Sulphydryl groups in photosynthetic energy conservation. IV. Inhibition of the ATPase of chloroplast coupling factor 1 by sulphydryl reagents

1. o-Iodosobenzoate and 2,2′-dithio bis-(5-nitropyridine) inhibited by about fifty per cent the ATPase activity of heat-activated chloroplast coupling factor 1 only when present during the heating but were without effect when added before or after the activation. Reversion of this inhibition was only obtained by a second heat treatment with 10 mM dithioerythritol.2. The inhibition of the Ca²⁺-ATPase of coupling factor 1 by o-iodosobenzoate or 2,2′-dithio bis-(5-nitropyridine) was not additive with similar inhibitions obtained with the alkylating reagents iodoacetamide and N-ethylmaleimide.3. The heat-activated ATPase of o-iodosobenzoate-treated coupling factor 1 had a higher K_m for ATP, without modification of V. The modified enzyme was desensitized against the allosteric inhibitor ADP.     

Sulfhydryl groups in photosynthetic energy conservation. VI. Subunit distribution of sulfhydryl groups and disulfide bonds in chloroplast coupling factor and ATPase activity

The subunit distribution of sulfhydryl groups and disulfide bonds of spinach chloroplasts coupling factor I has been determined. Native coupling factor I with a latent ATPase activity has eight sulfhydryl groups distributed 4 : 2 : 0 : 0 : 2 in the alpha, beta, gamma, delta and epsilon subunits, respectively. Heat treatment of coupling factor I, in addition to the activation of its ATPase activity, induces a dithiol-disulfide interchange between the gamma and the alpha subunit, changing the sulfhydryl groups' distribution to 2 : 2 : 2 : 0 : 2. Reduction of disulfide bonds of coupling factor I by dithioerythritol during heat treatment gives a subunit distribution of 4 : 4 : 4 : 0 : 2, suggesting that native coupling factor I has three disulfide bonds, two in the gamma subunit and one in one of the beta subunits. The results suggest an asymmetric redox state of some of the subunits of coupling factor I and an asymmetric positioning of some of them in the molecular structure of coupling factor I.     

Sulphydryl groups in photosynthetic energy conservation III. Inhibition of photophosphorylation in spinach chloroplasts by CdCl2

Low concentrations of CdCl₂ inhibited cyclic and non-cyclic photophosphorylation in spinach chloroplasts and depressed coupled electron transport to the basal level. The basal electron transport and the electron flow stimulated by several uncouplers, except arsenate, were not affected. The inhibitory effects of cadmium were reversed by dithiols like dithioerythritol or 2,3-dimercaptopropanol. The light-induced pH rise of chloroplast suspensions and the trypsin-activated Ca-ATPase were not affected by cadmium at the concentrations that inhibited ATP synthesis but the latter was affected by higher concentrations.     

Sulphydryl groups in photosynthetic energy conservation. V. Localization of the new disulfide bridges formed by o-iodosobenzoate in coupling factor of spinach chloroplasts.

1. Chemical modification by o-iodosobenzoate of soluble chloroplast coupling factor 1 (CF1) during heat activation resulted in inhibition of its Ca-ATPase activity and in the formation of two new intrapeptide disulfide bridges as suggested by: (a) the disappearance of three out of four accessible thiol groups, two from gamma and one from a beta subunit as a consequence of CF1 modification by o-iodosobenzoate; (b) the total free sulphydryl groups of CF1 were reduced from 8 to 4 after modification of CF1 by o-iodosobenzoate. Two groups disappeared from beta and two from gamma subunits; (c) a second heating step of CF1 in the presence of 10 mM dithioerythritol reversed the inhibition of the ATPase and reduced both the newly formed disulfide bridges and those present in native CF1. 2. Modification of chloroplasts in the light with o-iodosobenzoate resulted in the inhibition of photophosphorylation and ATPase. CF1 isolated and purified from these chloroplasts had its Ca-ATPase activity inhibited and two new disulfide bridges. The total number of free sulphydryl groups was reduced from 8 to 4 and three accessible groups disappeared from beta and gamma subunits.     

Inhibition of chloroplast coupling factor ATPase by 5'-p-fluorosulfonylbenzoyl adenosine.

Incubation of chloroplast coupling factor with 5′-p-fluorosulfonylbenzoyl adenosine in the 1 to 2 mM range inhibits subsequently measured ATPase activity. The inhibition is probably due to covalent binding since it survives ammonium sulfate fractionation and dialysis. The kinetics of the inhibited enzyme with respect to substrate show a decrease in V_max with no change in K_m for ATP. The presence of ATP or ADP together with the inhibitor provides some protection against inhibition. The results suggest a possible covalent attack at a nucleotide binding site, leading to inhibition of activity.     

Sulphydryl groups in photosynthetic energy conservation. I. Light-dependent inhibition of photophosphorylation by the sulphydryl reagent 2-2'dithio bis-(5-nitropyridine).

1. The sulphydryl reagent 2-2'dithio bis-(5-nitropyridine) (DTNP) inhibited photophosphorylation when the chloroplasts were preincubated with the reagent in the light. A maximum inhibition of about 50% was obtained in the presence of pyocyanine and MgCl 2 at 0.3 mumol DTNP per mg chlorophyll and was completed in about 40 s of preillumination. 2. Dithioerythritol, ADP plus Pi (or arsenate) and uncouplers prevented the inhibition when present during the preillumination while phloridzin, Dio-9 and discarine B were ineffective. Low concentrations of ADP or ATP afforded partial protection but other nucleotides had no effect. 3. DTNP inhibited the coupled electron transport rate to the basal level and had no effect on the uncoupled electron transport. The stimulation of proton uptake and inhibition of electron transport by ATP was prevented by DTNP. 4. The trypsin-activated but not the light- and dithioerythritol-triggered ATPase was inhibited by light preincubation of chloroplasts with DTNP. 5. Reversal of DTNP inhibition of photophosphorylation was obtained by a second preillumination in the presence of thiol groups. 6. More DTNP reacted with chloroplasts in the light than in the dark. Two mol of thione were formed in the light per mol of DTNP disappeared. 7. The results suggested that DTNP inhibition is related to the oxidation by DTNP of chloroplast vicinal dithiols probably exposed by a light-induced conformational change.     

Involvement of sulfhydryl groups in the activation mechanism of the ATPase activity of chloroplast coupling factor 1

Activation of the ATPase activity and the exposition of a new adenine nucleotide binding site of chloroplast coupling factor 1 (CF1) by dithioerythritol at 25 degrees C were reversed by oxidants. The ATPase activity elicited by heat (63 degrees C, 4 min) was slightly inhibited by oxidants and was partially additive with the activity induced by dithioerythritol. Titration of the thiols of CF1 and determination of their subunit distribution before and after activation by dithioerythritol show an increase of the free groups from 8 to 10 with the appearance of the 2 new thiols on the gamma subunit. These thiols were available to reagents in nondenatured enzyme and were reoxidized to a disulfide bond by iodosobenzoate or CuCl2. It is concluded that the mechanisms of CF1 activation by dithioerythritol and by heat are different and that the former involves a net reduction of a disulfide bond of the gamma subunit.     

Tentoxin-induced energy-independent adenine nucleotide exchange and ATPase activity with chloroplast coupling factor 1.

1. The effect of energy transfer inhibitors on energy-dependent exchange of tightly bound adenine nucleotides with washed, broken spinach thylakoids has been studied. Energy transfer inhibitors that inhibit the ATPase activity of soluble chloroplast coupling factor 1 (CF1) (e.g. phloridzin and tentoxin) do not inhibit energy-dependent adenine nucleotide exchange. Energy transfer inhibitors that block proton flux through the hydrophobic protein proton channel (CF0) (e.g. dicyclohexylcarbodiimide and triphenyltin chloride) also block light-dependent adenine nucleotide exchange. 2. Tentoxin, at relatively high concentrations, stimulates an energy-independent exchange of adenosine diphosphate. 3. High concentrations of tentoxin elicit a Ca2+-dependent ATPase activity with soluble CF1, but has no effect on the Ca2+-dependent ATPase activity of membrane-bound CF1. 4. The trypsin-activated, Ca2+-dependent, membrane-bound ATPase is not affected by high concentrations of tentoxin, whereas the dithiothreitol-activated, Mg2+-dependent ATPase is markedly inhibited. 5. The reconstitution of chloroplasts, partially depleted in CF1, with soluble CF1 is correlated with the loss of tentoxin-induced, Ca2+-dependent ATPase activity associated with soluble CF1.     

Inhibition of membrane-bound chloroplast coupling factor 1 by a dephosphorylated derivative of dialdehyde ADP

Periodate-oxidized ADP, if left in aqueous solution, loses its phosphates by beta-elimination. This dephosphorylated dialdehyde compound caused rapid and irreversible inhibition of membrane-bound spinach chloroplast coupling factor 1 (CF1). Inhibition was 2.5 times faster in the light than in the dark. A high concentration of uncoupler eliminated the light stimulation. Light could be replaced by an acid-base transition. Therefore, the dialdehyde reacts with a site or sites on CF1 that become exposed by a high-energy state-induced conformational change. The substrate nucleotides ADP, ATP, GDP, and GTP protected against inhibition while Pi and the non-substrate nucleotides AMP, GMP, CTP, and UTP did not. The protection by GTP was competitive and magnesium-dependent, suggesting that the dialdehyde binds to a nucleotide-binding site. However, the corresponding UDP and CDP dialdehyde derivatives also inhibited CF1 and showed the light-stimulation effect, indicating that the adenine is not important for the binding. These derivatives could be binding to a nucleotide-binding site or to another reactive site that becomes exposed during the light-induced conformational change. In the latter case the protection by substrate nucleotides would be due to prevention of the energy-dependent conformational change.     

The stimulation of coupling factor 1 ATPase by tentoxin.

Tentoxin at 10--1000 micrometer causes a marked species-selective stimulation of coupling factor 1 Ca2+-dependent ATPase activity (Ka 6.3 . 10(3) M-1). This effect decreases the Km for ATP to about 0.3 mM and increases V 2.75-fold. Above 1.6 micrometer tentoxin the rate of coupled electron transport was reduced to basal without uncoupling.     

Fluorescence energy transfer between ligand binding sites on chloroplast coupling factor 1.

The method of fluorescence energy transfer is used to measure the distance from the tight nucleotide binding sites to the 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole reactive sites on solubilized spinach chloroplast coupling factor 1 (CF1). The fluorescent adenine nucleotide analogs 1,N-6-ethenoadenosine diphosphate and 1,N-6-ethenoadenylyl imidodiphosphate were used as donors and 4-nitrobenzo-2-oxa-1,3-diazole bound to a tyrosine group and to an amino group were used as acceptors of energy transfer. Using three different donor-acceptor pairs, the distance measured varied from 38 to 43 A assuming both donor sites are equidistant from the acceptor site. A model is proposed for the location of the tight nucleotide binding sites and the active site on the alpha and beta subunits of CF1.     

Mechanism of phosphorylation catalyzed by chloroplast coupling factor 1. Stereochemistry

The reaction mechanism and substrate specificity of soluble chloroplast coupling factor 1 (CF1) from spinach were determined by using the purified isomers of chromium-nucleotide complexes either as substrates for the enzyme or as inhibitors of the Ca2+-dependent ATPase activity. The isolation of CrADP( [32P]Pi) formed upon the addition of the enzyme to [32P]Pi and lambda-bidentate CrADP and the observation that the lambda-bidentate CrADP epimer was 20-fold more effective in inhibiting the Ca2+-dependent ATPase activity than was the delta epimer suggest that the substrate of phosphorylation catalyzed by CF1 is the lambda-bidentate metal ADP epimer. Tridentate CrATP was hydrolyzed by soluble CF1 to CrADP(Pi) at an initial rate of 3.2 mumol (mg of CF1)-1 min-1, indicating that the tridentate metal ATP is the substrate for ATP hydrolysis. From these results a mechanism for the phosphorylation of ADP catalyzed by coupling factor 1 is proposed whereby the bidentate metal ADP isomer associates with the enzyme, phosphate inserts into the coordination sphere of the metal, and the oxygen of the beta-phosphate of ADP attacks the inorganic phosphate by an SN2 type reaction. The resulting product is the tridentate ATP ligand.     

Chemical cross-linking studies of chloroplast coupling factor 1.

Cross-linking reagents have been used to link covalently adjacent subunits of solubilized spinach chloroplast coupling factor 1, which is a latent ATPase. 1,5-Difluoro-2,4-dinitrobenzene, dimethyl-3,3'-dithiobispropionimidate, and dimethylsuberimidate are able to form bridges of 3 to 11 A between amino groups, and hydrogen peroxide and the o-phenanthroline-cupric ion complex catalyze the oxidation of intrinsic sulfhydryl groups. The five individual subunit bands (alpha, beta, gamma, delta, and epsilon) and several new aggregate bands can be separated by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The same four fastest moving aggregate bands, as characterized by their mobilities, migrate more slowly than the heaviest subunit band and appear with all of the cross-linkers employed. The subunit composition of the aggregate bands has been determined through the use of the reversible cross-linkers, dimethyldithiobispropionimidate, (o-phenanthroline)2Cu(II), and H2O2, and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis in which aggregates are separated in the first dimension, the disulfide cross-links are cleaved, and the individual subunits present in the aggregates are separated in the second dimension. The subunits are detected by Coomassie brilliant blue staining and by labeling some of the sulfhydryl groups of the gamma and epsilon subunits with radioactive N-ethylmaleimide. The results obtained indicate that the alpha and beta subunits can cross-link directly with each of the other subunits, that two beta subunits are adjacent, and that gamma epsilon, gamma epsilon 2, alpha delta, and beta delta aggregates are present. A minimal subunit stoichiometry consistent with these results is alpha 2 beta 2 gamma delta epsilon 2. A possible structural model of the coupling factor is derived from the data. Similar, but less extensive, experiments have been carried out with the heat-activated coupling factor (which is an ATPase); no differences in the spatial arrangement of subunits are detected from the two-dimensional gel electrophoresis analysis of the cross-linked aggregates.     

Relations between divalent cation binding and ATPase activity in coupling factor from chloroplast.

Although 150 individual samples of milk from Italian water buffalo (Bubalus arnee) were examined by acid and alkaline gel electrophoresis, no polymorphism was observed for α-lactalbumin and β-lactoglobulin. After isolation and purification of these two proteins their amino acid compositions were determined and compared with those of the corresponding bovine proteins. The sequence alignments of 36 and 17 amino-acids from the N-terminal ends and 2 amino-acids from the C-terminal ends of buffalo α-lactalbumin and β-lactoglobulin, respectively, have been established. Our results indicate that buffalo α-lactalbumin differs from its cow B counterpart by a substitution Asn/Gly at position 17 and by another substitution, likely Glu/Gln or Asp/Asn, at an unknown position. Buffalo β-lactoglobulin is homologous to the bovine B variant. Three substitutions differentiate the two proteins: Ile/Leu and Val/Ile at positions 1 and 162 respectively; a further one, Gln/Ile, has not yet been located. According to these results the B variant of bovine β-lactoglobulin might be the wild type of the Bos genus.     

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.     

Synergistic activation of an Mg-specific ATPase activity in chloroplast coupling factor by octylglucoside and tentoxin

(1) Octylglucoside stimulates an Mg²⁺-specific ATPase activity with CF₁ preparations from different higher plants and the alga Chlamydomonas reinhardii. (2) Tentoxin at high concentrations (10^?4–10^?3 M) in the presence of octylglucoside further stimulates the Mg²⁺-ATPase activity of CF₁ from tentoxin-sensitive species and inhibits the activity of CF₁ from tentoxin-resistant species. The extent of tentoxin stimulation and inhibition varies among species. A maximal stimulation of over 2-fold was obtained with spinach CF₁ and a maximal inhibition of 50% was obtained with C. reinhardii CF₁. In Nicotiana spp., tentoxin had only a marginal effect on the Mg²⁺-ATPase activity induced by octylglucoside.