Membrane-bound Coupling Factor Research Articles

The Arabidopsis Protein CGLD11 Is Required for Chloroplast ATP Synthase Accumulation

ATP synthases in chloroplasts (cpATPase) and mitochondria (mtATPase) are responsible for ATP production during photosynthesis and oxidative phosphorylation, respectively. Both enzymes consist of two multisubunit complexes, the membrane-bound coupling factor O and the soluble coupling factor 1. During cpATPase biosynthesis, several accessory factors facilitate subunit production and orchestrate complex assembly. Here, we describe a new auxiliary protein in Arabidopsis thaliana, which is required for cpATPase accumulation. AtCGLD11 (CONSERVED IN THE GREEN LINEAGE AND DIATOMS 11) is a protein without any known functional domain and shows dual localization to chloroplasts and mitochondria. Loss of AtCGLD11 function results in reduced levels of cpATPase and impaired photosynthetic performance with lower rates of ATP synthesis. In yeast two-hybrid experiments, AtCGLD11 interacts with the β subunits of the cpATPase and mtATPase. Our results suggest that AtCGLD11 functions in F1 assembly during cpATPase biogenesis, while its role in mtATPase biosynthesis may not, or not yet, be essential.

Characteristics of the Mg2+-ATPase Activity Associated with the Membrane-Bound Maize Coupling Factor

The membrane-bound coupling factor of maize mesophyll thylakoids is a latent ATPase. Mg(2+)-ATPase activity can be induced in the light with either dithiothreitol or low concentrations of trypsin. Maize thylakoids that are activated with light plus trypsin exhibit considerably higher levels of activity in Na(2)SO(3)-dependent Mg(2+)-ATPase assays compared to thylakoids that are light and dithiothreitol activated (1400 micromoles per milligram of chlorophyll per hour versus 200 micromoles per milligram of chlorophyll per hour). Treatment with light and dithiothreitol or light plus trypsin were also required to demonstrate high levels of octyl glucoside-dependent Mg(2+)-ATPase activity in maize mesophyll thylakoids. Only small differences in octyl glucoside-dependent Mg(2+)-ATPase activity were observed in preparations that were activated in the light with either trypsin or dithiothreitol. Mg(2+)-ATPase activity can also be induced in maize mesophyll chloroplasts by illuminating intact preparations under appropriate conditions. Little or no ATPase activity was observed in the absence of illumination or in the presence of light plus methyl viologen. The active state decayed in the dark with a t((1/2)) of 6 to 7 minutes at room temperature. Based on the effect of the thiol oxidant, o-iodosobenzoate, and the uncoupler, nigericin, on the kinetics of deactivation of ATPase activity in intact maize chloroplasts, it appears that the activation process requires a transmembrane proton gradient and reduction of a key disulfide bridge in the gamma of chloroplast coupling factor one.

Function of tightly bound nucleotides on membrane-bound chloroplast coupling factor.

The kinetic behavior of tightly bound nucleotides on chloroplast coupling factor from spinach was determined under phosphorylating and nonphosphorylating conditions. Chloroplast coupling factor 1 (CF1) was labeled with tightly bound radioactive ADP and/or ATP at two specific sites and reconstituted with thylakoid membranes depleted of CF1 by treatment with NaBr. The initial incorporation and dissociation of ADP from one of the sites requires light but occurs at the same rate under phosphorylating and non-phosphorylating conditions. The initial rate is considerably slower than the rate of ATP synthesis, but nucleotide exchange is very rapid during steady-state ATP synthesis. A direct correspondence between this nucleotide binding site and a site on soluble CF1 that hydrolyzes ATP was demonstrated. A second site binds MgATP very tightly; the MgATP does not dissociate during ATP synthesis nor does its presence alter the rate of ATP synthesis. This is analogous to the behavior found for soluble CF1 during ATP hydrolysis. These results demonstrate that the tight-binding nucleotide sites on soluble CF1 and membrane-bound coupling factor are essentially identical in terms of binding properties and kinetic behavior during ATP hydrolysis and synthesis.

Evidence for two tightly bound nucleotides on thylakoid-bound chloroplast coupling factor 1 (CF 1): One ADP — Exchangeable upon illumination - And one non-exchangeable MgATP

Isolated chloroplast coupling factor 1 contains a tightly bound ADP (which is exchangeable for labeled medium ADP or ATP) but contains two tightly bound nucleotides (one ADP and one ATP) after incubation with radioactive ATP in the presence of magnesium ions (Bruist, M.F. and Hammes, G.G. (1981) Biochemistry 20, 6298–6305). On the membrane-bound enzyme, only one nucleotide (mainly ADP) was found to be exchangeable during incubation with labeled ATP in the light (Bickel-Sandkötter, S. and Strotmann, H. (1981) FEBS Lett. 125, 188–192). This paper demonstrates that membrane-bound CF 1 contains another tight binding site, occupied with non-exchangeable ATP in addition to the tight ADP site (exchangeable in the light). During isolation, the incubation of CF 1 with EDTA causes the loss of the tightly bound ATP so that the normal isolation procedure yields an enzyme species with only one tightly bound nucleotide molecule (ADP). If EDTA is omitted during isolation, the additional ATP can be detected easily by the luciferin-luciferase method. Reconstitution of isolated CF 1 containing two tightly bound nucleotides with CF 1-free membranes results in a membrane-bound coupling factor with two radioactive nucleotides. Because the exchange of this ATP is very slow, it is assumed that the tightly bound ATP has structural rather than catalytic or regulatory functions.

Characterization of nucleotide binding sites on the membrane-bound chloroplast ATP synthase (coupling factor CF 1)

Phosphorylation of ADP and nucleotide exchange by membrane-bound coupling factor CF 1 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.

Rotational mobility and domain flexibility of membrane-bound bacterial coupling factor as detected with the triplet probe eosin-isothiocyanate

The membrane-bound coupling factor (BF 1) of chromatophores from the photosynthetic bacerium Rhodopseudomonas sphaeroides was covalently labeled with the triplet probe eosin-isothiocyanate. The labeled enzyme was isolated and functionally reconstituted into depleted chromatophores from the same bacterium. ATPase and ATP synthase activities of the reconstituted vesicles were strongly dependent on the labeling conditions, decreasing at increasing load of eosin molecules per BF 1. When labeling was carried out in the dark and in the presence of ATP, one molecule of eosin isothiocyanate was bound per BF 1 and the activities catalyzed by the reconstituted and labeled enzyme were as high as in untreated chromatophores. Upon light energization of the chromatophore membrane, a large conformational change of BF 1 could be detected by using the triplet probe as a spectroscopic tool. The domain flexibility and rotational mobility of the reconstituted coupling enzyme were directly related to the enhancement of the ATPase activity induced by light. Both the light-stimulated ATPase activity and conformational changes could be prevented by addition of ADP or oligomycin and affected to the same extent by uncouplers and inhibitors of electron transport. Moreover, the detected conformational changes were reversible in time, appearing with a half-time of 10 ms upon illumination of the chromatophores, and disappearing with a half-time of 70 ms in the dark. The results obtained prove the feasibility of the spectroscopic technique in detecting conformational changes of the membrane-bound BF 1, similarly to what already has been observed for chloroplast coupling factor (Wagner, R. and Junge, W. (1980) FEBS Lett. 114, 327–333), and add to the possibility of characterizing, by this method, energy transduction at a molecular level.

Photoaffinity labeling of the tight ADP binding site of the chloroplast coupling factor one (CF 1): The effect on the CF 1-ATPase activity

Chloroplast thylakoid membranes contain tightly bound ADP which is intimately involved in the mechanism of photophosphorylation. The photoaffinity analog 2-azido-ADP binds tightly to spinach thylakoid membrane-bound coupling factor one (CF 1) and, in a manner similar to ADP, inhibits the light-triggered ATPase activity (Czarnecki, J.J., Abbott, M.S. and Selman, B.R. (1983) Eur. J. Biochem. 136, 19–24). Ultraviolet irradiation of thylakoid membranes containing noncovalently, tightly bound 2-azido[β- 32P]ADP results in the inactivation of both the methanol-stimulated MgATPase activity of the membrane-bound CF 1 and the octylglucoside-dependent MgATPase activity of the solubilized enzyme. There is a linear correlation between the loss of enzyme activity and the covalent incorporation of the photoaffinity analog. Full inactivation of catalytic activity is estimated to occur upon incorporation of 1.07 mol analog and 0.65 mol analog per mol enzyme for the methanol- and octylglucoside-stimulated activities, respectively. Since 2-azido-ADP modifies only the β subunit of the CF 1 and since there are probably three β subunits per CF 1, these results indicate strong cooperativity among β subunits and between the site of tightly bound nucleotides and the catalytic sites.

Localization of the high-affinity binding site for ATP on the membrane-bound chloroplast ATP synthase.

The photoaffinity analog 2-azido-ADP has been used to investigate the high-affinity binding site(s) for ATP on the chloroplast thylakoid membrane. Photophosphorylation of 2-azido-ADP results in the rapid formation of 2-azido-ATP, which remains tightly bound to the membranes after extensive washing. The kinetic parameters of the tight binding of ATP and of 2-azido-ATP are similar (apparent Km = 1-2 microM; maximum extent = 0.2-0.4 nmol/mg of chlorophyll). Ultraviolet irradiation of washed thylakoid membranes containing tightly bound 2-azido-[gamma-32P]ATP induces covalent incorporation of the label exclusively into the beta subunit of the chloroplast coupling factor one. Previous results have shown that the tight binding site for ADP is also located on the beta subunit of the ATP synthase (Czarnecki, J. J., Abbott, M. S., and Selman, B. R. (1983) Eur. J. Biochem. 136, 19-24). To further characterize the tight binding sites for ADP and ATP, the membrane-bound coupling factor has been covalently modified with either tightly bound 2-azido-[gamma-32P]ATP or tightly bound 2-azido-[beta-32P]ADP. The photolabeled beta subunits have been isolated and subjected to partial proteolytic digestion and SDS-gel electrophoresis. The results of these experiments demonstrate that the tight binding sites for ADP and ATP are located on identical portions of beta subunit polypeptide.

Photoaffinity labeling with 2-azidoadenosine diphosphate of a tight nucleotide binding site on chloroplast coupling factor 1

An analog of ADP containing an azido group at the C-2 position of the purine ring has been synthesized and used as an affinity probe of the membrane-bound coupling factor 1 of spinach chloroplast thylakoid membranes. The 2-azido-ADP inhibited light-induced dark binding of ADP at the tight nucleotide binding site on the thylakoid membranes. The 2-azido-ADP itself bound tightly to the thylakoid membranes, with 1 muM as the concentration giving 50% maximum binding. Tight binding of the analog required the thylakoid membranes to be energized, and the nucleotide remained bound after repeated washings of the membranes. The maximum extent of tight binding of the analog (1,2-1.3 nmol/mg of chlorophyll) was stoichiometric with the known coupling factor 1 content of thylakoid membranes but somewhat higher than that observed for ADP (0.5-0.9 nmol per mg of chlorophyll). Tight binding of 2-azido-ADP was decreased by the simultaneous addition of ADP. UV photolysis of washed thylakoid membranes containing tightly-bound 2-azido-[beta-(32)P]ADP resulted in the covalent incorporation of label into the membranes. Isolation of the chloroplast coupling factor 1 from these membranes followed by NaDodSO(4) gel electrophoresis demonstrated that the analog was covalently bound to the beta subunit of the coupling factor complex.

Two types of kinetic regulation of the activated ATPase in the chloroplast photophosphorylation system.

The inhibition by light of chloroplast coupling factor ATPase is not due simply to competing photophosphorylation. This inhibition is only partially relieved by either an arsenate-pool trap for released phosphate, or a pyruvate kinase/phosphoenolpyruvate trap for ADP. Moreover, the amount of product return that does occur in the absence of trapping systems, ascertained by incorporation of 32Pi or [2-3H]ADP back into ATP during the hydrolysis reaction, is insufficient to account for the observed activity decrease. In intermediate pi:H2O oxygen exchange studies, the number of water oxygens incorporated into each molecule of Pi produced does not vary with light intensity during the ATPase assay. This indicates that the light-induced change in ATPase activity is not due to an alteration of rat constants involved in the forward and reverse partitioning of the E.ADP.Pi complex. In contrast, ammonium chloride, an uncoupler of photophosphorylation which stimulates membrane-bound coupling factor ATPase when added after light activation, causes a shift in the pattern of intermediate Pi:H2O oxygen exchange toward a lower number of water oxygens incorporated per Pi formed. The effect of NH4+ consistent with ATPase activity stimulation caused by enhanced partitioning forward of the E.products complex. These observations suggest the operation of two mechanisms of regulation of ATP ase activity during chloroplast de-energization. However, a direct effect of NH4+ on the coupling factor itself, independent of the membrane energization effect, cannot be ruled out by the present studies. Additional oxygen exchange experiments lead to the conclusion that the binding of ATP at a site catalyzing extensive ATP:H2O back exchange in the native chloroplast system ( Wimmer, M. J., and Rose, I. A. (1977) J. Biol. Chem. 252, 6769-6775) is different from the binding of ATP for net hydrolysis in the system activated for ATPase.

Effects of Hydroxamic Acids Isolated from Gramineae on Adenosine 5′-triphosphate Synthesis in Chloroplasts

Two hydroxamic acids isolated from maize extracts, 2,4-dihydroxy-7-methoxy-1,4-(2H)-benzoxazin-3(4H)-one (DIMBOA) and the 2-O-beta-d-glucopyranoside of DIMBOA, inhibit photophosphorylation by spinach chloroplasts. Both cyclic and noncyclic photophosphorylations were inhibited to the same extent. The concentrations producing 50% inhibition for DIMBOA and its glucoside were about 1 and 4 millimolar, respectively. These compounds inhibit coupled electron transport but do not affect basal or uncoupled electron transport. Both acids inhibit the ATPase activities of membrane-bound coupling factor 1 (CF(1)) and of purified CF(1). On the basis of these results, it is concluded that DIMBOA and its glucoside act as energy transfer inhibitors of photophosphorylation.

Inhibition of electron flow and energy transduction in isolated spinach chloroplasts by the herbicide dinoseb

The mode of action of dinoseb (2-sec-butyl-4-6-dinitrophenol) on chloroplast reactions was studied. Electron flow from water or from an artificial electron donor, diphenylcarbazide (DPC), to dichlorophenol indophenol (DCPIP) was inhibited at low concentrations of the herbicide (5–10 μM) suggesting a site for dinoseb inhibition at the oxidizing side of photosystem II (PS II). Ferricyanide photoreduction was also inhibited by dinoseb. Cyclic and non-cyclic photophosphorylation and Mg 2+-ATPase activity were inhibited by dinoseb, which indicates that this herbicide also acts as an energy transfer inhibitor. Among the above mentioned activities, non-cyclic photophosphorylation was the most sensitive to the inhibition by dinoseb. Ca 2+-ATPase activity of solubilized heat activated chloroplast coupling factor 1 (CF 1) was stimulated by dinoseb. However, the same activity was inhibited in chloroplasts, which perhaps reflect a difference in the mode of interaction of dinoseb with solubilized and membrane bound coupling factor.

Inhibition of energy-transducing functions of chloroplasts by spegazzinine

The effects of spegazzinine, a dihydroindole alkaloid, on various energy-transducing functions of chloroplasts were studied. The following observations were made, (i) Spegazzinine inhibited both cyclic and noncyclic photophosphorylation in isolated spinach chloroplast. The I 50 value was about 80 μ m. Over a concentration range which gave marked inhibition of ÀTP synthesis, there was no effect on basal or uncoupled electron flow or light-induced proton accumulation by isolated thylakoids, while the fraction of electron transport stimulated by coupled phosphorylation was reduced to the basal level by spegazzinine. (ii) The regulatory effect of low concentrations of ATP on proton movements and electron transport was diminished by the alkaloid, (iii) Spegazzinine also inhibited with similar efficiency the ATPase activities of membrane-bound coupling factor 1 (CF 1) and of purified CF 1. One mole of spegazzinine per mole of CF 1 seemed to be required to inhibit the ATPase activity, (iv) The allosteric effect of ADP on ATPase activity was not affected by spegazzinine. (v) On the basis of these results it is concluded that spegazzinine acts as an energy transfer inhibitor of hotophosphorylation and that its site of action may be at or near the catalytic site of ATPase.

Properties of ATPase activity in coupling factor from Chromatium strain D chromatophores.

Coupling factor extracted from chromatophores of the photosynthetic bacteria Chromatium strain D was partially purified. The enzyme catalyzed ATPase activity in the presence of Ca2+ and Mg2+ ions. Higher Vapp values were obtained when the activity was measured as a function of the divalent cation-ATP complex rather than as a function of either the divalent cation or ATP because the free components competitively inhibited the activity in the presence of the cation-ATP complex. The Km values were lower than or equal to the Ki values for free ATP indicating that the cation-ATP complex is bound tighter than the free ATP to the enzyme. Based on these results a possible mode of binding of substrate to the active site of the enzyme was suggested. A comparative study indicated no changes in the temperature dependance of ATPase activity when the enzyme was solubilized. However, possible conformation changes could have caused a decrease in the Km values for the (Ca-ATP)2- and (Mg-ATP)2- and in the Ki for free Mg2+ ions and ATP. The Ki for free Ca2+ ions increased on solubilization of the coupling factor. ATPase activity was inhibited by dicyclohexylcarbodiimide both in the soluble and in the membrane-bound coupling factor.

Salt inactivation as a mechanistic probe of membrane-bound chloroplast coupling factor 1.

Conditions are reported under which ATP protects membrane-bound coupling factor 1 against sodium bromide inactivation. The presence of Mg2+ was found to be obligatory for this protection. ADP and GTP also protected the enzyme against salt inactivation but to a much smaller extent. Other nucleotides tested were ineffective. At low ATP concentrations ADP prevented the effect of ATP and modified the saturation curve for ATP from hyperbolic to sigmoidal. Treatment of chloroplasts with 0.4 M MgCl2 or 2 M LiCl resulted in inactivation of photophosphorylation. In contrast to NaBr-depleted particles the MgCl2 or LiCl-depleted chloroplasts can be reconstituted by purified coupling factor 1. A binding site for Mg2+ and two different sites for ATP upon the coupling factor 1 are suggested to explain the mechanism of their protection against salt inactivation.

Energy-transducing membrane-bound coupling factor-ATPase from Mycobacterium phlei. I. Purification, homogeneity, and properties

The membrane-bound coupling factor from Mycobacterium phlei was solubilized from membrane vesicles by washing with low ionic strength buffer or 0.25 M sucrose. The solubilized enzyme exhibited coupling factor, latent ATPase, and succinate oxidation-stimulating activity. Purification by affinity chromatography using Sepharose coupled to ADP yielded a homogeneous preparation of latent ATPase which was purified about 200-fold with an 84% yield in a single step. Purified latent ATPase exhibited coupling factor activity but no succinate oxidation-stimulating activity. The molecular weight of latent ATPase was determined to be 250,000 +/- 10,000 by Sephadex G-200 chromatography. The ATPase was unmasked by trypsin treatment and activated by Mg2+ ion. However, trypsin treatment inactivated the coupling factor activity in the purified enzyme, indicating that the catalytic sites for ATPase and coupling activity are different. Unlike mitochondrial ATPase, latent ATPase from M. phlei was not cold-labile. Of the nucleoside triphosphates, UTP, ITP, and epsilon-ATP (1-N6-ethenoadenosine triphosphate) were hydrolyzed to a lesser extent compared to ATP. Kinetic data showed that ADP acted as a competitive inhibitor of latent ATPase activity with a Ki of 5 x 10(-3) M. Uncouplers of oxidative phosphorylation and respiratory inhibitors did not affect the latent ATPase activity, while sodium azide (0.1 mM) inhibited the latent ATPase activity.

Synergistic uncoupling of spinach chloroplasts by combinations of photophosphorylation inhibitors and carbonyl cyanide p-trifluoromethoxyphenylhydrazone

Combinations of low concentrations of carbonyl cyanide p-trifluoromethoxyphenylhy-drazone (FCCP) with suboptimal concentrations of Dio-9, phloridzin, ajmaline, and dihydrodiscarine B synergistically inhibited cyclic and noncyclic photophosphorylation in spinach chloroplasts but their effects on the light-triggered ATPase were additive rather than synergistic. The effect was reversed by washing and prevented by dithioerythritol and by cistein. Carbonyl cyanide m-chlorophenylhydrazone (CCP) could replace FCCP but uncouplers of other types like atebrin did not substitute for FCCP. Combinations of FCCP with the four inhibitors synergistically uncoupled ferricyanide reduction in the presence of ADP and P i but not in their absence. The synergistic uncoupling was not observed on the light-dependent pH rise of chloroplast suspensions. Association of FCCP with any of the inhibitors completely abolished the stimulation of proton uptake or the inhibition of electron transport induced by low concentrations of ATP. This synergistic and peculiar uncoupling can not be ascribed to a modification of membrane permeability. One possible explanation is that the effect requires a conformational state of the membrane-bound coupling factor 1 (CF 1) induced by phosphorylating conditions which would facilitate the interaction of inhibitors and FCCP with the membrane.

Delayed light studies on photosynthetic energy conversion. VIII. Evidence from millisecond emission of chloroplasts for two adenylate binding sites on membrane-bound coupling factor, CF 1

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 (CF 1) with EDTA, and by antibody to CF 1. Readdition of CF 1 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 CF 1. 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, CF 1. 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 CF 1.

Chemical Modification of the Inner Mitochondrial Membrane

Abstract 1. Exposure of mitochondria and submitochondrial particles from bovine heart to [35S]diazobenzenesulfonate yielded information on the asymmetrical distribution of membrane components. Radioactive coupling factor 1 (ATPase) was isolated from submitochondrial particles and radioactive cytochrome c from mitochondria. The specific radioactivities were comparable to those observed when the soluble proteins were exposed to [35S]diazobenzenesulfonate. The low radioactivity of coupling factor 1 isolated from mitochondria and of cytochrome c isolated from submitochondrial particles is evidence for the lack of permeation of the radioactive marker from one side of the membrane to the other. 2. Cytochrome oxidase was labeled from both sides of the membrane, but the specific radioactivity was about 6-fold lower than that of a solubilized cytochrome oxidase treated with [35S]diazobenzenesulfonate. This finding indicates a transmembranous location of cytochrome oxidase. 3. Spectral and enzymatic analyses of mitochondria and submitochondrial particles after cross-linking with polylysine show that cytochrome a is located on the same side as cytochrome c and suggest that cytochrome a3 is located on the same side as coupling factor 1. 4. Exposure of membrane-bound coupling factor 1 to [35S]-diazobenzenesulfonate resulted in an asymmetrical labeling of its subunits which was distinctly different from the labeling pattern after exposure of the soluble coupling factor.

Light-dependent Inhibition of Photophosphorylation by N-Ethylmaleimide

Abstract The treatment of chloroplasts with 1 mm N-ethylmaleimide (NEM) in the light, but not in the dark, resulted in a partial, permanent inhibition of photophosphorylation. Noncyclic electron flow coupled to phosphorylation was also inhibited on incubation of chloroplasts with NEM in the light, but this inhibition was reversed by ammonium chloride. The uncouplers, carbonyl cyanide m-chlorophenylhydrazone and ammonium chloride, largely prevented the inhibition. Adenosine triphosphate and adenosine diphosphate partially protected phosphorylation from inhibition by light and NEM. Light-induced hydrogen ion uptake was unaffected by NEM except that the uptake stimulated by adenosine triphosphate was inhibited. The calcium-dependent adenosine triphosphatase of coupling factor 1 was also inhibited in chloroplasts treated with NEM in the light prior to extraction of the coupling factor. It is suggested that light causes a conformational change in the membrane-bound coupling factor, allowing the reaction of a group (or groups) with NEM. The sensitivity of the inhibition to adenine nucleotides suggests that these nucleotides modify the conformation of coupling factor 1 in the light.