Chloroplast F1 Research Articles

Acetylation and phosphorylation control both local and global stability of the chloroplast F1 ATP synthase

ATP synthases (ATPases) are enzymes that produce ATP and control the pH in the cell or cellular compartments. While highly conserved over different species, ATPases are structurally well-characterised but the existence and functional significance of many post-translational modifications (PTMs) is not well understood. We combined a range of mass spectrometric techniques to unravel the location and extent of PTMs in the chloroplast ATP synthase (cATPase) purified from spinach leaves. We identified multiple phosphorylation and acetylation sites and found that both modifications stabilise binding of ε and δ subunits. Comparing cross-linking of naturally modified cATPase with the in vitro deacetylated enzyme revealed a major conformational change in the ε subunit in accord with extended and folded forms of the subunit. Locating modified residues within the catalytic head we found that phosphorylated and acetylated residues are primarily on α/β and β/α interfaces respectively. By aligning along different interfaces the higher abundance acetylated residues are proximal to the regulatory sites while the lower abundance phosphorylation sites are more densely populated at the catalytic sites. We propose that modifications in the catalytic head, together with the conformational change in subunit ε, work in synergy to fine-tune the enzyme during adverse conditions.

Redox regulation of CF1-ATPase involves interplay between the γ-subunit neck region and the turn region of the βDELSEED-loop

The soluble F1 complex of ATP synthase (FoF1) is capable of ATP hydrolysis, accomplished by the minimum catalytic core subunits α3β3γ. A special feature of cyanobacterial F1 and chloroplast F1 (CF1) is an amino acid sequence inserted in the γ-subunit. The insertion is extended slightly into the CF1 enzyme containing two additional cysteines for regulation of ATPase activity via thiol modulation. This molecular switch was transferred to a chimeric F1 by inserting the cysteine-containing fragment from spinach CF1 into a cyanobacterial γ-subunit [Y. Kim et al., redox regulation of rotation of the cyanobacterial F1-ATPase containing thiol regulation switch, J Biol Chem, 286 (2011) 9071–9078]. Under oxidizing conditions, the obtained F1 tends to lapse into an ADP-inhibited state, a common regulation mechanism to prevent wasteful ATP hydrolysis under unfavorable circumstances. However, the information flow between thiol modulation sites on the γ-subunit and catalytic sites on the β-subunits remains unclear. Here, we clarified a possible interplay for the CF1-ATPase redox regulation between structural elements of the βDELSEED-loop and the γ-subunit neck region, i.e., the most convex part of the α-helical γ-termini. Critical residues were assigned on the β-subunit, which received the conformation change signal produced by disulfide/dithiol formation on the γ-subunit. Mutant response to the ATPase redox regulation ranged from lost to hypersensitive. Furthermore, mutant cross-link experiments and inversion of redox regulation indicated that the γ-redox state might modulate the subunit interface via reorientation of the βDELSEED motif region.

Resolving the Negative Potential Side (n-side) Water-accessible Proton Pathway of F-type ATP Synthase by Molecular Dynamics Simulations

The rotation of F(1)F(o)-ATP synthase is powered by the proton motive force across the energy-transducing membrane. The protein complex functions like a turbine; the proton flow drives the rotation of the c-ring of the transmembrane F(o) domain, which is coupled to the ATP-producing F(1) domain. The hairpin-structured c-protomers transport the protons by reversible protonation/deprotonation of a conserved Asp/Glu at the outer transmembrane helix (TMH). An open question is the proton transfer pathway through the membrane at atomic resolution. The protons are thought to be transferred via two half-channels to and from the conserved cAsp/Glu in the middle of the membrane. By molecular dynamics simulations of c-ring structures in a lipid bilayer, we mapped a water channel as one of the half-channels. We also analyzed the suppressor mutant cP24D/E61G in which the functional carboxylate is shifted to the inner TMH of the c-protomers. Current models concentrating on the "locked" and "open" conformations of the conserved carboxylate side chain are unable to explain the molecular function of this mutant. Our molecular dynamics simulations revealed an extended water channel with additional water molecules bridging the distance of the outer to the inner TMH. We suggest that the geometry of the water channel is an important feature for the molecular function of the membrane part of F(1)F(o)-ATP synthase. The inclination of the proton pathway isolates the two half-channels and may contribute to a favorable clockwise rotation in ATP synthesis mode.

C-Terminal Mutations in the Chloroplast ATP Synthase γ Subunit Impair ATP Synthesis and Stimulate ATP Hydrolysis

Two highly conserved amino acid residues, an arginine and a glutamine, located near the C-terminal end of the gamma subunit, form a "catch" by hydrogen bonding with residues in an anionic loop on one of the three catalytic beta subunits of the bovine mitochondrial F1-ATPase [Abrahams, J. P., Leslie, A. G., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628]. The catch is considered to play a critical role in the binding change mechanism whereby binding of ATP to one catalytic site releases the catch and induces a partial rotation of the gamma subunit. This role is supported by the observation that mutation of the equivalent arginine and glutamine residues in the Escherichia coli F1 gamma subunit drastically reduced all ATP-dependent catalytic activities of the enzyme [Greene, M. D., and Frasch, W. D. (2003) J. Biol. Chem. 278, 5194-5198]. In this study, we show that simultaneous substitution of the equivalent residues in the chloroplast F1 gamma subunit, arginine 304 and glutamine 305, with alanine decreased the level of proton-coupled ATP synthesis by more than 80%. Both the Mg2+-dependent and Ca2+-dependent ATP hydrolysis activities increased by more than 3-fold as a result of these mutations; however, the sulfite-stimulated activity decreased by more than 60%. The Mg2+-dependent, but not the Ca2+-dependent, ATPase activity of the double mutant was insensitive to inhibition by the phytotoxic inhibitor tentoxin, indicating selective loss of catalytic cooperativity in the presence of Mg2+ ions. The results indicate that the catch residues are required for efficient proton coupling and for activation of multisite catalysis when MgATP is the substrate. The catch is not, however, required for CaATP-driven multisite catalysis or, therefore, for rotation of the gamma subunit.

Mutations within the C-Terminus of the γ Subunit of the Photosynthetic F1-ATPase Activate MgATP Hydrolysis and Attenuate the Stimulatory Oxyanion Effect

Two highly conserved amino acid residues near the C-terminus within the gamma subunit of the mitochondrial ATP synthase form a "catch" with an anionic loop on one of the three beta subunits within the catalytic alphabeta hexamer of the F1 segment [Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628]. Forming the catch is considered to be an essential step in cooperative nucleotide binding leading to gamma subunit rotation. The analogous residues, Arg304 and Gln305, in the chloroplast F1 gamma subunit were changed to leucine and alanine, respectively. Each mutant gamma was assembled together with alpha and beta subunits from Rhodospirillum rubrum F1 into a hybrid photosynthetic F1 that carries out both MgATPase and CaATPase activities and ATP-dependent gamma rotation [Tucker, W. C., Schwarcz, A., Levine, T., Du, Z., Gromet-Elhanan, Z., Richter, M. L. and Haran, G. (2004) J. Biol. Chem. 279, 47415-47418]. Surprisingly, changing Arg304 to leucine resulted in a more than 2-fold increase in the kcat for MgATP hydrolysis. In contrast, changing Gln305 to alanine had little effect on the kcat but completely abolished the well-known stimulatory effect of the oxyanion sulfite on MgATP hydrolysis. The MgATPase activities of combined mutants with both residues substituted were strongly inhibited, whereas the CaATPase activities were inhibited, but to a lesser extent. The results indicate that the C-terminus of the photosynthetic F1 gamma subunit, like its mitochondrial counterpart, forms a catch with the alpha and beta subunits that modulates the nucleotide binding properties of the catalytic site(s). The catch is likely to be part of an activation mechanism, overcoming inhibition by free mg2+ ions, but is not essential for cooperative nucleotide exchange.

Characterization of a phosphate binding domain on the alpha-subunit of chloroplast ATP synthase using the photoaffinity phosphate analogue 4-azido-2-nitrophenyl phosphate.

The photoaffinity phosphate analogue 4-azido-2 nitrophenyl phosphate (ANPP) was shown previously (Pougeois, R., Lauquin, G. J.-M., and Vignais, P. V. (1983) Biochemistry 22, 1241-1245) to bind covalently and specifically to a single catalytic site on one of the three beta-subunits of the isolated chloroplast coupling factor 1 (CF(1)). Modification by ANPP strongly inhibited ATP hydrolysis activity. In this study, we examined labeling of membrane-bound CF(1) by ANPP by exposing thylakoid membranes to increasing concentrations of the reagent. ANPP exhibited saturable binding to two sites on CF(1), one on the beta-subunit and one on the alpha-subunit. Labeling by ANPP resulted in the complete inhibition of both ATP synthesis and ATP hydrolysis by the membrane-bound enzyme. Labeling of both sites by ANPP was reduced by more than 80% in the presence of P(i) (> or = 10 mM) and ATP (> or = 0.5 mM). ADP was less effective in competing with ANPP for binding, giving a maximum of approximately 35% inhibition at concentrations > or = 2 mM. ANPP-labeled tryptic peptides of the alpha-subunit were isolated and sequenced. The majority of the probe was contained in three peptides corresponding to residues Gln(173) to Arg(216), Gly(217) to Arg(253), and His(256) to Arg(272) of the alpha-subunit. In the mitochondrial F(1) (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), all three analogous peptides are located within the nucleotide binding pocket and within close proximity to the gamma-phosphate binding site. The data indicate, however, that the azidophenyl group of bound ANPP is oriented at approximately 180 degrees in the opposite direction to the adenine binding site with reference to the phosphate binding site on the alpha-subunit. The study has confirmed that ANPP is a bona fide phosphate analogue and suggests that it specifically targets the gamma-phosphate binding site within the nucleotide binding pockets on the alpha- and beta-subunits of CF(1). The study also indicates that in the resting state of the chloroplast F(1)-F(0) complex both the alpha- and beta-subunits are structurally asymmetric.

ATPase Activity of a Highly Stable α3β3γ Subcomplex of Thermophilic F1 Can Be Regulated by the Introduced Regulatory Region of γ Subunit of Chloroplast F1

A mutant F(1)-ATPase alpha(3)beta(3)gamma subcomplex from the thermophilic Bacillus PS3 was constructed, in which 111 amino acid residues (Val(92) to Phe(202)) from the central region of the gamma subunit were replaced by the 148 amino acid residues of the homologous region from spinach chloroplast F(1)-ATPase gamma subunit, including the regulatory stretch, and were designated as alpha(3)beta(3)gamma((TCT)) (Thermophilic-Chloroplast-Thermophilic). By the insertion of this regulatory region into the gamma subunit of thermophilic F(1), we could confer the thiol modulation property to the thermophilic alpha(3)beta(3)gamma subcomplex. The overexpressed alpha(3)beta(3)gamma((TCT)) was easily purified in large scale, and the ATP hydrolyzing activity of the obtained complex was shown to increase up to 3-fold upon treatment with chloroplast thioredoxin-f and dithiothreitol. No loss of thermostability compared with the wild type subcomplex was found, and activation by dithiothreitol was functional at temperatures up to 80 degrees C. alpha(3)beta(3)gamma((TCT)) was inhibited by the epsilon subunit from chloroplast F(1)-ATPase but not by the one from the thermophilic F(1)-ATPase, indicating that the introduced amino acid residues from chloroplast F(1)-gamma subunit are important for functional interaction with the epsilon subunit.

Kinetic Analysis of Tentoxin Binding to Chloroplast F1-ATPase: A MODEL FOR THE OVERACTIVATION PROCESS

The mechanism of action of tentoxin on the soluble part (chloroplast F1 H+-ATPase; CF1) of chloroplast ATP synthase was analyzed in the light of new kinetic and equilibrium experiments. Investigations were done regarding the functional state of the enzyme (activation, bound nucleotide, catalytic turnover). Dialysis and binding data, obtained with 14C-tentoxin, fully confirmed the existence of two tentoxin binding sites of distinct dissociation constants consistent with the observed Kinhibition and Koveractivation. This strongly supports a two-site model of tentoxin action on CF1. Kinetic and thermodynamic parameters of tentoxin binding to the first site (Ki = 10 nM; kon = 4.7 x 10(4) s-1.M-1) were determined from time-resolved activity assays. Tentoxin binding to the high affinity site was found independent on the catalytic state of the enzyme. The analysis of the kinetics of tentoxin binding on the low affinity site of the enzyme showed strong evidence for an interaction between this site and the nucleotide binding sites and revealed a complex relationship between the catalytic state and the reactivation process. New catalytic states of CF1 devoid of epsilon-subunit were detected: a transient overstimulated state, and a dead end complex unable to bind a second tentoxin molecule. Our experiments led to a kinetic model for the reactivation phenomenon for which rate constants were determined. The implications of this model are discussed in relation to the previous mechanistic hypotheses on the effect of tentoxin.

The use of HPLC for the Study of Chloroplast ATPase Enzymatic Activity and ATP Binding

The advantages of the use of HPLC for the purification of the activated chloroplast F1 ATPase, for the measurement of enzymatic activity (by ADP-ATP separation on anion exchange column) and for ATP binding measurement (by the Hummel and Dreyer chromatographic method on gel filtration columns) are pointed out, by comparison with conventional methods such as ATP regenerating method or centrifugation on Sephadex columns. The enzymatic data obtained at various ATP and Mg2+ concentrations and with different Mg2+ chelators and the ATP binding data led to the conclusion that metal free ATP is the true substrate of the enzyme and that Mg2+ is the activator. MgATP also binds to the enzymatic sites but with a weaker affinity (KD = 180 M) than ATP (KD = 14 M) and is not hydrolysed. Moreover, no cooperativity between the sites is needed to account for the results. A schema is proposed in which the protonated form of the lysine of the enzymatic site interacts with the negatively charged γ phosphate of ATP. Complexation with Mg2+ would prevent this interaction. These results and their interpretation are discussed and compared with those of the literature.

Fluorescence resonance energy transfer mapping of subunit delta in spinach chloroplast F1 ATPase.

Despite the considerable progress in the field of F0F1-ATPases caused by solving the 2.8-A structure of mitochondrial F1 ATPase [Abrahams, J. P., Leslie, A. G. W., Lutter, R. & Walker, J. E. (1994) Nature 370, 621-628], little is known about the position and function of the enzyme's small subunits which were not resolved in the X-ray analysis. We have previously genetically engineered Cys residues into the delta subunit of chloroplast F1 and used these mutant subunits in cross-linking studies [Lill, H., Hensel, F., Junge, W. & Engelbrecht, S. (1996) J. Biol. Chem. 271, 32737-32742]. In this work, various fluorophores have been introduced into the mutant delta subunits and used in fluorescence-resonance energy-transfer measurements. The resulting distances were fitted into the framework of existing data. Subunit delta was found to be located between two alpha/beta couples, stretching from the level of the nucleotide binding sites up to a position close to the N-termini of subunits alpha and beta. These results corroborate and further refine the previously found location of spinach CF1 delta at the periphery and membrane-distal part of CF1, where it may constitute a part of a stator in the rotatory machinery of F0F1.

Photoinactivation of the F1-ATPase from Spinach Chloroplasts by Dequalinium Is Accompanied by Derivatization of Methionine β183

In contrast to the F1-ATPases from bovine mitochondria and the thermophilic Bacillus PS3, which are reversibly inhibited by dequalinium in the absence of irradiation, the Mg2+-ATPase activity of heat- or dithiothreitol-activated chloroplast F1 (CF1) from spinach chloroplasts is slightly stimulated by dequalinium. Conversely, dequalinium is a partial inhibitor (maximal inhibition is 85-90%) of the Ca2+-ATPase of CF1 activated by heat, dithiothreitol, or octylglucoside. The Mg2+- and Ca2+-ATPase activities of CF1 respond differently in the presence of lauryl dimethylamine oxide (LDAO) in the assay medium. Whereas the Mg2+-ATPase activity of heat- or dithiothreitol-activated CF1 is stimulated up to 14-fold by increasing concentrations of LDAO, the Ca2+-ATPase is inhibited in a biphasic manner by increasing concentrations of LDAO. In the presence of LDAO, dequalinium does not stimulate the heat-activated Mg2+-ATPase over that promoted by LDAO alone. That dequalinium slightly stimulates Mg2+-ATPase activity although it inhibits Ca2+-ATPase activity can be reconciled by assuming that dequalinium binds to two sites in CF1, a stimulatory site that also binds LDAO and an inhibitory site. By acting as a partial inhibitor of the Mg2+-ATPase activity that it activates, the combined effect of dequalinium is modest stimulation. Irradiation of heat- or dithiothreitol-activated CF1 or the alpha3beta3gamma subcomplex of CF1 in the presence of 12 microM dequalinium led to rapid photoinactivation. ATP and ADP, separately or in combination with Mg2+, protect against photoinactivation. After photoinactivating the alpha3beta3gamma subcomplex of CF1 with [14C]dequalinium, tryptic and peptic digests of the isolated, derivatized beta subunit were fractionated by high performance liquid chromatography. Sequencing of the isolated, radioactive tryptic and peptic peptides revealed that Metbeta183, which is at or near the catalytic site, is derivatized in a single beta subunit when CF1 is photoinactivated with [14C]dequalinium.

Cross-linking of chloroplast F0F1-ATPase subunit epsilon to gamma without effect on activity. Epsilon and gamma are parts of the rotor.

Cys residues were directed into positions 17, 28, 41 and 85 of a Cys6-->Ser mutant of subunit epsilon of spinach chloroplast F0F1 ATP synthase. Wild-type and engineered epsilon were expressed in Escherichia coli, purified in the presence of urea, refolded and reassembled with spinach chloroplast F1 lacking the epsilon subunit [F1(-epsilon)]. Cys-containing epsilon variants were modified with a sulfhydryl-reactive photolabile cross-linker. Photocross-linking of epsilon to F1(-epsilon) yielded the same SDS gel pattern of cross-link products independent of the presence or absence of Mg2+ x ADP, phosphate and Mg2+ x ATP. Epsilon (wild type) [Ser6,Cys28]epsilon and [Ser6,Cys41]epsilon were cross-linked with subunit gamma. With chloroplast F0F1 the same cross-link pattern was obtained, except for one extra cross-link, probably between [Ser6,Cys28]epsilon and F0 subunit III. [Ser6,Cys17]epsilon and [Ser6,Cys85]epsilon did not produce cross-links. Cross-linking of epsilon, [Ser6,Cys28]epsilon, [Ser6,Cys41]epsilon to gamma in soluble chloroplast F1 impaired the ability of epsilon to inhibit Ca2+-ATPase activity. The Mg2+-ATPase activity of soluble F1 (measured in the presence of 30% MeOH) was not affected by cross-linking epsilon with gamma. Functional reconstitution of photophosphorylation in F1-depleted thylakoids was observed with F1 in which gamma was cross-linked to [Ser6,Cys28]epsilon or [Ser6,Cys41]epsilon but not with wild-type epsilon. In view of the intersubunit rotation of gamma relative to (alphabeta)3, which is driven by ATP hydrolysis, gamma and epsilon would seem to act concertedly as parts of the 'rotor' relative to the 'stator' (alphabeta)3.

Subunit movement during catalysis by F1-F0-ATP synthases.

The catalytic portion (F1) of ATP synthases have the subunit composition alpha 3, beta 3, gamma, delta, epsilon. This composition imparts structural asymmetry to the entire complex that results in differences in nucleotide binding affinity among the six binding sites. Evidence that two or more sites participate in catalysis, alternating their properties, led to the notion that the interactions of individual alpha beta pairs with the small subunit must change as binding sites properties alternate. A rotation of the gamma subunit within the alpha 3 beta 3 hexamer has been proposed as a means of alternating the properties of catalytic sites. Evidence argues that the rotation of the complete gamma subunit during ATP hydrolysis is not mandatory for activity. The gamma subunit of chloroplast F1 may be cleaved into three large fragments that remain bound to F1. This cleavage enhances ATPase activity without loss of evidence of site-site interactions. Complexes of alpha 3 beta 3 have been shown to have significant ATPase activity in the absence of gamma. Mg2+ATP affects the interaction of gamma with the different beta subunits, and induces other changes in F1, but whether these changes are induced by catalysis, or are fast enough to be involved in the catalytic turnover of the enzyme has not been established. Likewise, changes in structure and in binding site properties induced in thylakoid membrane bound CF1 by formation of an electrochemical proton gradient may activate the enzyme rather than be apart of catalysis. Mechanisms other than rotary catalysis should be considered.

Investigating the structure of nucleotide binding sites on the chloroplast F1-ATPase using electron spin resonance spectroscopy

The relative structure and binding properties of nucleotide binding sites of the latent, nonactivated chloroplast F1(CF1)ATPase have been investigated by employing ESR spectroscopy using 2-N3-2′,3′-SL-ATP (2-N3-SL-ATP), a spin-labeled photoaffinity analog of ATP. These results are compared to data obtained in analogous experiments using CF1 that was either depleted of its ε-subunit or activated by different methods. Nonactivated (na) CF1 in complex with 2-N3-SL-ATP exhibits ESR spectra typical for enzyme-bound spin labels. At increased 2-N3-SL-ATP concentration, a second spectral component for enzyme-bound spin label is observed. The line-shape of the second signal indicates an environment of the enzyme-bound radical that differs from the spin-labeled nucleotide bound first. It can be explained by an enzyme-bound radical bound in a way that allows for higher mobility, e.g. a nucleotide binding site in an “open” or “loose” conformation. Maximal binding of about 5 mol 2-N3-SL-ANP per mol of enzyme has been reached. Similar results are obtained when using enzyme that has been either previously depleted of the ε-subunit or treated with the reducing agent dithiothreitol (DTT) in the cold. Upon heat-activation of CF1 in the presence of ATP and the presence or absence of the reducing agent DTT, the line-shape of the ESR spectra is observed to be quite different from the non-heat-treated enzyme forms. The “loose” or “open” nucleotide binding site described above (or at least an environment of the enzyme similar to this site) is observed to be accessible to 2-N3-SL-ATP even at substoichiometric concentrations of the nucleotide analog. The results presented indicate that the enzyme form of CF1 generated after heat treatment in the presence of ATP with or without DTT exhibits altered binding specificities mainly with respect to the sequence of occupation of two different types of nucleotide binding sites.

Synthesis, structure, and properties of MeSer1-tentoxin, a new cyclic tetrapeptide which interacts specifically with chloroplast F1 H(+)-ATPase differentiation of inhibitory and stimulating effects.

A new tentoxin analogue, in which the L-methyl alanine residue is substituted by L-methylserine, has been prepared following the synthetic pathway recently described for the synthesis of tentoxin [Cavelier, F., & Verducci, J. (1995) Tetrahedron Lett. 36, 4425-4428]. Using two-dimensional homonuclear proton nuclear magnetic resonance and structural analysis, we observed that MeSer1-tentoxin, like tentoxin, adopts several conformations in aqueous solution and presents self-aggregative properties. This analogue was found to be conformationally similar to the natural toxin. It showed the same efficiency as tentoxin in inhibition of ATPase activity of the isolated chloroplast F1 proton ATPase (CF1) as well as in inhibition of the ATP synthase activity of the membrane-bound enzyme (CF0CF1) in thylakoids and proteoliposomes. At concentrations above 10 microM, MeSer1-tentoxin did not reactivate CF1 to a high extent, contrary to tentoxin. It appeared, however, to bind in the same way, since the reactivating effect of tentoxin was inhibited by MeSer1-tentoxin. These results show that it is possible, using tentoxin analogues, to separate inhibitory and activating effects on the chloroplast ATPase, despite the limited chemical difference between the two toxins.

The role of Mg2+ in the hydrolytic activity of the isolated chloroplast ATPase: Study by high-performance liquid chromatography

The influences of total magnesium ion concentration at different total ATP concentrations, and of total ATP concentration, for different total magnesium ion concentrations, on the enzymatic rate of the isolated chloroplast F1 ATPase, have been followed by a chromatographic method consisting in the separation and determination of ADP. From the various series of curves, it is concluded that the experimental results (position of the maxima, Km values) are better fitted by a mechanism involving the activation of the enzyme by magnesium ion and hydrolysis of free ATP, rather than by the classical mechanism, for which the enzyme hydrolyzes the MgATP complex and is inhibited by Mg2+. Although the equations giving the reaction rate are similar in the two cases, the calculated values of Km are widely different. The value obtained from the classical mechanism does not agree with KD, the dissociation constant of the enzyme-substrate complex, measured by the Hummel and Dreyer method. Moreover, when the total ATP concentration tends toward the total magnesium ion concentration, the nucleotide binding to the enzyme tends toward zero, although it should be maximum if MgATP were the true substrate. Finally, the inhibitory effect of Na+ is more easily explained as a competition between this ion and the activating Mg2+, than by the classical mechanism.

Properties Of The Plasma Membrane Atpases Of The Halophilic Archaebacteria Haloferax Mediterranei And Haloferax Volcanii

Both, Haloferax mediterranei and Haloferax volcanii membranes contain ATPases which are capable of hydrolyzing ATP in presence of Mg2+ or Mn2+. The ATPases require high concentrations of NaCl, a pH value of 9, and high temperatures up to 60 °C. Free manganese ions inhibited the enzyme activity of either ATPase. The ATPases of Hf. mediterranei and Hf. volcanii, respectively, show different sensitivities to inhibitors of ATP hydrolysis. ATP hydrolysis of isolated Hf. mediterranei ATPase was inhibited by NaN3, which was reported to be specific for F-ATPases, by nitrate and N-ethylmaleimide (NEM), which are specific inhibitors of V-ATPases. ATP hydrolysis of Haloferax mediterranei membranes was not inhibited by DCCD , but [14C]DCCD was bound to a 14 kDa peptide of the isolated, partially purified enzyme. Furthermore, the ATPase was inactivated by preincubation with 7-chloro-4-nitrobenzofurazan (NBD-Cl). The ATPase activity of Hf. volcanii membranes was inhibited by NEM but not by nitrate and NaN3. SDS gel electrophoresis of the partially purified enzyme of Haloferax mediterranei showed putative ATPase subunits of 53.5, 49, 42, 22, 21, 14, 12, and 7.5 kDa. Immunoblots showed cross reactivity between a 53 kDa peptide and anti-β (chloroplast F1), as well as between 53, 50 and 47 kDa peptides and an ATPase antibody of Methanosarcina barkeri. The results will be discussed in context with the placement of the archaebacterial ATPases (A-ATPases) between F- and V-ATPases

A PLANT BIOCHEMIST'S VIEW OF H+-ATPases AND ATP SYNTHASES.

My twenty-five year fascination with membrane ATPases grew out of my experiences in the laboratories of André Jagendorf and Efraim Racker. André introduced me to photosynthetic phosphorylation and Ef, to whose memory this article is dedicated, convinced me that ATPases had much to do with ATP synthesis. Astounding progress has been made in the H+-ATPase field in just two decades. By the early 1970s, it was generally recognized that oxidative and photosynthetic ATP synthesis were catalyzed by membrane enzymes that could act as H+-ATPases and that the common intermediate between electron transport and phosphorylation is the electrochemical proton gradient. At that time, it had been shown that a cation-stimulated ATPase activity was associated with plasma membrane preparations from plant roots. The endomembrane or vacuolar ATPases were unknown. The application of improved biochemical methods for membrane isolation and purification, as well as membrane protein reconstitutions, led rapidly to the conclusion that there are three major classes of membrane H+-ATPases, P, V and F. P-ATPases, which will not be considered further in this article, are phosphorylated during their catalytic cycle and have a much simpler polypeptide composition than V- or F-ATPases. The plasma membrane H+-ATPase of plant, yeasts and fungal cells is one example of this class of enzymes (see Pedersen and Carafoli, 1987, for a comparison of plasma membrane ATPases). Biochemical and gene sequencing analysis have revealed that V- and F-ATPases resemble each other structurally, but are distinct in function and origin. The 'V' stands for vacuolar and the 'F' for F1Fo. F1 was the first factor isolated from bovine heart mitochondria shown to be required for oxidative phosphorylation. Fo was so named because it is a factor that conferred oligomycin sensitivity to soluble F1. Other F-ATPases are often named to indicate their sources. For example, chloroplast F1 is denoted CF1 (see Racker, 1965, for early work on F1). Recent successes in reconstitution of vacuolar ATPase have led to a V1Vo nomenclature for this enzyme as well. The term 'ATP synthase' is now in general use to describe F-ATPases. This term emphasizes the facts that although F-ATPases function to synthesize ATP, they do not catalyze, normally, ATP hydrolysis linked to proton flux. In contrast, V-ATPases are very unlikely to operate as ATP synthases. Thus, F-ATPases are proton gradient consumers, whereas V-ATPases generate proton gradients at the expense of hydrolysis. In this brief review, I will compare the structures of F- and V-ATPases. Also, I give some insight into the mechanisms that help prevent wasteful ATP hydrolysis by the chloroplast ATP synthase (CF1Fo).

Structural mapping of cysteine-63 of the chloroplast ATP synthase beta subunit.

The single sulfhydryl residue (cysteine-63) of the beta subunit of the chloroplast ATP synthase F1 (CF1) was accessible to labeling reagents only after removal of the beta subunit from the enzyme complex. This suggests that cysteine-63 may be located at an interface between the beta and the alpha subunits of CF1, although alternative explanations such as a conformational change in beta brought about by its release from CF1 cannot be ruled out. Cysteine-63 was specifically labeled with [(diethylamino)methylcoumarinyl]-maleimide, and the distance between this site and trinitrophenyl-ADP at the nucleotide binding site on beta was mapped using fluorescence resonance energy transfer. Cysteine-63 is located in a hydrophobic pocket, 42 A away from the nucleotide binding site on beta.

Characteristics of the combination of inhibitory magnesium and azide with the F1 ATPase from chloroplasts

The interactions between ADP, Mg2+, and azide that result in the inhibition of the chloroplast F1 ATPase (CF1) have been explored further. The binding of the inhibitory Mg2+ with low Kd is shown to occur only when tightly bound ADP is present at a catalytic site. Either the tightly bound ADP forms part of the Mg(2+)-binding site or it induces conformational changes creating the high-affinity site for inhibitory Mg2+. Kinetic studies show that CF1 forms two catalytically inactive complexes with Mg2+. The first complex results from Mg2+ binding with a Kd for Mg2+ dissociation of about 10-15 microM, followed by a slow conversion to a complex with a Kd of about 4 microM. The rate-limiting step of the CF1 inactivation by Mg2+ is the initial Mg2+ binding. When medium Mg2+ is chelated with EDTA, the two complexes dissociate with half-times of about 1 and 7 min, respectively. Azide enhances the extent of Mg(2+)-dependent inactivation by increasing the affinity of the enzyme for Mg2+ 3-4 times and prevents the reactivation of both complexes of CF1 with ADP and Mg2+. This results from decreasing the rate of Mg2+ release; neither the rate of Mg2+ binding to CF1 nor the rate of isomerization of the first inactive complex to the more stable form is affected by azide. This suggests that the tight-binding site for the inhibitory azide requires prior binding of both ADP and Mg2+.