[Effects of Medium Viscosity Increasing Agents on ATP Synthesis in Chloroplast Thylakoids].

The effect of an increase in the medium viscosity on cyclic photophosphorylation in chloroplast thylakoids and on Ca2+ -dependent ATP hydrolysis by the chloroplast coupling factor CF, was studied. With 0.1-0.2 mM ADP used it was found that the rate of ATP synthesis decreases after addition of various agents that increase the medium viscosity (sucrose, dextran 40 or polyethylene glycol 6000 provided that these agents cause neither uncoupling nor electron transport inhibition in the absence of ADP. Dextran and polyethylene glycol inhibited ATP synthesis by 50% when their concentrations were much lower (6-10%) than that of sucrose (30-40%), while 50% inhibition of Ca2+ -dependent ATP hydrolysis by CFI-ATPase was observed at higher concentrations of dextran and polyethylene glycol (9-13%) and lower concentrations of sucrose (about 20%). For ADP, the effective Michaelis constant (KM) was shown to increase 2-3-fold with the increasing viscosity; meanwhile the maximal rate of cyclic photophosphorylation remained virtually unchanged. The dependence of K(M) on the medium viscosity can serve as a criterion for the process of diffusion-controlled photophosphorylation. Possible mechanisms of ADP and ATP diffusion are discussed.

Distinct redox behaviors of chloroplast thiol enzymes and their relationships with photosynthetic electron transport in Arabidopsis thaliana.

The thiol/disulfide redox network mediated by the thioredoxin (Trx) system in chloroplasts ensures light-responsive control of diverse crucial functions. Despite the suggested importance of this system, the working dynamics against changing light environments remains largely unknown. Thus, we directly assessed the in vivo redox behavior of chloroplast Trx-targeted thiol enzymes in Arabidopsis thaliana. In a time-course analysis throughout a day period that was artificially mimicked to natural light conditions, thiol enzymes showed a light-dependent shift in redox state, but the patterns were distinct among thiol enzymes. Notably, the ATP synthase CF(1-γ) subunit was rapidly reduced even under low-light conditions, whereas the stromal thiol enzymes fructose 1,6-bisphosphatase, sedoheptulose 1,7-bisphosphatase, and NADP-malate dehydrogenase were gradually reduced/re-oxidized along with the increase/decrease in light intensity. Photo-reduction of thiol enzymes was suppressed by the impairment of photosynthetic linear electron transport using DCMU and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, but sensitivity to the impairment was uneven between CF(1-γ) and other stromal thiol enzymes. These different dependencies of photo-reduction on electron transport, rather than the redox state of Trx and the circadian clock, could readily explain the distinct diurnal redox behaviors of thiol enzymes. In addition, our results indicate that the cyclic electron transport around PSI is also involved in redox regulation of some thiol enzymes. Based on these findings, we propose an in vivo working model of the redox regulation system in chloroplasts.

A novel mechanism of P-type ATPase autoinhibition involving both termini of the protein.

The activity of many P-type ATPases is found to be regulated by interacting proteins or autoinhibitory elements located in N- or C-terminal extensions. An extended C terminus of fungal and plant P-type plasma membrane H(+)-ATPases has long been recognized to be part of a regulatory apparatus involving an autoinhibitory domain. Here we demonstrate that both the N and the C termini of the plant plasma membrane H(+)-ATPase are directly involved in controlling the pump activity state and that N-terminal displacements are coupled to secondary modifications taking place at the C-terminal end. This identifies the first group of P-type ATPases for which both ends of the polypeptide chain constitute regulatory domains, which together contribute to the autoinhibitory apparatus. This suggests an intricate mechanism of cis-regulation with both termini of the protein communicating to obtain the necessary control of the enzyme activity state.

Singlet oxygen inhibits ATPase and proton translocation activity of the thylakoid ATP synthase CF1CFo.

Singlet oxygen ((1)O(2)) produced in plants during photosynthesis has a strong damaging effect not only on both photosystems but also on the whole photosynthetic machinery. This is also applicable for the adenosine triphosphate (ATP) synthase. Here we describe the impact of (1)O(2) generated by the photosensitizer Rose Bengal on the ATP hydrolysis and ATP-driven proton translocation activity of CF1CFo. Both activities were reduced dramatically within 1min of exposure. Interestingly, it is shown that oxidized thylakoid ATP synthase is more susceptible to (1)O(2) than CF1CFo in its reduced state, a new insight on the mechanism of (1)O(2) interaction with the gamma subunit.

Inhibition of the ATPase activity of the catalytic portion of ATP synthases by cationic amphiphiles.

Melittin, a cationic, amphiphilic polypeptide, has been reported to inhibit the ATPase activity of the catalytic portions of the mitochondrial (MF1) and chloroplast (CF1) ATP synthases. Gledhill and Walker [J.R. Gledhill, J.E. Walker. Inhibition sites in F1-ATPase from bovine heart mitochondria, Biochem. J. 386 (2005) 591-598.] suggested that melittin bound to the same site on MF1 as IF1, the endogenous inhibitor polypeptide. We have studied the inhibition of the ATPase activity of CF1 and of F1 from Escherichia coli (ECF1) by melittin and the cationic detergent, cetyltrimethylammonium bromide (CTAB). The Ca2+- and Mg2+-ATPase activities of CF1 deficient in its inhibitory epsilon subunit (CF1-epsilon) are sensitive to inhibition by melittin and by CTAB. The inhibition of Ca2+-ATPase activity by CTAB is irreversible. The Ca2+-ATPase activity of F1 from E. coli (ECF1) is inhibited by melittin and the detergent, but Mg2+-ATPase activity is much less sensitive to both reagents. The addition of CTAB or melittin to a solution of CF1-epsilon or ECF1 caused a large increase in the fluorescence of the hydrophobic probe, N-phenyl-1-naphthylamine, indicating that the detergent and melittin cause at least partial dissociation of the enzymes. ATP partially protects CF1-epsilon from inhibition by CTAB. We also show that ATP can cause the aggregation of melittin. This result complicates the interpretation of experiments in which ATP is shown to protect enzyme activity from inhibition by melittin. It is concluded that melittin and CTAB cause at least partial dissociation of the alpha/beta heterohexamer.

Inhibition of spinach chloroplast F0F1 by an Fe2+/ascorbate/H2O2 system.

Plant chloroplasts are particularly threatened by free radical attack. We incubated purified soluble spinach chloroplast F(0)F(1) (CF(0)F(1), EC 3.6.3.34) with an Fe(2+)/H(2)O(2)/ascorbate system, and about 60% inactivation of the ATPase activity was reached after 60 min. Inactivation was not prevented by omission of H(2)O(2), by addition of catalase or superoxide dismutase, nor by the scavengers mannitol, DMSO, or BHT. No evidence for enzyme fragmentation or oligomerization was detected by SDS-PAGE. The chloroplast ATP synthase is resistant to attack by the reactive oxygen species commonly found at the chloroplast level. DTT in the medium completely prevented the inhibition, and its addition after the inhibition partially recovered the activity of the enzyme. CF(0)F(1) thiol residues were lost upon oxidation. The rate of thiol modification was faster than the rate of enzyme inactivation, suggesting that the thiol residues accounting for the inhibition may be hindered. Enzyme previously oxidized by iodobenzoate was not further inhibited by the oxidative system. The production of ascorbyl radical was identified by EPR and is possibly related to CF(0)F(1) inactivation. It is thus suggested that the ascorbyl radical, which accumulates under plant stress, might regulate CF(0)F(1).

Proton flux through the chloroplast ATP synthase is altered by cleavage of its gamma subunit.

Electron transport, the proton gradient and ATP synthesis were determined in thylakoids that had been briefly exposed to a low concentration of trypsin during illumination. This treatment cleaves the gamma subunit of the ATP synthase into two large fragments that remain associated with the enzyme. Higher rates of electron transport are required to generate a given value of the proton gradient in the trypsin-treated membranes than in control membranes, indicating that the treated membranes are proton leaky. Since venturicidin restores electron transport and the proton gradient to control levels, the proton leak is through the ATP synthase. Remarkably, the synthesis of ATP by the trypsin-treated membranes at saturating light intensities is only slightly inhibited even though the proton gradient is significantly lower in the treated thylakoids. ATP synthesis and the proton gradient were determined as a function of light intensity in control and trypsin-treated thylakoids. The trypsin-treated membranes synthesized ATP at lower values of the proton gradient than the control membranes. Cleavage of the gamma subunit abrogates inhibition of the activity of the chloroplast ATP synthase by the epsilon subunit. Our results suggest that overcoming inhibition by the epsilon subunit costs energy.

[Covalent binding of 1,N(6)-ethenoadenosine diphosphate to catalytic and noncatalytic sites of chloroplast ATP-synthase].

The catalytic and noncatalytic sites of the chloroplast coupling factor (CF1) were selectively modified by incubation with the dialdehyde derivative of the fluorescent adenosine diphosphate analogue 1,N(6)-ethenoadenosine diphosphate. The modified CF1 was reconstituted with EDTA-treated chloroplast thylakoid membranes. The influence of light-induced transmembrane proton gradient and of phosphate ions on the fluorescence of 1,N(6)-ethenoadenosine diphosphate covalently bound to catalytic sites of reconstituted CF1 (ATP-synthase) was studied. Upon illumination of thylakoid membranes with saturating white light, the quenching of fluorescence of covalently bound 1,N(6)-ethenoadenosine diphosphate was observed. The quenching was reversed by the addition of inorganic phosphate to the reaction mixture in the dark. Repeated illumination induced the quenching once again: however, the addition of phosphate ions did not affect the fluorescence intensity now. When 1,N(6)-ethenoadenosine diphosphate was covalently bound to noncatalytic sites of ATP-synthase, no similar fluorescent changes were observed. The interrelation of the observed changes of 1,N(6)-ethenoadenosine diphosphate fluorescence and the mechanism of energy-dependent changes in the structure of the catalytic site of ATP-synthase is discussed.

Substitutions of the conserved Gly47 affect the CF1 inhibitor and proton gate functions of the chloroplast ATP synthase epsilon subunit.

The conserved residue Gly47 of the chloroplast ATP synthase beta subunit was substituted with Leu, Arg, Ala and Glu by site-directed mutagenesis. This process generated the mutants epsilon G47L, epsilon G47R, epsilon G47A and epsilon G47E, respectively. All the beta variants showed lower inhibitory effects on the soluble CF1(-epsilon) Ca2+-ATPase compared with wild-type epsilon. In reduced conditions, epsilon G47E and epsilon G47R had a lower inhibitory effect on the oxidized CF1(-epsilon) Ca2+-ATPase compared with wild-type epsilon. In contrast, epsilon G47L and epsilon G47A increased the Ca2+-ATPase activity of soluble oxidized CF1(-epsilon). The replacement of Gly47 significantly impaired the interaction between the subunit epsilon and gamma in an in vitro binding assay? Further study showed that all epsilon variants were more effective in blocking proton leakage from the thylakoid membranes. This enhanced ATP synthesis of the chloroplast and restored ATP synthesis activity of the reconstituted membranes to a level that was more efficient than that achieved by wild-type epsilon. These results indicate that the conserved Gly47 residue of the epsilon subunit is very important for maintaining the structure and function of the epsilon subunit and may affect the interaction between the epsilon subunit, beta subunit of CF1 and subunit III of CFo, thereby regulating the ATP hydrolysis and synthesis, as well as the proton translocation role of the subunit III of CFo.

The C-terminal domain of the epsilon subunit of the chloroplast ATP synthase is not required for ATP synthesis.

The epsilon subunit of the ATP synthases from chloroplasts and Escherichia coli regulates the activity of the enzyme and is required for ATP synthesis. The epsilon subunit is not required for the binding of the catalytic portion of the chloroplast ATP synthase (CF1) to the membrane-embedded part (CFo). Thylakoid membranes reconstituted with CF1 lacking its epsilon subunit (CF1-epsilon) have high ATPase activity and no ATP synthesis activity, at least in part because the membranes are very leaky to protons. Either native or recombinant epsilon subunit inhibits ATPase activity and restores low proton permeability and ATP synthesis. In this paper we show that recombinant epsilon subunit from which 45 amino acids were deleted from the C-terminus is as active as full-length epsilon subunit in restoring ATP synthesis to membranes containing CF1-epsilon. However, the truncated form of the epsilon subunit was significantly less effective as an inhibitor of the ATPase activity of CF1-epsilon, both in solution and bound to thylakoid membranes. Thus, the C-terminus of the epsilon subunit is more involved in regulation of activity, by inhibiting ATP hydrolysis, than in ATP synthesis.

The carboxyl terminus of the epsilon subunit of the chloroplast ATP synthase is exposed during illumination.

The epsilon subunit of the chloroplast ATP synthase is an inhibitor of activity of the enzyme. Recombinant forms of the epsilon subunit from spinach chloroplasts lacking the last 10, 32, or 45 amino acids were immobilized onto activated Sepharose. A polyclonal antiserum raised against the epsilon subunit was passed over these immobilized protein columns, and the purified antibodies which were not bound recognized the portions of the epsilon subunit missing from the recombinant form present on the column. The full polyclonal antiserum can strip the epsilon subunit from the ATP synthase in illuminated thylakoid membranes [Richter, M. L., and McCarty, R. E. (1987) J. Biol. Chem. 262, 15037-15040]. Exposure of illuminated thylakoid membranes to antibodies recognizing the last 32 amino acids of the epsilon subunit collapses the proton gradient and hinders ATP synthesis with similar efficiency as the full polyclonal preparation. These results indicate that antibodies against the last 32 amino acids of the epsilon subunit are capable of stripping the subunit from the ATP synthase in illuminated membranes. Neither of these effects was seen when the membranes were exposed to the antibodies in the dark. This is direct evidence that the chloroplast ATP synthase undergoes a conformational shift during its activation by the electrochemical proton gradient which specifically alters the conformation of the carboxyl-terminal domain of the epsilon subunit from protected to solvent-exposed. The relation between this shift and activation of the enzyme by the electrochemical proton gradient is discussed.

Introduction of a carboxyl group in the loop of the F0 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 (deltaGp) and the proton gradient (deltamuH+) 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.