M.J.C. Scholts

Energy metabolism in the cyanobacterium Plectonema boryanum. Participation of the thylakoid photosynthetic electron transfer chain in the dark respiration of NADPH and NADH

The oxidation of NADPH and NADH was studied in the light and in the dark using sonically derived membrane vesicles and osmotically shocked spheroplasts. These two types of cell-free membrane preparations mostly differ in that the cell and thylakoid membranes are scrambled in the former type and that they are more or less separated in the latter type of preparations. In the light, using both kinds of preparations, each of NADPH and NADH donates electrons via the plastoquinone-cytochrome bc redox complex (Qbc redox complex) to the thylakoid membrane-bound cytochrome c-553 preoxidized by a light flash and to methylviologen via Photosystem I. NADPH donates electrons to the thylakoid membrane via a weakly rotenone-sensitive dehydrogenase to a site that is situated beyond the 3(3′,4′-dichlorophenyl)-1,1-dimethylurea sensitive site and before plastoquinone. Ferredoxin and easily soluble cytoplasmic proteins are presumably not involved in light-mediated NADPH oxidation. Inhibitors of electron transfer at the Qbc redox complex as the dinitrophenylether of 2-iodo-4-nitrothymol, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone and 2-n-heptyl-4-hydroxy-quinone-N-oxide are effective, but antimycin A and KCN are not. The oxidation of NADH showed comparable sensitivity to these inhibitors. However, the oxidation of NADH is antimycin-A-sensitive regardless of the kind of membrane preparation used, indicating that in this case electrons are donated to a different site on the thylakoid membrane. In the dark, NADPH and NADH donate electrons at sites that behave similar to those of light-mediated oxidation, indicating that the initial steps of electron transfer are situated at the thylakoid membranes. However, NADPH oxidation is in some cases not sensitive to inhibitors active at the Qbc redox complex. It is concluded that O2 reduction takes place at two different sites, one partly developed in vitro, situated near the rotenone-sensitive NADPH dehydrogenase, and another, highly KCN-sensitive one, situated beyond the Qbc redox complex and used in vivo. The terminal oxygen-reducing step of NADPH and NADH oxidation in the dark showed a preparation-dependent sensitivity for KCN, more than 80% inhibition in sonically derived membrane vesicles and less than 30% inhibition in osmotically shocked spheroplasts. From this result we tentatively conclude that the highly KCN-sensitive oxidase is not necessarily located at the thylakoid membrane and could be located at the cytoplasmic membrane.

Dependence of the proton translocation stoichiometry of cyanobacterial and chloroplast H+-ATP synthase on the membrane composition

Abstract The high proton translocation stoichiometry (approx. 9 H+ / ATP) of ATPase proteoliposomes reconstituted from a thermophilic cyanobacterium (Van Walraven et al. (1986) FEBS Lett. 208, 138–142) has also been observed with chloroplast ATP synthase when reconstituted with cyanobacterial lipids. Both enzyme complexes in isolated and reconstituted form show highest stable trypsin-activated ATP hydrolysis activity at the same temperature (55°C). Also, both isolated ATP synthases require the same reconstitution procedure for maximal coupling quality. The proton translocation stoichiometry has been deduced from the relation between the initial rates of ATP hydrolysis at varying sizes of the electrochemical potential gradient ( Δ gm H + ). A Δ gm H + was imposed by valinomycin-induced K+ diffusion potentials or by base-pulses which were equally efficient in inhibiting ATP hydrolysis. Kinetic experiments with the use of the pH indicator Cresol red confirm the high proton translocation stoichiometry of both types of ATPase proteoliposome. Functional co-reconstitution of both types of ATPase proteoliposome with cyanobacterial cytochrome b6f complex leads to a decrease in proton translocation stoichiometry to about 7 H+ / ATP. Cyanobacterial membrane vesicles take up 4.4 protons per ATP hydrolyzed. A value of 4.5 H+ / ATP is observed with chloroplasts in equilibrium (Graber, P., Junesch, U. and Thulke, G. (1986) in Progress in Photosynthesis Research (Biggins, J., ed.), pp. 177–184, Martinus Nijhoff, Dordrecht). These results indicate that the proton translocation stoichiometry of the ATP synthase depends on the membrane composition. The consequence of this finding for the mechanism of proton translocation and the possible physiological relevance are discussed.

Energy metabolism in the cyanobacterium Plectonema boryanum. Oxidative phosphorylation and respiratory pathways

Abstract The filamentous cyanobacterium Plectonema boryanum catalyzes efficient dark oxidative phosphorylation of exogenous ADP during NADPH consumption after a lysozyme treatment of only 30 min and subsequent dilution in hypoosmotic medium. It is shown that the thylakoid membranes and membrane areas bearing the terminal oxidase (presumably the cell membrane with cytochrome c:O2 oxidoreductase) and easily soluble cytoplasmic proteins are involved in KCN-sensitive dark oxidative phosphorylation. The dinitrophenyl ether of 2-iodo-4-nitrothymol, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone and KCN are inhibitors of dark respiratory ATP synthesis. Dependent on the physiological condition, other more or less KCN-insensitive respiratory pathways towards O2 may be present. A tentative scheme of the respiratory pathways is proposed.

Activation of the H+-ATP synthases of a thermophilic cyanobacterium and chloroplasts — a comparative study

The activation requirements of the ATP synthases of the thermophilic cyanobacterium Synechococcus 6716, studied in coupled membrane vesicles and in the isolated or reconstituted complex, and of the ATP synthase of spinach chloroplasts were compared. It was found that methanol, heat treatment or dithiothreitol did not activate ATP hydrolysis in Synechococcus 6716. In contrast to the chloroplast enzyme, activation could only be accomplished with sulfite, octyl glucoside, a proton electrochemical potential difference and trypsin. The lack of activation by dithiothreitol, heat and methanol in the cyanobacterial ATP synthase can be explained by the absence of three cysteine residues in the regulatory γ subunit of the F 1 part. The threshold value of the proton electrochemical potential difference at which ATP synthesis occurs at low G p was about 9.5 kJ mol −1 for the cyanobacterial ATP synthase. This is similar to the threshold value of the reduced form of the enzyme in chloroplasts. With cyanobacterial membrane vesicles, an H + / ATP stoichiometry slightly exceeding 4 was obtained in ATP hydrolysis as well as in ATP synthesis, measured as a function of an artificially applied proton electrochemical potential difference. These findings are discussed in terms of a single structural difference between the cyanobacterial and the chloroplast enzyme. When comparing the enzyme of Synechococcus 6716 with that of chloroplasts, our results indicate that the difference in activation requirements of both ATP synthases resides in a different arrangement of the γ and e subunits.

Activation of the H+-ATP synthase in thylakoid vesicles from the cyanobacterium Synechococcus 6716 by Δ\̄gmH+. Including a comparison with chloroplasts, and introducing a new method to calibrate light-induced Δ\̄gmH+

Abstract Activation of ATP hydrolysis activity in well-coupled membrane vesicles of the thermophilic cyanobacterium Synechococcus 6716 has been studied. In addition to a basal (dark) activity dependent on culture age, the cyanobacterial thylakoid ATP synthase is activated by Δ gm H + . Activation of this enzyme has been studied quantitatively and the results are compared with literature data regarding activation of the chloroplast ATP synthase. We found that activation behaviour both in chloroplasts and in cyanobacterial membrane vesicles may be characterized by the value of Δ gm H + where 50% activation occurs ( Δ gm a ) and a Hill-type coefficient (p) that estimates the number of protons involved. The activating Δ gm H + was induced either by the acid-base pulse method, or by illumination. To calibrate the size of light-induced Δ gm H + , a method has been developed whereby at each light intensity the value of ΔGp is determined where the ATP synthase reaction is in equilibrium. Δ gm H + at equilibrium then is calculated from this ΔGp and the H + ATP ratio. The value of p for the cyanobacterial ATP synthase is similar to that of the chloroplast enzyme (between 1 and 2). The cyanobacterial enzyme resembles the thiol-modulated (reduced) form of the chloroplast ATP synthase in that Δ gm a is low (14.1 kJ mol−1) and that the Δ gm H + -activated state is quite stable. As in the thiol-modulated chloroplast ATP synthase, decay of the activated state is accelerated by ADP. We conclude that the Synechococcus 6716 membrane vesicles are an excellent system in which to study Δ gm H + activation of ATP synthase with no need for thiol modulation.

Measurement of diffusion potentials in liposomes. Origin and properties of the threshold level in the oxonol VI response

Abstract A model is presented for the response of the membrane potential probe oxonol VI on diffusion potentials in liposomes. In this model the dependence of the probe response on the initial ion gradient is explained in terms of internal volume, internal ion concentration, membrane capacity and initial membrane potential. It is found that in the presence of an initial membrane potential (positive outside) there is a threshold value of the ion gradient needed for a probe response, which increases when the internal volume or the internal ion concentration decrease. The model is confirmed by experiments with liposomes of different sizes and internal KCl concentrations, prepared from asolectin or lipids isolated from the thermophilic cyanobacterium Synechococcus 6716. The significance of the model for threshold values observed in other energy-dependent phenomena is discussed.

pH-dependent Ca2+ binding to the F0 c-subunit affects proton translocation of the ATP synthase from Synechocystis 6803.

It was shown before (Wooten, D. C., and Dilley, R. A. (1993) J. Bioenerg. Biomembr. 25, 557–567; Zakharov, S. D., Li, X., Redko, T. P., and Dilley, R. A. (1996) J. Bioenerg. Biomembr. 28, 483–493) that pH dependent reversible Ca2+ binding near the N- and C-terminal end of the 8 kDa subunit c modulates ATP synthesis driven by an applied pH jump in chloroplast and E. coli ATP synthase due to closing a “proton gate” proposed to exist in the F0 H+ channel of the F0F1 ATP synthase. This mechanism has further been investigated with the use of membrane vesicles from mutants of the cyanobacterium Synechocystis 6803. Vesicles from a mutant with serine at position 37 in the hydrophilic loop of the c-subunit replaced by the charged glutamic acid (strain plc 37) has a higher H+/ATP ratio than the wild type and therefore shows ATP synthesis at low values of ΔμH+. The presence of 1 mM CaCl2 during the preparation and storage of these vesicles blocked acid–base jump ATP formation when the pH of the acid side (inside) was between pH 5.6 and 7.1, even though the ΔpH of the acid–base jump was thermodynamically in excess of the necessary energy to drive ATP formation at an external pH above 8.28. That is, in the absence of added CaCl2, ATP formation did occur under those conditions. However, when the base stage pH was 7.16 and the acid stage below pH 5.2, ATP was formed when Ca2+ was present. This is consistent with Ca2+ being displaced by H+ ions from the F0 on the inside of the thylakoid membrane at pH values below about 5.5. Vesicles from a mutant with the serine of position 3 replaced by a cysteine apparently already contain some bound Ca2+ to F0. Addition of 1 mM EGTA during preparation and storage of those vesicles shifted the otherwise already low internal pH needed for onset of ATP synthesis to higher values when the external pH was above 8. With both strains it was shown that the Ca2+ binding effect on acid–base induced ATP synthesis occurs above an internal pH of about 5.5. These results were corroborated by 45Ca2+- ligand blot assays on organic solvent soluble preparations containing the 8 kDa F0 subunit c from the S-3-C mutant ATP synthase, which showed 45Ca2+ binding as occurs with the pea chloroplast subunit III. The phosphorylation efficiency (P/2e), at strong light intensity, of Ca2+ and EGTA treated vesicles from both strains were almost equal showing that Ca2+ or EGTA have no other effect on the ATP synthase such as a change in the proton to ATP ratio. The results indicate that the Ca2+ binding to the F0 H+ channel can block H+ flux through the channel at pH values above about 5.5, but below that pH protons apparently displace the bound Ca2+, opening the CF0 H+ channel between the thylakoid lumen and H+ conductive channel.

The H+/ATP ratio of the ATP synthase from the cyanobacterium Synechococcus 6716 varies with growth temperature and light intensity

Abstract The proton translocation stoichiometry (H+/ATP ratio) and other bioenergetic features were investigated in membrane vesicles from the moderately thermophile Synechococcus 6716 grown at 38°C and 50°C with saturating light intensity, and at 38°C with limiting light intensity. At 50°C growth is slower but proceeds to a higher cell density than at 38°C. Increasing the growth temperature from 38°C to 50°C resulted in an altered membrane fatty acid composition, with increased length and saturation of the acyl chains. At 38°C and lower light intensity chain length was somewhat decreased and saturation increased to a small extent. Membrane vesicles from cells grown at 50°C performed cyclic photophosphorylation at lower light intensities and lower threshold Δ μ H + than vesicles from cells grown at 38°C. The 50°C vesicles also displayed a diminished light-induced proton uptake, but ATP synthesis activity and the attained ΔGp remained constant. Moreover, ATP synthesis became more resistant to uncoupling. From acid–base transition induced ATP synthesis experiments the H+/ATP ratios were determined to be 3.9, 3.1 and 3.3 for membrane vesicles from cells grown at 50°C, 38°C and light-limited 38°C, respectively. In vesicles from cells grown at 50°C, ATP hydrolysis is inhibited by a lower valinomycin-induced K+-diffusion potential than in vesicles from cells grown at 38°C. A molecular mechanism to explain changes in H+/ATP as well as the physiological implications are discussed.

Membrane vesicles fromSynechocystis 6803 showing proton and electron transport and high ATP synthase activities.

A simple procedure for the preparation of well-coupled and stable membrane vesicles from the transformable cyanobacteriumSynechocystis 6803 is described with the primary aim of producing vesicles suitable for the study of photosynthetic electron transport and phosphorylation. Spheroplasts were obtained from the cyanobacterium by lysozyme treatment and stored untill prior to measurement, thylakoid vesicles were obtained by osmotic shock. These vesicles showed very high and stable ATP synthesis rates either driven by light or by acid-base transition, and also performed light-induced ATP hydrolysis and linear electron transport. Formation of a proton gradient is studied by aminoacridines.

Introduction of a carboxyl group in the loop of the Fo c -subunit affects the H+/ATP coupling ratio of the ATP synthase from Synechocystis 6803.

The proton translocation stoichiometry (H+/ATP ratio) was investigated in membrane vesicles from a Synechocystis 6803 mutant in which the serine at position 37 in the hydrophilic loop of the c-subunit from the wild type was replaced by a negatively charged glutamic acid residue (strain plc37). At this position the c-subunit of chloroplasts and the cyanobacterium Synechococcus 6716 already contains glutamic acid. H+/ATP ratios were determined with active ATP synthase in thermodynamic equilibrium between phosphate potential (ΔGp) and the proton gradient (ΔμH+) induced by acid–base transition. The mutant displayed a significantly higher H+/ATP ratio than the control strain (wild type with kanamycin resistance) at pH 8 (4.3 vs. 3.3); the higher ratio also being observed in chloroplasts and Synechococcus 6716. Furthermore, the pH dependence of the H+/ATP of strain plc37 resembles that of Synechococcus 6716. When the pH was increased from 7.6 to 8.4, the H+/ATP of the mutant increased from 4.2 to 4.6 whereas in the control strain the ratio decreased from 3.8 to 2.8. Differences in H+/ATP between the mutant and the control strain were confirmed by measuring the light-induced phosphorylation efficiency (P/2e), which changed as expected, i.e., the P/2e ratio in the mutant was significantly less than that in the wild type. The need for more H+ ions used per ATP in the mutant was also reflected by the significantly lower growth rate of the mutant strain. The results are discussed against the background of the present structural and functional models of proton translocation coupled to catalytic activity of the ATP synthase.