightly bound lipids. Extraction and analysis of these lipids show that they are almost exclusively sulpholipids. The calculated amount of bound sulpholipids in spinach and in Dunaliella salina CF₀-CF₁ preparations are 5 and 20 mols/mol enzyme, respectively. Attempts to exchange the bound lipids with other lipids or with detergents have failed, indicating a very strong association with CF₀-CF₁.     

The effects of tetraphenylboron on energy-linked reactions in spinach chloroplasts

1. The inhibitory action of tetraphenylboron, a lipid-soluble anion, on the proton uptake, the photophosphorylation and the light-induced quenching of the fluorescence of 9-aminoacridine by spinach chloroplasts was studied.2. The inhibition of the three processes by tetraphenylboron was transient; the proton uptake was affected to a much smaller extent than either the photophosphorylation or the fluorescence quenching.3. The inhibitory effects of tetraphenylboron on the proton uptake and the fluorescence quenching of 9-aminoacridine were qualitatively the same in CF₁-depleted chloroplasts, that were recoupled with N,N′-dicyclohexylcarbodiimide (DCCD).4. The reversal of the fluorescence quenching of 9-aminoacridine upon addition of tetraphenylboron in the light was found to be very fast, being completed within the response time of the apparatus.5. The presence of tetraalkylammonium salts in the incubation medium prevented the inhibitory effect of tetraphenylboron.6. Tetraphenylboron disappeared from the chloroplast suspension in a light-dependent irreversible way; in the dark no ‘ptake’ of tetraphenylboron could be detected.7. The effects of tetraphenylboron may be explained by the presence of groups with a high affinity for tetraphenylboron in the membrane; these groups become protonated upon illumination of the chloroplasts.     

Thiol modulation of the chloroplast protonmotive ATPase and its effect on photophosphorylation

Thiol modulation of the chloroplast protonmotive ATPase (CF₀-CF₁) by preillumination of broken chloroplasts in the presence of dithiothreitol (or preillumination of intact chloroplasts in the absence of added thiols) had the following effects on photophosphorylation. (1) When assayed at pH 8 and saturating light, the initial rate of photophosphorylation was increased by 10–40%. There was an accompanying increase in the rate of coupled electron transport with no significant change in the overall $\text{P}\text{2}\text{e}$ ratio. (2) On lowering the pH of the assay medium to pH 7, the stimulatory effect of thiol modulation on photophosphorylation and coupled electron flow was enhanced. At pH 7, there was also a small increase in $\text{P}\text{2}\text{e}$ ratio. (3) Addition of a non-saturating amount of uncoupler to the assay medium enhanced the stimulatory effect of thiol modulation on photophosphorylation. In the presence of 1 mM NH₄Cl, there was only a small increase in coupled electron flow and a correspondingly larger increase in $\text{P}\text{2}\text{e}$ ratio. (4) Lowering the light intensity, or inhibiting electron transport, diminished the stimulatory effect of thiol modulation on photophosphorylation, coupled electron transport and $\text{P}\text{2}\text{e}$ ratio. (5) Under all the above conditions, the ΔpH maintained across the thylakoid membrane was lower after thiol modulation, even when photophosphorylation markedly increased in rate. (6) Thiol modulation of CF₀-CF₁ increased the observed Michaelis constant for ADP (K_m(ADP)) and the apparent maximum rate (V_app of photophosphorylation by the same factor, so that ratio $\text{V}{}_{\mathrm{<mtext>app</mtext>}}\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ was not altered. $\text{V}{}_{\mathrm{<mtext>app</mtext>}}\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ was also unaffected by changing the medium pH, but was significantly decreased upon addition of uncouplers to the medium. These results indicate that the observed rate of ATP synthesis catalysed by thiol demodulated chloroplasts is limited kinetically by the fraction (α) of enzyme molecules that are active during photophosphorylation. A model based on a dual pH optimum requirement for activation of CF₀-CF₁ is presented to explain the dependence of α on ΔpH. Thiol modulation of CF₀-CF₁ is proposed to stimulate photophosphorylation by causing the enzyme to become active over a lower range of ΔpH, thereby reducing the kinetic limitation on ATP synthesis imposed by the activation process.     

Light dependence of the decay of the proton gradient in broken chloroplasts

The initial rates and steady-state values of proton uptake by broken chloroplasts have been measured as functions of light intensity at various concentrations of chlorophyll, pyocyanine, supporting electrolyte, buffer, as well as pH and temperature. Kinetic analysis of the data shows that the rate of decay of proton gradient due to backward leakage depends on light intensity. Under steady illumination, the decay constant k_L is equal to k_D + mR₀, where R₀ is the initial rate of proton uptake which is a function of light intensity, k_D is the decay constant in the dark and m is a parameter which is independent of light intensity. Treatment of chloroplasts with lysolecithin, neutral detergent, 2,4-dinitrophenol, or valinomycin in the presence of K⁺ increases k_D without affecting m. Treatment with N,N′-dicyclohexylcarbodiimide or adenylyl imidodiphosphate under appropriate conditions decreases m without affecting k_D. Treatment with glutaraldehyde makes k_L independent of light intensity and hence m = 0. These results suggest that the light-dependent part (mR₀) of k_L is due to leakage of protons through the coupling factor (CF₁-CF₀) complex which can open or close depending on light intensity and that the light-independent part (k_D) of the decay constant k_L is due to proton leakage elsewhere.     

The effect of chloroplast coupling factor removal on thylakoid membrane ion permeability

Removal of coupling factor protein (CF₁) from spinach thylakoid membranes results in an enhancement of proton permeability but has no effect on chloride or potassium permeability. Anion permeability was measured by the rate of thylakoid packed volume changes. Potassium permeability was monitored by turbidity changes, packed thylakoid volume changes and ion flux studies using ⁸⁶Rb⁺ as a tracer. ⁴⁵Ca²⁺ was used to measure divalent cation fluxes. CF₁-depleted chloroplasts had an unaltered rate of Ca²⁺ uptake, but the rate of Ca²⁺ efflux appeared to be increased. Calcium efflux rates could also be increased by the addition of a proton specific uncoupler, FCCP.     

Effect of pyridine homologues on the basal rate of electron transport and H+/e − ratio in chloroplasts

Low concentrations of hydrophobic pyridine homologues (1 mM) were found to increase the rate of the Hill reaction in chloroplasts without significantly affecting either the steady-state proton uptake or the rate of proton leakage in the dark. By assuming that the organic base can be bound to two types of independent binding sites in the thylakoid membrane with dissociation constantsK ₁ andK ₂ respectively, the kinetic data can be treated quantitatively. The values ofK ₁ andK ₂ determined by the treatment are in the same relative order as the hydrophobicities of the pyridine homologues:K ₁=1.16 mM andK ₂=54 mM for pyridine; 0.6 and 38 mM for 4-picoline; 0.27 and 31 mM for 4-ethylpyridine, 0.10 and 4.2 mM for 4-t-butylpyridine; 0.08 and 3.2 mM for 4-n-butylpyridine. The rates of oxygen generation and proton uptake by illuminated chloroplasts with either ferricyanide or 1,4-benzoquinone as the electron acceptor were also measured in the presence of various pyridine homologues. Low concentration of pyridine homologues were found to decrease the H⁺/e ^– ratio. This last observation seems to substantiate an indirect coupling mechanism between electron transport and proton translocation.Abbreviations Chl chlorophyll - CF₀ - CF₁ the coupling factor complex of chloroplast - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Tricine N-tris-(hydroxymethyl)methylglycine     

Calcium gating of H+ fluxes in chloroplasts affects acid-base-driven ATP formation

In previous work, calcium ions, bound at the lumenal side of the CF₀H⁺ channel, were suggested to keep a H⁺ flux gating site closed, favoring sequestered domain H⁺ ions flowing directly into the CF₀-CF₁ and driving ATP formation by a localized gradient. Treatments expected to displace Ca⁺⁺ from binding sites had the effect of allowing H⁺ ions in the sequestered domains to equilibrate with the lumen, and energy coupling showed delocalized characteristics. The existence of such a gating function implies that a closed-gate configuration would block lumenal H⁺ ions from entering the CF₀-CF₁ complex. In this work that prediction was tested using as an assay the dark, acid-base jump ATP formation phenomenon driven by H⁺ ions derived from succinic acid loaded into the lumen.Chlorpromazine, a photoaffinity probe for many proteins having high-affinity Ca⁺⁺-binding sites, covalently binds to the 8-kDa CF₀ subunit in the largest amounts when there is sufficient Ca⁺⁺ to favor the localized energy coupling mode, i.e., the gate closed configuration. Photoaffinity-bound chlorpromazine blocked 50% or more of the succinate-dependent acid-base jump ATP formation, provided that the ionic conditions during the UV photoaffinity treatment were those which favor a localized energy coupling pattern and a higher level of chlorpromazine labeling of the 8-kDa CF₀ subunit. Thylakoids held under conditions favoring a delocalized energy coupling mode and less chlorpromazine labeling of the CF₀ subunit did not show any inhibition of acid-base jump ATP formation.Chlorpromazine and calmidazolium, another Ca⁺⁺-binding site probe, were also shown to block redox-derived H⁺ initially released into sequestered domains from entering the lumen, at low levels of domain H⁺ accumulation, but not at higher H⁺ uptake levels; ie., the closed gate state can be overcome by sufficiently acidic conditions. That is consistent with the observation that the inhibition of lumenal succinate-dependent ATP formation by photoaffinity-attached chlorpromazine can be reversed by lowering the pH of the acid stage from 5.5 to 4.5.The evidence is consistent with the concept that Ca⁺⁺ bound at the lumenal side of the CF₀ H⁺ channel can block H⁺ flux from either direction, consistent with the existence of a molecular structure in the CF₀ complex having the properties of a gate for H⁺ flux across the inner boundary of the CF₀. Such a gate could control the expression of localized or delocalized energy coupling gradients.     

ATP binding to noncatalytic sites of chloroplast coupling factor CF1

A kinetic analysis of ATP binding to noncatalytic sites of chloroplast coupling factor CF₁ was made. The ATP binding proved to be unaffected by reduction of the disulfide bridge of the CF₁ -subunit. The first-order equation describing nucleotide binding to noncatalytic sites allowed for two vacant nucleotide binding sites different in their kinetics. As suggested by nucleotide concentration dependence of the rate of nucleotide binding, the tight binding was preceded by rapid reversible binding of nucleotides. Preincubation of CF₁ with Mg²⁺ resulted in a decreased rate of ATP binding. ATP dissociation from noncatalytic sites was described by the first order equation for similar sites with a dissociation rate constant k _d (ATP) 10^–3 min^–1. Noncatalytic sites of CF₁ were shown to be not homogeneous. One of them retained the major part of endogenous ADP after precipitation of CF₁ with ammonium sulfate. Its two other sites differed in kinetic parameters and affinity for ATP. Anions of phosphate, sulfite, and especially, pyrophosphate inhibited the interaction between ATP and the noncatalytic sites.     

Effect of pyridine homologues on proton flux through the CF0 · CF1 complex and photophosphorylation in chloroplasts

At concentrations below 1 mM, hydrophobic pyridine homologues decrease the rate of photophosphorylation and light-stimulated hydrolysis of ATP and light-activated exchange of the tightly bound nucleotides in chloroplasts, but increase the rate of the Hill reaction. Unlike uncoupling agents, the presence of the organic base at such low concentrations decreases the rate of light-dependent leakage and has no effect on the efficiency of two-stage photophosphorylation in broken chloroplasts. By assuming that the organic base is bound to independent equivalent sites in the thylakoid membrane, a simple expression can be derived which relates the observed rates of photophosphorylation and light-stimulated hydrolysis of ATP quantitatively to the concentration of the organic base in solution and gives dissociation equilibrium constants which are on the order of the relative hydrophobicities of the pyridine homologues. A possible mechanistic model for the CF₀ · CF₁ complex is proposed which could serve as the basis for a unified interpretation of the kinetics of proton translocation in illuminated chloroplasts, the steady-state rate of photophosphorylation, the light-stimulated ATPase activity, and the light-activated exchange of tightly bound adenine nucleotides.Abbreviations AMPPNP adenylylimidodiphosphate - Chl chlorophyll - CF₀ · CF₁ the coupling factor complex of chloroplasts - DCCD N,N-dicyclohexylcarbodiimide - DTT dithiothreitol - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - TCA trichloroacetic acid - Tricine N-tris-(hydroxymethyl)methylglycine     

The reversibility of freeze/thaw injury to spinach thylakoids; restoration of light-induced proton pumping,membrane-conformational changes and proton gradient formation

Spinach thylakoids have been frozen under a variety of salt / sucrose concentrations to remove varying amounts of peripheral membrane proteins, including the water-soluble part of the coupling factor complex (CF₁). This leads to a defined degree of uncoupling by exposing the CF₀ proton channel. The ability of thylakoids, subjected to this treatment, to reconstitute light-induced proton pumping, membrane-conformational changes and proton gradient formation when treated with DCCD, an energy transfer inhibitor which interacts with the CF₀ proton channel, thus reducing the proton permeability of the membrane, has been investigated. Full reconstitution of proton pumping and ΔpH formation could be obtained in thylakoids in which up to 75% of the coupling factor complex had been removed by the freezing regime. Even under the most severe conditions employed, in which over 80% of the CF₁ was removed from the membrane, there was still between 25 and 50% reconstitution of proton pumping. Reconstitution of membrane conformational changes as monitored by 90° scattering changes showed a strong positive correlation to the restoration of proton pumping. Reconstitution of slower, light-induced transmittance changes, in contrast, exhibited a more variable response. Little reconstitution of the slow transmittance changes was found under conditions which removed more than 60–70% of the coupling factor complex.     

Proton channel of the chloroplast ATP synthase,CF0: Its time-averaged single-channel conductance as function of pH,temperature, isotopic and ionic medium composition

Summary The proton-driven ATP synthase of chloroplasts is composed of two elements, CF₀ and CF₁. The membrane bound CF₀ conducts protons and the peripheral CF₁ interacts with nucleotides. By flash spectrophotometric techniques applied to thylakoid membranes from which about 50% of total CF₁ was removed, we have previously determined the protonic (timeaveraged) single-channel conductance of CF₀. Being in the order of 1 pS, it was sufficiently large to support the proposed role of CF₀ as a low-impedance access for protons to the coupling site in CF₀CF₁. On the other hand, it was too large to be readily reconciled with current concepts of proton supply to and proton conduction through the channel.We studied the time-averaged single-channel conductance of CF₀ under variation of pH, pD, ionic composition, temperature, and water/membrane structure with the following results: (i) CF₀ was proton-specific even against a background of 300mm monovalent or 30mm divalent catins. (ii) While the conductance of CF₀ was pH/pD-independent in the range from 5.6–8.0, in D₂O it was lower by a constant factor of 1.7 than in H₂O (iii) Addition of glycerol diminished the conductance and abolished the isotope effect. (iv) The Arrhenius activation energy was 42 kJ/mol and thus intermediate between the ones found for the water-filled pore, gramicidin (30 kJ/mol), and the mobile carrier, valinomycin (65 kJ/mol).The results implied that CF₀ is endowed with an extremely proton-specific (10⁷-fold) selectivity filter. Its conductance is very high, and its conduction cycle is not necessarily rate limited by a protolytic reaction. The mechanisms of rapid proton supply to the channel mouth and of proton conduction remained enigmatic.     

Delayed light studies on photosynthetic energy conversion. VIII. Evidence from millisecond emission of chloroplasts for two adenylate binding sites on membrane-bound coupling factor,CF1

Effects of adenylates on chloroplast delayed light emission, at millisecond dark times, are inverse to the previously characterized effects of adenylates on electron transport rates. Either ADP alone or ATP alone increase intensity of delayed light, while ADP plus P_i decrease it. ADP alone requires the presence of an electron acceptor to have this effect on delayed light, but ATP does not.All three adenylate effects are abolished by uncoupling with gramicidin, by partial removal of photophosphorylation coupling factor (CF₁) with EDTA, and by antibody to CF₁. Readdition of CF₁ re-established the adenylate effects in EDTA-stripped membranes. The three adenylate effects are differentially sensitive to pH, and pH differentially affected their abolition by antibody to CF₁. The two adenylate effects shown in the absence of P_i are exhibited at lower adenylate concentrations than the ADP plus P_i effect, and are also less sensitive to phloridzin.These results are discussed in terms of probable adenylate effects on membrane-bound chloroplast coupling factor, CF₁. At least two ADP binding sites would differ with respect to adenylate concentration for half maximal binding; pH of optimal binding capacity; phloridzin sensitivity; and functional regulation of electron transport, proton uptake, and energy storage within the membrane as measured by delayed light emission. It remains unclear whether the high affinity ADP binding site is identical to a high affinity ATP binding site on CF₁.