Effect of cetyltrimethylammonium on ATP hydrolysis and proton translocation in the F0-F1 H+-ATP synthase of mitochondria

The amphiphylic alkyl cation cetyltrimethylammonium inhibits the catalytic activity of soluble and membrane-bound F₁ in a noncompetitive fashion. In sonic submitochondrial particles the Dixon plot showed a peculiar pattern with upward deviation at cetyltrimethylammonium concentration higher than 80µM. In membrane-bound F₁ the inhibition by cetyltrimethylammonium was potentiated by the F₀ inhibitor ologomycin. Cetyltrimethylammonium also inhibited the oligomycin-sensitive proton conductivity in F₁-containing particles but was without any effect in F₁-depleted particles. Also this inhibitory effect was potentiated by oligomycin. These results indicate functional cooperative interactions between F₀ and F₁.     

A functionally inactive, cold-stabilized form of the Escherichia coli F1Fo ATP synthase

An unusual effect of temperature on the ATPase activity of E. coli F₁F_o ATP synthase has been investigated. The rate of ATP hydrolysis by the isolated enzyme, previously kept on ice, showed a lag phase when measured at 15 °C, but not at 37 °C. A pre-incubation of the enzyme at room temperature for 5 min completely eliminated the lag phase, and resulted in a higher steady-state rate. Similar results were obtained using the isolated enzyme after incorporation into liposomes. The initial rates of ATP-dependent proton translocation, as measured by 9-amino-6-chloro-2-methoxyacridine (ACMA) fluorescence quenching, at 15 °C also varied according to the pre-incubation temperature. The relationship between this temperature-dependent pattern of enzyme activity, termed thermohysteresis, and pre-incubation with other agents was examined. Pre-incubation of membrane vesicles with azide and Mg²⁺, without exogenous ADP, resulted in almost complete inhibition of the initial rate of ATPase when assayed at 10 °C, but had little effect at 37 °C. Rates of ATP synthesis following this pre-incubation were not affected at any temperature. Azide inhibition of ATP hydrolysis by the isolated enzyme was reduced when an ATP-regenerating system was used. A gradual reactivation of azide-blocked enzyme was slowed down by the presence of phosphate in the reaction medium. The well-known Mg²⁺ inhibition of ATP hydrolysis was shown to be greatly enhanced at 15 °C relative to at 37 °C. The results suggest that thermohysteresis is a consequence of an inactive form of the enzyme that is stabilized by the binding of inhibitory Mg-ADP.     

Involvement of ATP synthase residues alphaArg-376, betaArg-182, and betaLys-155 in Pi binding

alphaArg-376, betaLys-155, and betaArg-182 are catalytically important ATP synthase residues that were proposed to be involved in substrate Pi binding and subsequent steps of ATP synthesis [Senior, A.E., Nadanaciva, S. and Weber, J. (2002) Biochim. Biophys. Acta 1553, 188-211]. Here, it was shown using purified Escherichia coli F(1)-ATPase that whereas Pi protected wild-type from reaction with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, mutations betaK155Q, betaR182Q, betaR182K, and alphaR376Q abolished protection. Therefore, in ATP synthesis initial binding of substrate Pi in open catalytic site betaE is supported by each of these three residues.     

Met23Lys mutation in subunit gamma of FOF1-ATP synthase from Rhodobacter capsulatus impairs the activation of ATP hydrolysis by protonmotive force

H⁺-F_OF₁-ATP synthase couples proton flow through its membrane portion, F_O, to the synthesis of ATP in its headpiece, F₁. Upon reversal of the reaction the enzyme functions as a proton pumping ATPase. Even in the simplest bacterial enzyme the ATPase activity is regulated by several mechanisms, involving inhibition by MgADP, conformational transitions of the ε subunit, and activation by protonmotive force. Here we report that the Met23Lys mutation in the γ subunit of the Rhodobacter capsulatus ATP synthase significantly impaired the activation of ATP hydrolysis by protonmotive force. The impairment in the mutant was due to faster enzyme deactivation that was particularly evident at low ATP/ADP ratio. We suggest that the electrostatic interaction of the introduced γLys23 with the DELSEED region of subunit β stabilized the ADP-inhibited state of the enzyme by hindering the rotation of subunit γ rotation which is necessary for the activation.     

A more robust version of the Arginine 210-switched mutant in subunit a of the Escherichia coli ATP synthase

Previous work has shown that the essential R210 of subunit a in the Escherichia coli ATP synthase can be switched with a conserved glutamine Q252 with retention of a moderate level of function, that a third mutation P204T enhances this function, and that the arginine Q252R can be replaced by lysine without total loss of activity. In this study, the roles of P204T and R210Q were examined. It was concluded that the threonine in P204T is not directly involved in function since its replacement by alanine did not significantly affect growth properties. Similarly, it was concluded that the glutamine in R210Q is not directly involved with function since replacement by glycine results in significantly enhanced function. Not only did the rate of ATP-driven proton translocation increase, but also the sensitivity of ATP hydrolysis to inhibition by N,N′-dicyclohexylcarbodiimide (DCCD) rose to more than 50%. Finally, mutations at position E219, a residue near the proton pathway, were used to test whether the Arginine-switched mutant uses the normal proton pathway. In a wild type background, the E219K mutant was confirmed to have greater function than the E219Q mutant, as has been shown previously. This same unusual result was observed in the triple mutant background, P204T/R210Q/Q252R, suggesting that the Arginine-switched mutants are using the normal proton pathway from the periplasm.     

Analysis of ionic channels by a flash spectrophotometric technique applicable to thylakoid membranes: CF0, the proton channel of the chloroplast ATP synthase,and, for comparison,gramicidin

Summary We previously introduced a flash spectrophotometric method to analyze proton conduction by CF₀ in vesicles derived from thylakoid membranes (H. Lill, S. Engelbrecht, G. Schönknecht & W. Junge, 1986,Eur. J. Biochem.160:627–634). The unit conductance of CF₀, as revealed by this technique, was orders of magnitude higher than that theoretically expected for a hydrogen-bonded chain. We scrutinized the validity of this method. Small vesicles were derived from thylakoids by EDTA treatment. The intrinsic electric generators in the membrane were stimulated by short flashes of light and the relaxation of the voltage via ionic channels was measured through electrochromic absorption changes of intrinsic pigments. The voltage decay was stimulated by a statistical model. As the vesicle-size distribution had only a minor influence, the simulation required only two fit parameters, the first proportional to the unit conductance of an active channelG, and the second denoting the average number of active channels per vesiclen. This technique was applied to CF₀, the proton channel of the chloroplast ATP synthase, and to gramicidin, serving as a standard. For both channels we found the above two fit parameters physically meaningful. They could be independently varied in predictable wasy, i.e.n by addition of known inhibitors of F₀-type proton channels andG via the temperature. for gramicidin, the unit conductance (2.7 pS) was within the range described in the literature. This established the competence of this method for studies on the mechanism of proton conduction by CF₀, whose conductance so far has not been accessible to other, more conventional approaches. The time-averaged unit conductance of CF₀ was about 1 pS, equivalent to the turnover of 6×10⁵ H⁺/(CF₀·sec) at 100 mV driving force.     

ATP synthesis without R210 of subunit a in the Escherichia coli ATP synthase

Interactions between subunit a and oligomeric subunit c are essential for the coupling of proton translocation to rotary motion in the ATP synthase. A pair of previously described mutants, R210Q/Q252R and P204T/R210Q/Q252R [L.P. Hatch, G.B. Cox and S.M. Howitt, The essential arginine residue at position 210 in the a subunit of the Escherichia coli ATP synthase can be transferred to position 252 with partial retention of activity, J. Biol. Chem. 270 (1995) 29407-29412] has been constructed and further analyzed. These mutants, in which the essential arginine of subunit a, R210, was switched with a conserved glutamine residue, Q252, are shown here to be capable of both ATP synthesis by oxidative phosphorylation, and ATP-driven proton translocation. In addition, lysine can replace the arginine at position 252 with partial retention of both activities. The pH dependence of ATP-driven proton translocation was determined after purification of mutant enzymes, and reconstitution into liposomes. Proton translocation by the lysine mutant, and to a lesser extent the arginine mutant, dropped off sharply above pH 7.5, consistent with the requirement for a positive charge during function. Finally, the rates of ATP synthesis and of ATP-driven proton translocation were completely inhibited by treatment with DCCD (N,N′-dicyclohexylcarbodiimide), while rates of ATP hydrolysis by the mutants were not significantly affected, indicating that DCCD modification disrupts the F₁-F_o interface. The results suggest that minimal requirements for proton translocation by the ATP synthase include a positive charge in subunit a and a weak interface between subunit a and oligomeric subunit c.     

The size of the population of weakly coupled chlorophyll pigments involved in thylakoid photoinhibition determined by steady-state fluorescence spectroscopy

On the basis of experiments with singlet quenchers and in agreement with previous data, it is suggested that a population of energetically weakly coupled chlorophylls may play a central role in photoinhibition in vivo and in vitro. In the present study, we have used steady state fluorescence techniques to gain direct evidence for these uncoupled chlorophylls. Due to the presence of their emission maxima, near 650 nm and more prominently in the 670-675 nm interval both chlorophylls b and a seem to be involved. A straightforward mathematical model is developed to describe the data which allows us to conclude that the uncoupled/weakly coupled population size is in the range of 1-3 molecules per photosystem.     

Interactions involved in grasping and locking of the inhibitory peptide IF1 by mitochondrial ATP synthase

When mitochondria become deenergized, futile ATP hydrolysis is prevented by reversible binding of an endogenous inhibitory peptide called IF1 to ATP synthase. Between initial IF1 binding and IF1 locking the enzyme experiences large conformational changes. While structural studies give access to analysis of the dead-end inhibited state, transient states have thus far not been described. Here, we studied both initial and final states by reporting, for the first time, the consequences of mutations of Saccharomyces cerevisiae ATP synthase on its inhibition by IF1. Kinetic studies allowed the identification of amino acids or motifs of the enzyme that are involved in recognition and/or locking of IF1 α-helical midpart. This led to an outline of IF1 binding process. In the recognition step, protruding parts of α and especially β subunits grasp IF1, most likely by a few residues of its α-helical midpart. Locking IF1 within the αβ interface involves additional residues of both subunits. Interactions of the α and β subunits with the foot of the γ subunit might contribute to locking and stabilizing of the dead-end state.     

Disulfide-bond formation in the H+-pyrophosphatase of Streptomyces coelicolor and its implications for redox control and enzyme structure

Redox control of disulfide-bond formation in the H+-pyrophosphatase of Streptomyces coelicolor was investigated using cysteine mutants expressed in Escherichia coli. The wild-type enzyme, but not a cysteine-less mutant, was reversibly inactivated by oxidation. To determine the residues involved in oxidative inactivation, different cysteine residues were replaced. Analysis with a cysteine-modifying reagent revealed that the formation of a disulfide bond between cysteines 253 and 621 was responsible for enzyme inactivation. This result suggests that residues in different cytoplasmic loops are close to each other in the tertiary structure. Both cysteine residues are conserved in K+-independent (type II) H+-pyrophosphatases.     

Spermine and spermidine inhibition of photosystem II: Disassembly of the oxygen evolving complex and consequent perturbation in electron donation from TyrZ to P680 and the quinone acceptors QA to QB

Polyamines are implicated in plant growth and stress response. However, the polyamines spermine and spermidine were shown to elicit strong inhibitory effects in photosystem II (PSII) submembrane fractions. We have studied the mechanism of this inhibitory action in detail. The inhibition of electron transport in PSII submembrane fractions treated with millimolar concentrations of spermine or spermidine led to the decline of plastoquinone reduction, which was reversed by the artificial electron donor diphenylcarbazide. The above inhibition was due to the loss of the extrinsic polypeptides associated with the oxygen evolving complex. Thermoluminescence measurements revealed that charge recombination between the quinone acceptors of PSII, Q_A and Q_B, and the S₂ state of the Mn-cluster was abolished. Also, the dark decay of chlorophyll fluorescence after a single turn-over white flash was greatly retarded indicating a slower rate of Q_A⁻ reoxidation.     

Characterization of wave phenomena in the relaxation of flash-induced chlorophyll fluorescence yield in cyanobacteria

Fluorescence yield relaxation following a light pulse was studied in various cyanobacteria under aerobic and microaerobic conditions. In Synechocystis PCC 6803 fluorescence yield decays in a monotonous fashion under aerobic conditions. However, under microaerobic conditions the decay exhibits a wave feature showing a dip at 30–50 ms after the flash followed by a transient rise, reaching maximum at ~ 1 s, before decaying back to the initial level. The wave phenomenon can also be observed under aerobic conditions in cells preilluminated with continuous light. Illumination preconditions cells for the wave phenomenon transiently: for few seconds in Synechocystis PCC 6803, but up to one hour in Thermosynechocystis elongatus BP-1. The wave is eliminated by inhibition of plastoquinone binding either to the Q_B site of Photosystem-II or the Q_o site of cytochrome b₆f complex by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea or 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, respectively. The wave is also absent in mutants, which lack either Photosystem-I or the NAD(P)H-quinone oxidoreductase (NDH-1) complex. Monitoring the redox state of the plastoquinone pool revealed that the dip of the fluorescence wave corresponds to transient oxidation, whereas the following rise to re-reduction of the plastoquinone pool. It is concluded that the unusual wave feature of fluorescence yield relaxation reflects transient oxidation of highly reduced plastoquinone pool by Photosystem-I followed by its re-reduction from stromal components via the NDH-1 complex, which is transmitted back to the fluorescence yield modulator primary quinone electron acceptor via charge equilibria. Potential applications of the wave phenomenon in studying photosynthetic and respiratory electron transport are discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

An HPLC-based method of estimation of the total redox state of plastoquinone in chloroplasts, the size of the photochemically active plastoquinone-pool and its redox state in thylakoids of Arabidopsis

We have described a direct, high-performance liquid chromatography-based method of estimation of the total level of plastoquinone (PQ) in leaves, the redox state of total (photoactive and non-photoactive) PQ, as well as the redox state of the PQ-pool that is applicable to any illumination conditions. This method was applied to Arabidopsis thaliana leaves but it can be applied to any other plant species. The obtained results show that the level of total PQ was 25 ± 3 molecules/1000 chlorophyll (Chl) molecules in relation to foliar total Chl content. The level of the photoactive PQ, i.e., the PQ-pool, was about 31% of the total PQ present in Arabidopsis leaves that corresponds to about 8 PQ molecules/1000 Chl molecules. The reduction level of the non-photoactive PQ fraction, present outside thylakoids in chloroplasts, was estimated to account for about 49%. The measurements of the redox state of the PQ-pool showed that the pool was reduced during the dark period in about 24%, and during the light period (150 μmol/m²·s) the reduction of the PQ-pool increased to nearly 100%. The obtained results were discussed in terms of the activity of chlororespiration pathways in Arabidopsis and the regulatory role of the redox state of PQ-pool in various physiological and molecular processes in plants.     

Non-photochemical quenching of chlorophyll a fluorescence by oxidised plastoquinone: new evidences based on modulation of the redox state of the endogenous plastoquinone pool in broken spinach chloroplasts

Twenty-five years ago, non-photochemical quenching of chlorophyll fluorescence by oxidised plastoquinone (PQ) was proposed to be responsible for the lowering of the maximum fluorescence yield reported to occur when leaves or chloroplasts were treated in the dark with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of electron flow beyond the primary quinone electron acceptor (Q_A) of photosystem (PS) II [C. Vernotte, A.L. Etienne, J.-M. Briantais, Quenching of the system II chlorophyll fluorescence by the plastoquinone pool, Biochim. Biophys. Acta 545 (1979) 519-527]. Since then, the notion of PQ-quenching has received support but has also been put in doubt, due to inconsistent experimental findings. In the present study, the possible role of the native PQ-pool as a non-photochemical quencher was reinvestigated, employing measurements of the fast chlorophyll a fluorescence kinetics (from 50 μs to 5 s). The about 20% lowering of the maximum fluorescence yield F_M, observed in osmotically broken spinach chloroplasts treated with DCMU, was eliminated when the oxidised PQ-pool was non-photochemically reduced to PQH₂ by dark incubation of the samples in the presence of NAD(P)H, both under anaerobic and aerobic conditions. Incubation under anaerobic conditions in the absence of NAD(P)H had comparatively minor effects. In DCMU-treated samples incubated in the presence of NAD(P)H fluorescence quenching started to develop again after 20-30 ms of illumination, i.e., the time when PQH₂ starts getting reoxidised by PS I activity. NAD(P)H-dependent restoration of F_M was largely, if not completely, eliminated when the samples were briefly (5 s) pre-illuminated with red or far-red light. Addition to the incubation medium of HgCl₂ that inhibits dark reduction of PQ by NAD(P)H also abolished NAD(P)H-dependent restoration of F_M. Collectively, our results provide strong new evidence for the occurrence of PQ-quenching. The finding that DCMU alone did not affect the minimum fluorescence yield F₀ allowed us to calculate, for different redox states of the native PQ-pool, the fractional quenching at the F₀ level (Q₀) and to compare it with the fractional quenching at the F_M level (Q_M). The experimentally determined Q₀/Q_M ratios were found to be equal to the corresponding F₀/F_M ratios, demonstrating that PQ-quenching is solely exerted on the excited state of antenna chlorophylls.     

Remodeling of tobacco thylakoids by over-expression of maize plastidial transglutaminase

Transglutaminases (TGases, EC 2.3.2.13) are intra- and extra-cellular enzymes that catalyze post-translational modification of proteins by establishing ?-(γ-glutamyl) links and covalent conjugation of polyamines. In chloroplast it is well established that TGases specifically polyaminylate the light-harvesting antenna of Photosystem (PS) II (LHCII, CP29, CP26, CP24) and therefore a role in photosynthesis has been hypothesised (Della Mea et al. [23] and refs therein). However, the role of TGases in chloroplast is not yet fully understood. Here we report the effect of the over-expression of maize (Zea mays) chloroplast TGase in tobacco (Nicotiana tabacum var. Petit Havana) chloroplasts. The transglutaminase activity in over-expressers was increased 4 times in comparison to the wild-type tobacco plants, which in turn increased the thylakoid associated polyamines about 90%. Functional comparison between Wt tobacco and tgz over-expressers is shown in terms of fast fluorescence induction kinetics, non-photochemical quenching of the singlet excited state of chlorophyll a and antenna heterogeneity of PSII. Both in vivo probing and electron microscopy studies verified thylakoid remodeling. PSII antenna heterogeneity in vivo changes in the over-expressers to a great extent, with an increase of the centers located in grana-appressed regions (PSIIα) at the expense of centers located mainly in stroma thylakoids (PSIIβ). A major increase in the granum size (i.e. increase of the number of stacked layers) with a concomitant decrease of stroma thylakoids is reported for the TGase over-expressers.     

ATP and magnesium drive conformational changes of the Na/K-ATPase cytoplasmic headpiece

Conformational changes of the Na⁺/K⁺-ATPase isolated large cytoplasmic segment connecting transmembrane helices M4 and M5 (C45) induced by the interaction with enzyme ligands (i.e. Mg²⁺ and/or ATP) were investigated by means of the intrinsic tryptophan fluorescence measurement and molecular dynamic simulations. Our data revealed that this model system consisting of only two domains retained the ability to adopt open or closed conformation, i.e. behavior, which is expected from the crystal structures of relative Ca²⁺-ATPase from sarco(endo)plasmic reticulum for the corresponding part of the entire enzyme. Our data revealed that the C45 is found in the closed conformation in the absence of any ligand, in the presence of Mg²⁺ only, or in the simultaneous presence of Mg²⁺ and ATP. Binding of the ATP alone (i.e. in the absence of Mg²⁺) induced open conformation of the C45. The fact that the transmembrane part of the enzyme was absent in our experiments suggested that the observed conformational changes are consequences only of the interaction with ATP or Mg²⁺ and may not be related to the transported cations binding/release, as generally believed. Our data are consistent with the model, where ATP binding to the low-affinity site induces conformational change of the cytoplasmic part of the enzyme, traditionally attributed to E2 → E1 transition, and subsequent Mg²⁺ binding to the enzyme-ATP complex induces in turn conformational change traditionally attributed to E1 → E2 transition.     

Some Photosystem II properties depending on the D1 protein variants in Thermosynechococcus elongatus

Cyanobacteria have multiple psbA genes encoding PsbA, the D1 reaction center protein of the Photosystem II complex which bears together with PsbD, the D2 protein, most of the cofactors involved in electron transfer reactions. The thermophilic cyanobacterium Thermosynechococcus elongatus has three psbA genes differently expressed depending on the environmental conditions. Among the 344 residues constituting each of the 3 possible PsbA variants there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2 and 27 between PsbA2 and PsbA3. In this review, we summarize the changes already identified in the properties of the redox cofactors depending on the D1 variant constituting Photosystem II in T. elongatus. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Domain Features of the Peripheral Stalk Subunit H of the Methanogenic A1AO ATP Synthase and the NMR Solution Structure of H1-47

A series of truncated forms of subunit H were generated to establish the domain features of that protein. Circular dichroism analysis demonstrated that H is divided at least into a C-terminal coiled-coil domain within residues 54-104, and an N-terminal domain formed by adjacent α-helices. With a cysteine at the C-terminus of each of the truncated proteins (H_1-47, H_1-54, H_1-59, H_1-61, H_1-67, H_1-69, H_1-71, H_1-78, H_1-80, H_1-91, and H_47-105), the residues involved in formation of the coiled-coil interface were determined. Proteins H_1-54, H_1-61, H_1-69, and H_1-80 showed strong cross-link formation, which was weaker in H_1-47, H_1-59, H_1-71, and H_1-91. A shift in disulfide formation between cysteins at positions 71 and 80 reflected an interruption in the periodicity of hydrophobic residues in the region ₇₁AEKILEETEKE₈₁. To understand how the N-terminal domain of H is formed, we determined for the first time, to our knowledge, the solution NMR structure of H_1-47, which revealed an α-helix between residues 15-42 and a flexible N-terminal stretch. The α-helix includes a kink that would bring the two helices of the C-terminus into the coiled-coil arrangement. H_1-47 revealed a strip of alanines involved in dimerization, which were tested by exchange to single cysteines in subunit H mutants.     

Parallel expression of alternate forms of psbA2 gene provides evidence for the existence of a targeted D1 repair mechanism in Synechocystis sp. PCC 6803

The D1 protein of Photosystem II (PSII) is recognized as the main target of photoinhibitory damage and exhibits a high turnover rate due to its degradation and replacement during the PSII repair cycle. Damaged D1 is replaced by newly synthesized D1 and, although reasonable, there is no direct evidence for selective replacement of damaged D1. Instead, it remains possible that increased turnover of D1 subunits occurs in a non-selective manner due for example, to a general up-regulation of proteolytic activity triggered during damaging environmental conditions, such as high light. To determine if D1 degradation is targeted to damaged D1 or generalized to all D1, we developed a genetic system involving simultaneous dual expression of wild type and mutant versions of D1 protein. Dual D1 strains (nS345P:eWT and nD170A:eWT) expressed a wild type (WT) D1 from ectopic and a damage prone mutant (D1-S345P, D1-D170A) from native locus on the chromosome. Characterization of strains showed that all dual D1 strains restore WT like phenotype with high PSII activity. Higher PSII activity indicates increased population of PSII reaction centers with WT D1. Analysis of steady state levels of D1 in nS345P:eWT by immunoblot showed an accumulation of WT D1 only. But, in vivo pulse labeling confirmed the synthesis of both S345P (exists as iD1) and WT D1 in the dual strain. Expression of nS345P:eWT in FtsH2 knockout background showed accumulation of both iD1 and D1 proteins. This demonstrates that dual D1 strains express both forms of D1, yet only damage prone PSII complexes are selected for repair providing evidence that the D1 degradation process is targeted towards damaged PSII complexes. Since the N-terminus has been previously shown to be important for the degradation of damaged D1, the possibility that the highly conserved cysteine 18 residue situated in the N-terminal domain of D1 is involved in the targeted repair process was tested by examining site directed mutants of this and the other cysteines of the D1 protein. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.