Specificity of nucleotide binding sites in isolated chloroplast coupling factor (CF1)

The binding of various nucleotides to chloroplast coupling factor CF₁ was studied by two dialysis techniques. It was found that the number of nucleoside diphosphate sites and their specificities for the base moiety is dependent on the magnesium concentration. In the presence of 50 μM added MgCl₂, the protein has a single strong site/mol protein with K_d = 0.5 μM for ADP and high specificity (K_d > 20 μM for ?ADP, GDP, CDP). In the presence of 5 mM MgCl₂, the protein has two independent tight ADP sites (K_d = 0.4 μM) of low specificity (K_d ≈ 0.8, 2, and 2 μrmM, respectively for ?ADP, GDP, and CDP). These results are compared with the specificity of the partial reactions for photophosphorylation.     

Characterization of nucleotide binding sites on the membrane-bound chloroplast ATP synthase (coupling factor CF1)

Phosphorylation of ADP and nucleotide exchange by membrane-bound coupling factor CF₁ are very fast reactions in the light, so that a direct comparison of both reactions is difficult. By adding substrate ADP and phosphate to illuminated thylakoids together with the uncoupler FCCP, the phosphorylation time is limited and the amount of ATP formed can be reduced to less than 1 ATP per enzyme. Low concentrations of medium nucleotides during illumination increase the amount of ATP formed during uncoupling presumably by binding to the tight nucleotide binding site (further designated as ‘site A’) with an affinity of 1 to 7 μM for ADP and ATP. ATP formation itself shows half-saturation at about 30 μM. Loosely bound nucleotides are exchanged upon addition of nucleotides with uncoupler (Schumann, J. (1984) Biochim. Biophys. Acta 766, 334–342). Release depends binding of nucleotides to a second site. The affinity of this site for ADP (in the presence of phosphate) is about 30 μM. It is assumed that phosphorylation and induction of exchange both occur on the same site (site B). During ATP hydrolysis, an ATP molecule is bound to site A, while on another site, ATP is hydrolyzed rapidly. The affinity of ADP for the catalytic site (70 μM) is in the same range as the observed Michaelis constant of ADP during phosphorylation; it is assumed that site B is involved in ATP hydrolysis. Site A exhibits some catalytic activity; it might be that site A is involved in ATP formation in a dual-site mechanism. For ATP hydrolysis, however, direct determination of exchange rates showed that the exchange rate of ATP bound to site A is about 30-times lower than ATP hydrolysis under the same conditions.     

Properties of the solvent-stimulated ATPase activity of chloroplast coupling factor 1 from Chlamydomonas reinhardii

The effects of solvents on the ATPase activity of chloroplast coupling factor 1 (CF₁) isolated from wild-type Chlamydomonas reinhardii have been studied. Of the solvents examined, the following order summarizes their maximal ability to stimulate the ATPase activity of CF₁: ethanol > methanol>allyl alcohol >n-propanol > acetone≈dioxane > ethylene glycol. Glycerol inhibits the CF₁ activity at all concentrations. In the absence of organic solvents, 50% of the activity of the enzyme is irreversibly lost after a 10 min incubation at 65–70°C. Ethanol (23%) causes a 30°C drop in the temperature required for 50% inactivation. ATP partially stabilizes the CF₁ in the presence, but not in the absence, of ethanol. In the absence of organic solvents, both free Mg²⁺ and ADP inhibit the CF₁-ATPase. Mg²⁺ is a noncompetitive inhibitor with respect to MgATP, and the kinetic constants are: V, 6.3 μmol ATP hydrolyzed/mg protein per min; K_m(MgATP), 0.23 mM; K_ii(Mg²⁺), 27 μM; and K_is(Mg²⁺), 50 μM. In the presence of ethanol, double-reciprocal plots are no longer linear and have a Hill coefficient of about 1.8±0.1. V increases about 10–12-fold. The pattern of inhibition by Mg²⁺ appears to change from noncompetitive to competitive with respect to MgATP. In addition, ADP no longer inhibits the MgATPase activity of CF₁.     

The activation and inactivation of the Dunaliella salina chloroplast coupling factor 1 (CF1) in vivo and in situ

Preillumination of intact cells of the eukaryotic, halotolerant, cell-wall-less green alga Dunaliella salina induces a dark ATPase activity the magnitude of which is about 3–5-fold higher than the ATPase activity observed in dark-adapted cells. The light-induced activity arises from the activation and stabilization in vivo of chloroplast coupling factor 1 (CF₁). This activity, 150–300 μmol ATP hydrolyzed/mg Chl per h, rapidly decays (with a half-time of about 6 min at room temperature) in intact cells but only slowly decays (with a half-time of about 45 min at room temperature) if the cells are lysed by osmotic shock immediately after illumination. The activated form of the ATPase in lysed cells is inhibited if the membranes are treated with ferri- but not ferrocyanide, suggesting that the stabilization of the activated form of CF₁ is due to the reduction of the enzyme in vivo in the light.     

N-Ethylmaleimide inhibition of the catalytic activities of the Dunaliella salina coupling factor 1 (CF1) and the restoration of the inhibition of the CF1 ATPase activity by N-ethylmaleimide

The sensitivity of the catalytic activities of the D. salina chloroplast coupling factor 1 (CF₁) to chemical modification by N-ethylmaleimide has been investigated. (i) When D. salina thylakoid membranes are treated with N-ethylmaleimide, both photophosphorylation and the inducible CF₁ ATPase activity are partially (approx. 60%) inhibited. The inhibition of both activities does not require the presence of a proton-motive force, and the inhibition of photophosphorylation is directly related to the N-ethylmaleimide-covalent modification of CF₁ as shown by (a) the time-course for the inhibition and (b) the maximal extent of inhibition. (ii) Treatment of the purified, latent, D. salina CF₁ with low concentrations of N-ethylmaleimide also results in the partial (approx. 60%) inhibition of the inducible ATPase activity (I₅₀ ≈ 50 μM). The inhibition does not require the presence of the chemical modifier during the activation of the enzyme. (iii) N-ethylmaleimide-induced inhibition of the ATPase activity of either membrane-bound or solubilized CF₁ is partially reversed by either (a) prolonged incubation at low concentrations of N-ethylmaleimide or (b) short incubation times at high concentrations of N-ethylmaleimide. The results are interpreted as indicating multiple binding sites on the D. salina CF₁ that have different rates of reactivity with N-ethylmaleimide. Those sites (or site) that react rapidly with N-ethylmaleimide cause(s) an inhibition of both ATP synthase and ATPase activities, whereas those sites (or site) that react more slowly partially restore(s) the original-ATPase activity. The effects of N-ethylmaleimide on the catalytic activity of D. salina CF₁ are probably mediated by N-ethylmaleimide-induced conformational changes of the enzyme.     

Tentoxin An uncompetitive inhibitor of lettuce chloroplast coupling factor 1

The interaction of tentoxin $\text{[cyclo}\text{(-}\text{l}\text{-leucyl}\text{-N-}\text{methyl-(Z)-dehydrophenyl-analyl-glycyl}\text{-N-}\text{methyl-}\text{l}\text{-alanyl-)}\text{]}$ with solubilized lettuce chloroplast coupling factor 1 was characterized by direct binding studies, measurement of the time course of ATPase inhibition, and steady-state enzyme kinetics. Neither substrates, products or Ca²⁺ competed with the tentoxin binding site, nor did they induce any large change in tentoxin affinity. The inhibition of lettuce chloroplast coupling factor 1 ATPase was found to be the time dependent, and at equilibrium the affinities estimated by equilibrium ultrafiltration and enzyme inhibition were similar (1.8 · 10⁸M^?1). The steady-state kinetics best fit an uncompetitive pattern suggesting that the inhibited steps follow an irreversible step occurring after ATP binding.     

Kinetics of nucleotide binding to chloroplast coupling factor (CF1)

Studies of the kinetics of association and dissociation of the formycin nucleotides FTP and FDP with CF₁ were carried out using the enhancement of formycin fluorescence. The protein used, derived from lettuce chloroplasts by chloroform induced release, contains only 4 types of subunit and has a molecular weight of 280 000.In the presence of 1.25 mM MgCl₂, 1 mol of ATP or FTP is bound to the latent enzyme, with K_d = 10^?7 or 2 · 10^?7, respectively. The fluorescence emission (λ_max 340 nm) of FTP is enhanced 3-fold upon binding, and polarization of fluorescence is markedly increased. The fluorescence changes have been used to follow FTP binding, which behaves as a bimolecular process with K₁ = 2.4 · 10⁴ M^?1 · s^?1. FTP is displaced by ATP in a process apparently involving unimolecular dissociation of FTP with k_?1 = 3 · 10^?3 s^?1. The ratio of rates is comparable to the equilibrium constant and no additional steps have been observed.The protein has 3 sites for ADP binding. Rates of ADP binding are similar in magnitude to those for FTP. ADP and ATP sites are at least partly competitive with one another.The kinetics of nucleotide binding are strikingly altered upon activation of the protein as an ATPase. The rate of FTP binding increases to at least 10⁶ M^?1 · s^?1. This suggests that activation involves lowering of the kinetic barriers to substrate and product binding-dissociation and has implications for the mechanism of energy transduction in photophosphorylation.     

The interaction of phenylglyoxal with soluble and membrane-bound chloroplast coupling factor 1

The rate of inhibition of cyclic photophosphorylation in chloroplast thylakoids by the arginine reagent phenylglyoxal was enhanced in the light, i.e., under conditions where membrane energization occurred. Uncouplers, but not energy-transfer inhibitors, prevented the effect of light. Chemical modification of chloroplast thylakoids by phenylglyoxal under dark or in light conditions affected differently the light-induced exchange of tightly bound ADP. In both cases the exchange was less inhibited than photophosphorylation. Complete inhibition of ATPase activity of soluble CF₁ was correlated with the incorporation of 8 mol [¹⁴C]phenylglyoxal per mol enzyme. About 50% of the incorporated radioactivity was lost at different rates depending on the buffer present and suggesting a change in the stoichiometry of the adduct from 2:1 to 1:1. Inhibition of ATPase and photophosphorylating activities of chloroplasts by modification with [¹⁴C]phenylglyoxal in the dark was associated with the incorporation of 1 and 2 mol reagent per mol membrane-bound CF₁, respectively. In the light the rate of incorporation was enhanced and both reactions were inactivated when 2 mol $\text{[}{}^{14}\text{C]phenylglyoxal}\text{CF}{}_1$ were bound. In all the labelling experiments the radioactivity was mainly recovered from the α- and β-subunits.     

Disposition of water in chloroplast coupling factor 1 (CF1) A neutron scattering analysis

Small-angle neutron scattering experiments were performed in dilute aqueous solutions of chloroplast F₁-ATPase. By contrast variation in ¹H₂O/²H₂O mixtures and when using different concentrations of glycerol in ²H₂O, structural information on the spatial distribution of dry protein and water was obtained. The maximum distance within latent and active CF₁ was 12 nm. the shape of CF₁ was globular. The total volume of CF₁ was 900 nm³, and its dry volume (excluding the volume of one water molecule per two exchangeable hydrogen atoms) was 400 nm³. A volume of 670 nm³ was inaccessible to glycerol at low glycerol concentrations (less than 25%). At higher concentrations (up to 50%) a volume of 460 nm³ was excluded to glycerol. Within the resolution of our experiment (1.6 nm) there was no evidence for particular water-rich regions or of secluded water spaces or any particular places for glycerol exchange. Upon thiol activation of the latent enzyme only small changes in structure were detectable just at the limits of the experimental error. They suggest an enhancement of the surface roughness.     

Inhibition of chloroplast coupling factor by naphthylglyoxal

The trypsin-activated Ca²⁺ -ATPase of spinach chloroplast membranes was completely inhibited by treatment with naphthylglyoxal, a fluorescent compound that should bind covalently to arginine residues. The inhibition followed apparent first-order kinetics. The apparent order of reaction with respect to inhibitor concentration gave values near unity, suggesting that inactivation is a consequence of modifying one arginine residue per active site. Partial protection against naphthylglyoxal was afforded by ADP and ATP, with either less or no protection by other nucleotide bases. At inhibition levels less than complete, the K_m for ATP was not affected but the V_max of the enzyme was diminished. The light-dependent exchange of tightly bound nucleotides on the membrane-bound enzyme was not inhibited by naphthylglyoxal treatment, indicating significant retention of the conformational response of the enzyme to the membrane high-energy state. Using [³H]naphthylglyoxal, the extent of inhibition was a linear function of the amount of naphthylglyoxal bound up to 60% inhibition. The curves extrapolated to 2 mol naphthylglyoxal bound, associated with complete inhibition of ATPase. The radioactive naphthylglyoxal was distributed equally between α- and β-subunits.     

The activation and inactivation of the Dunaliella salina chloroplast coupling factor 1 (CF1) in vivo and in situ

Preillumination of intact cells of the eukaryotic, halotolerant, cell-wall-less green alga Dunaliella salina induces a dark ATPase activity the magnitude of which is about 3–5-fold higher than the ATPase activity observed in dark-adapted cells. The light-induced activity arises from the activation and stabilization in vivo of chloroplast coupling factor 1 (CF₁). This activity, 150–300 μmol ATP hydrolyzed/mg Chl per h, rapidly decays (with a half-time of about 6 min at room temperature) in intact cells but only slowly decays (with a half-time of about 45 min at room temperature) if the cells are lysed by osmotic shock immediately after illumination. The activated form of the ATPase in lysed cells is inhibited if the membranes are treated with ferri- but not ferrocyanide, suggesting that the stabilization of the activated form of CF₁ is due to the reduction of the enzyme in vivo in the light.     

Energy-dependent exchange of adenine nucleotides on chloroplast coupling factor (CF1)

1. [¹⁴C]ADP is incorporated into washed broken chloroplasts in the light. The bound labelled nucleotides which cannot be removed by washing are almost exclusively related to coupling factor CF₁. [¹⁴C]ADP binding exhibits a monophasic concentration curve with a K_m of 2 μM.2. By illumination of the chloroplasts, previously incorporated labelled nucleotides are released. A fast release is obtained in the presence of unlabelled ADP and ATP, indicating an energy-dependent exchange. A slow and incomplete release is induced by light in the absence of unlabelled adenine nucleotides. Obviously, under those conditions, an adenine nucleotide depleted CF₁ conformation is established.3. Re-binding of [¹⁴C]ADP by depleted membranes is an energy-independent process. Even after solubilization of adenylate-depleted CF₁, [¹⁴C]ADP is incorporated into the protein. By re-binding of ADP in the dark, CF₁ is converted to a non-exchangeable form.4. Energy-dependent adenine nucleotide exchange on CF₁ is suggested to include three different conformational states of the enzyme: (1) a stable, non-exchangeable form which contains firmly bound nucleotides, is converted to (2), an unstable form containing loosely bound adenine nucleotides. This conformation allows adenylate exchange; it is in equilibrium with (3) a metastable, adenylate-depleted form. The transition from state (1) to state (2) is the energy-requiring step.     

The partial purification of a factor from Dunaliella salina that causes the rapid in situ inactivation of light-activated chloroplast coupling factor 1 (CF1)

A factor having the expected properties of the in vivo oxidant responsible for inactivating the in vivo light-activated chloroplast coupling factor 1 (CF₁) has been partially purified from cell-free extracts of Dunaliella salina. This factor is highly polar, weakly acidic, and relatively temperature stable. The ability of this factor to inactivate light-activated CF₁ is prevented if it is pretreated with reductants such as dithiothreitol. The factor has virtually no effect on the ethanol-induced, Mg²⁺ -dependent ATPase activity of the isolated CF₁.     

Light/dark labeling differences in chloroplast membrane polypeptides associated with chloroplast coupling factor 0

The fluorogenic reagent fluorescamine has been used to determine the labeling patterns of Type C spinach chloroplast membrane polypeptides. Membrane polypeptides labeled with fluorescamine were detected by scanning high resolution sodium dodecyl sulfate polyacrylamide gradient slab gels for fluorescence emission.Three membrane polypeptides show a decrease in the extent of labeling when chloroplast membranes are labeled in the light compared to when they are labeled in the dark. These polypeptides have apparent molecular weights of 32 000, 23 000 and 15 000.The decrease in labeling observed in the light is abolished or reduced by treatments which inactivate the light-generated transmembrane pH gradient. CF₁-depleted chloroplasts show neither a light-activated pH gradient nor a light/dark difference in labeling of these three polypeptides. Both a light-activated pH gradient and light/dark differences in labeling are observed in CF₁-depleted chloroplasts which have been treated with N,N′-dicyclohexylcarbodiimide.The same ammonium sulfate fractions of a 2% sodium cholate extract, which are believed to be enriched in the membrane-bound sector of the chloroplast ATPase (CF_o) are also found to be enriched in the 32 000, 23 000 and 15 000 molecular weight polypeptides. The three polypeptides are believed to be components of CF_o, and the light/dark labeling differences may indicate conformational changes within CF_o. Such conformational changes may reflect a mechanism which couples light-generated proton gradients to ATP synthesis.     

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.     

Cation requirement for the reconstitution of oligomycin-sensitive ATPase by means of soluble F1-ATPase and F1-depleted submitochondrial particles

Bovine heart submitochondrial particles depleted of F₁ by treatment with urea (‘F₁-depleted particles’) were incubated with soluble F₁-ATPase. The binding of F₁ to the particles and the concomitant conferral of oligomycin sensitivity on the ATPase activity required the presence of cations in the incubation medium. NH⁺₄, K⁺, Rb⁺, Cs⁺, Na⁺ and Li⁺ promoted reconstitution maximally at 40–74 mM, guanidinium⁺ and Tris⁺ at 20–30 mM, and Ca²⁺ and Mg²⁺ at 3–5 mM. The particles exhibited a negative ζ-potential, as determined by microelectrophoresis, and this was neutralized by mono- and divalent cations in the same concentration range as that needed to promote F₁ binding and reconstitution of oligomycin-sensitive ATPase. It is concluded that the cations act by neutralizing negative charges on the membrane surface, mainly negatively charged phospholipids. These results are discussed in relation to earlier findings reported in the literature with F₁-depleted thylakoid membranes and with submitochondrial particles depleted of both F₁ and the coupling proteins F₆ and oligomycin sensitivity-conferring protein.     

The binding of eosin-labeled subunit δ to the isolated chloroplast ATPase, CF1, as revealed by rotational diffusion in solution

We investigated the binding of subunit δ to solubilized chloroplast ATPase. Purified δ was covalently labeled with eosin 5-isothiocyanate and its rotational correlation time was determined by a photoselection technique as a function of added CF₁ (containing δ) and of CF₁(−δ) (lacking δ). In aqueous buffer the rotational correlation time of labeled δ was 33 ns. This is compatible with a rather elongated shape with the dimensions 2b = 100 Å/2a = 28 Å. Binding of δ to CF₁ decreased the rotational correlation time about 10-fold. The result was a biphasic decay of the laser flash-induced absorption anisotropy which was analyzed to yield the proportion of δ (bound to CF₁) relative to δ (free). CF₁(−δ), which completely lacked the δ-subunit, bound one δ (mol/mol) with high affinity (K_d ≈ 100 nM) and at least another δ with about 20-fold lower affinity. The δ-containing CF₁, revealed only the low-affinity site(s) for δ. This was compatible with a 1:1 stoichiometry of δ in isolated CF₁.     

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.