Light-induced exchange of nucleotides into coupling factor 1 in spinach chloroplast thylakoids.

The method of centrifugation of chloroplast thylakoids through silicone fluid, previously used to estimate the uptake of solutes by thylakoids, is shown to be an excellent method for measuring binding of nucleotides to thylakoids. This binding, which is probably an exchange (Harris, D. A. and Slater, E. C. (1975) Biochim. Biophys. Acta 387, 335-348), is enhanced by light and is sensitive to uncoupling. Half-maximal binding of adenosine 5'-triphosphate (ATP) or adenosine 5'-diphosphate (ADP) at 10 mjM was reached within less than 0.1 s. With illumination times sufficient to elicit maximal binding, saturation of the site(s) is approached at 20 muM nucleotide and dissociation constants of 5 muM and 7 muM were calculated for ADP and ATP, respectively. At saturation, the binding corresponds to 1 mol/mol of coupling factor 1 or less. Although the light-dependent binding of ADP does not require Mg2+, that of ATP is markedly enhanced by Mg2+. A 10-fold molar excess of guanosine di- or triphosphate or adenyl-5'-yl imidodiphosphate had little effect on the binding. Adenosine 5'-phosphosulfate, a competitive inhibitor of phosphorylation with respect to ADP, decreases the binding. Thylakoids, previously illuminated in the absence of added nucleotides, retain the capacity to bind ADP or ATP in the dark long after the H+ electrochemical gradient has decayed. The conformation of coupling factor 1 in darkened thylakoids following illumination in the absence of added nucleotides may thus differ from that in thylakoids either illuminated in the presence of nucleotides or kept in the dark. Approximately 20% of the ADP bound to coupling factor 1 in thylakoids is converted to ATP by a 2-s illumination. Bound inorganic phosphate, derived either from ATP or from inorganic phosphate itself, serves as the phosphoryl donor. Bound ADP may, therefore, be of catalytic significance in the mechanism of phosphorylation.     

Selective modification of coupling factor 1 in spinach chloroplast thylakoids by a fluorescent maleimide

N-(1-Anilinonaphthyl-4)maleimide (ANM) has been used to modify coupling factor 1 (CF1), the terminal coupling factor of photophosphorylation in chloroplasts. As with other monofunctional maleimides, incubation of thylakoids with ANM in the light, but not in the dark, causes energy transfer inhibition of photophosphorylation. In the dark, sites on both the gamma and epsilon subunits of CF1 are modified. The light-accessible site is also on the gamma subunit. Trypsin digestion of the enzyme after dithiothreitol activation reveals that the dark-and light-accessible sites on the gamma subunit are different amino acid residues. Fluorescence of ANM bound at the dark-and light-accessible sites has been measured after isolation of CF1 from thylakoids. The fluorescence emission maximum of ANM at the light-accessible site is blue-shifted and the quantum yield is increased 2-fold relative to ANM bound at dark-accessible sites. On the soluble enzyme, fluorescence polarization is high and equivalent for ANM bound at both dark-and light-accessible sites. Fluorescence energy transfer from a tryptophan in a hydrophilic region of the epsilon subunit to ANM bound to the epsilon subunit but not to the gamma subunit has been observed. The significance of these observations is discussed with respect to the structure of the gamma subunit and its role in conformational transitions within CF1 that occur during energization of the membrane.     

Acid-induced phosphorylation of adenosine 5'-diphosphate bound to coupling factor 1 in spinach chloroplast thylakoids.

Adenosine 5'-diphosphate, bound to coupling factor 1 (CF1) in spinach chloroplast thylakoids, is in part converted to adenosine 5'-triphosphate, upon injection of the thylakoids into strong acids in the dark. Bound phosphate serves as the phosphoryl donor for this uncoupler-insensitive conversion. Exposure of the thylakoids to heat or to urea prior to their injection into acid caused dissociation of ADP and prevents the apparent acid-induced synthesis of ATP. Conformational changes in CF1 may be elicited by acid denaturation which resemble those brought about by the proton electrochemical gradient across thylakoid membranes.     

Delta subunit of chloroplast coupling factor 1 inhibits proton leakage through coupling factor O

The ATP synthase of chloroplasts consists of a proton-conducting portion, CF0, and a catalytic portion, CF1. The smaller subunits of CF1, in particular delta, may play a key role in the coupling of proton transport to ATP synthesis. Purified subunit delta, when added to partially CF1-depleted thylakoid membranes, can restore photophosphorylation (Engelbrecht, S., and Junge, W. (1987) Eur. J. Biochem. 172, 213-218). We report here that it does so by blocking proton conduction through CF0. Thylakoids were CF1-depleted by incubation in hypoosmolar NaCl/EDTA solutions. Variation of the NaCl concentrations and of the incubation times not only changed the overall degree of CF1 depletion but also the subunit composition of solubilized CF1, namely CF1 containing delta and CF1(-delta). This was quantified by immunoelectrophoresis and by fast protein liquid chromatography. Proton conduction was measured by flash spectrophotometry by using standard electrochromic and pH-indicating absorption changes. The removal of integral CF1 was correlated with high electric conductance of thylakoid membranes, an increased extent of rapid proton leakage, and loss of ATP synthesis activity, which exceeded the percentual loss of CF1. The removal of predominantly CF1(-delta) resulted in comparatively lesser effects on the loss of ATP synthesis and on the extent and velocity of proton leakage. On the same line, addition of integral CF1 and of purified delta diminished the electric leak in CF1-depleted thylakoids. Both approaches, the controlled removal of CF1 and CF1(-delta), respectively, and addition of delta and CF1 showed that delta can act as a "stopcock" to the exposed proton channel CF0.     

Reconstitution of photophosphorylation in EDTA-treated thylakoids by added chloroplast coupling factor 1 (ATPase) and chloroplast coupling factor 1 lacking the delta subunit. Structural or functional?

Upon EDTA treatment thylakoids lose the chloroplast coupling factor 1 (CF1) part of their ATP synthase, CF0CF1, this exposes the proton channel, CF0. The previously established ability of the CF1 subunit delta to block the proton leak through CF0 prompted us to study (a) the ability of complete CF1 and, for comparison, CF1 lacking the delta subunit to block proton leakage and thereby to reconstitute structurally some photophosphorylation activity of the remaining CF0CF1 molecules and (b) their ability to form functional enzymes (functional reconstitution). In order to discriminate between activities caused by added CF1 or CF1(-delta) and remaining CF0CF1, the former were inhibited by chemical modification of subunit beta by N,N'-dicyclohexyl carbodiimide (DCCD) and the latter by tentoxin. We found that added CF1 acted both structurally and functionally while added DCCD-treated CF1 (DCCD-CF1) acted only structurally. In contrast to previous observations, CF1(-delta) and DCCD-CF1(-delta) also acted structurally although the reduction of proton leakage was smaller than with DCCD-CF1. Hence there was no functional reconstitution without subunit delta present. Previous studies indicated that only a small fraction of exposed CF0 is highly conducting and that this small fraction is distinguished by its high affinity for added CF1. The results of this study point rather to a wider distribution of CF0 conductance states and binding affinities.     

Quantitative aspects of adenosine triphosphate-driven proton translocation in spinach chloroplast thylakoids

After illumination in the presence of dithiothreitol, chloroplast thylakoids catalyze ATP hydrolysis and an exchange between ATP and Pi in the dark. ATP hydrolysis is linked to inward proton translocation. The relationships between ATP hydrolysis, ATP-Pi exchange, and proton translocation during the steady state were examined. The internal proton concentration was found to be proportional to the rate of ATP hydrolysis when these parameters were varied by procedures that do not alter the proton permeability of the thylakoid membranes. A linear relationship between the internal proton concentration and the rate of nonphosphorylating electron flow was previously verified. By determining the constant relating internal proton concentration to both ATP hydrolysis and electron flow, the proton/ATP ratio for the chloroplast ATPase complex was calculated to be 3.4 +/- 0.3. The presence of Pi, which allows ATP-Pi exchange to occur, lowers the internal proton concentration, but does not alter the relationship between the net rate of ATP hydrolysis and internal proton concentration. ATP-Pi exchange shows a dependence on the proton activity gradient very similar to that of ATP synthesis in the light. These results suggest that ATP-Pi exchange resembles photophosphorylation. In agreement with this idea, it is nucleoside diphosphate from the medium that is phosphorylated during exchange. Moreover, the energy-linked incorporation of Pi and ADP into ATP during exchange occurs at a similar rate. Thus, ATP synthesis from medium ADP and Pi takes place at the expense of the pH gradient generated by ATP hydrolysis.     

Lack of site-specificity of spinach chloroplast coupling factor 1

The irreversible inhibition of chloroplast phosphorylation by either sulfate anions, or N-ethylmaleimide, is energy dependent. Chloroplasts must first be illuminated in the presence of the inhibitors and a mediator of electron flow, for the subsequent phosphorylation to show any inhibition. Both inhibitors affect the chloroplast coupling factor 1.Electron transport only through Photosystem I can be used to activate either of these inhibitions. The subsequent inhibition in a second light reaction is the same whether ATP synthesis is supported by Photosystem I, or by Photosystem II electron transport. The reverse experiment, activating inhibition by electron transport only through Photosystem II, is possible in the case of sulfate. Again, the inhibition is expressed whether Photosystem II or Photosystem I electron flow supports ATP synthesis. We conclude that the two electron transport regions probably generate the same high energy state which is able to activate all members of a functionally uniform coupling factor population. These enzyme molecules must catalyze phosphorylation coupled to electron transport through either region of the chain. The results tend to discredit models requiring a separate group of coupling factor molecules unique to each part of the chain.     

Crystallization of coupling factor 1 (CF1) from spinach chloroplast.

Spinach chloroplast coupling factor (CF₁) was crystal-lized at 20°C from 0.05 M TRIS-PO₄, containing 4 mM ATP, 15mM KCl, 1.0 mM EDTA and 1.80 M (NH₄)₂SO₄, at pH 7.8. Some unit cell parameters were determined by electron microscopy and by X-ray diffraction. The cube shaped crystals have a tetragonal lattice, a = b = 135 Å, c = 280 Å with eight molecules per unit cell; possible space group P422 or P4₂2₁2, hence half a molecule in the asymmetric unit. Crystals grown at pH 7.5 in the absence of ATP have an orthorhombic lattice, a = 125 Å, b = 145 Å, c = 169 Å (C222₁), eight molecules per unit cell.     

Tentoxin-induced binding of adenine nucleotides to soluble spinach chloroplast coupling factor 1.

The effect of tentoxin on the binding of adenine nucleotides to soluble chloroplast coupling factor (CF1) has been studied and the following results have been obtained: 1. Tentoxin (400 micron) increases the maximum attainable tight binding of ADP to CF1. In the absence of tentoxin, the maximal binding observed by the method employed is about 0.3 nmol ADP/mg protein, whereas in the presence of tentoxin this ranges from 1.5 to 2.0 nmol ADP/mg protein. 2. Tentoxin-induced binding of ADP to CF1 is severely inhibited by divalent cations (50% inhibition at about 2 mM) but only weakly inhibited by monovalent cations (less than 50% inhibition at 100 mM). 3. The binding of ADP to CF1 induced by tentoxin is inhibited by ATP and adenylyl imidodiphosphate but is not inhibited by other nucleotides including AMP, GDP, CDP, IDP, or beta, gamma-methylene ATP. 4. The ADP-CF1 complex induced by tentoxin is quite stable. 75% remains bound to CF1 even after passage of the complex through a gel filtration column. An additional 25% can be removed by incubation in the presence of ADP, and all of the bound ADP can be removed only after incubation in the presence of both tentoxin and ADP. The latter result is interpreted as a tentoxin-induced exchange of bound ADP for medium ADP.     

Isolation and composition of the subunits of spinach chloroplast coupling factor protein.

A more convenient method for preparing large amounts of spinach chloroplast coupling factor is described, in which centrifugation of the EDTA-extracted chloroplasts is replaced by batchwise adsorption on DEAE-cellulose followed by filtration through Miracloth. Methods have been developed to purify the subunits from coupling factor dissociated by sodium dodecyl sulfate, involving hydroxylapatite chromatography followed by gel filtration with the detergent still present. The amino acid composition of the subunits purified by these methods was determined, with some differences noted in values for cysteine, tyrosine, phenylalanine, and methionine compared to previously published values. The stoichiometry of the subunits was estimated as 2:2:1:1:2 from their relative adsorption of dye after gel electrophoresis, compared to dye adsorbed by known amounts of the purified subunits. Estimates of subunit stoichiometry are rounded off to nearest whole numbers; actual preparations of coupling factor usually show less than complete amounts of the two smallest subunits.     

Modifications of the gamma subunit of chloroplast coupling factor 1 alter interactions with the inhibitory epsilon subunit.

The treatment of chloroplast coupling factor 1 (CF1) with dithiothreitol or with trypsin modifies the gamma subunit. Reduction of the gamma subunit disulfide bond in CF1 in solution with dithiothreitol enhances the dissociation of epsilon (Duhe, R. J., and Selman, B. R. (1990) Biochim. Biophys. Acta 1017, 70-78). The Ca(2+)-ATPase activity of either oxidized or reduced CF1 increases as the enzyme is diluted. Added epsilon subunit inhibits the Ca(2+)-ATPase activity of both forms of the diluted CF1, suggesting that epsilon dissociation is the cause of activation by dilution. Half-maximal activation occurred at much higher concentrations of the reduced CF1, indicating that reduction decreases the affinity for epsilon about 20-fold. Immunoblotting techniques show that there is only one epsilon subunit/CF1 in intact chloroplasts, in thylakoid membranes, and in solution. No epsilon is released from CF1 in thylakoids under conditions of ATP synthesis. The gamma subunit of CF1 in illuminated thylakoids is specifically cleaved by trypsin. CF1 purified from thylakoids treated with trypsin in the light is deficient in epsilon subunit, and has a high rate of ATP hydrolysis. Added epsilon neither inhibits the ATPase activity of, nor binds tightly to the cleaved enzyme.     

Selective modification of an alpha subunit of chloroplast coupling factor 1

Lucifer yellow (4-amino-N-[3-(vinylsulfonyl)phenyl]naphthalimide-3,6-disulf onate), a fluorescent probe that can react covalently with sulfhydryl or amino groups, has been used to modify chloroplast coupling factor 1 (CF1). Conditions are described under which Lucifer yellow selectively labels the alpha subunit of CF1 to the extent of about 1 mol of probe per mole of CF1. An especially reactive amino group is apparently labeled, and modification has little effect on the ATPase activity of the enzyme. Lucifer yellow is a useful probe for fluorescence energy transfer measurements. The distances between this probe and fluorescent and absorbing molecules attached to seven specific sites on the beta, gamma, and epsilon subunits were determined. These distances converge to a single location. In addition to providing further information about the structure of CF1, these results suggest that the alpha subunits of CF1 are not structurally equivalent.     

Reversibility of the cyanide inhibition of electron transport in spinach chloroplast thylakoids

The prior treatment of thylakoids with cyanide (30 mM) was shown to inhibit plastocyanin-dependent electron transport reactions. We find that cyanide inhibition of electron flow from either water or diaminodurene to methyl viologen, but not from water to ferricyanide, is partially reversed when the thylakoids are collected by centrifugation and resuspended in a cyanide-free medium. However, methyl viologen reduction in thylakoids pretreated with cyanide is sensitive to cyanide (～1 mM) added to the reaction mixtures, whereas that in control thylakoids is unaffected. The cyanide must be added in the dark. Electron transport to methyl viologen in chloroplasts pretreated with cyanide is also sensitive to inhibition by EDTA and bathocuproine sulfonate. Thus, KCN inhibition of electron transport in thylakoids is partially reversible. Moreover, the accessibility of plastocyanin to various reagents is probably altered by the KCN treatment.     

Superoxide anion permeability of phospholipid membranes and chloroplast thylakoids

The permeability of phospholipid membranes to the superoxide anion (O₂^?) was determined using soybean phospholipid vesicles containing FMN in the internal space. The efflux of O₂^? generated by the illumination of FMN was so slow that more than 90% of the radicals were spontaneously disproportionated within the vesicles before they could react with cytochrome c at the membrane exterior. The amount of diffused O₂^? was proportional to the intravesicular concentration of O₂^? over a range from 1 to 10 μm which was deduced from its disproportionation rate. The permeability coefficient of the phospholipid bilayer for O₂^? was estimated to be 2.1 × 10^?6 cm s^?1 at pH 7.3 and 25 ° C. Superoxide dismutase trapped inside vesicles was not reactive with extravesicular O₂^? unless Triton X-100 was added. O₂^? generated outside spinach chloroplast thylakoids did not interact with superoxide dismutase or cytochrome c which had been enclosed in the thylakoids. Thus, chloroplast thylakoids also showed little permeability to O₂^?.