Evidence for changes in the nucleotide conformation in the active site of H(+)-ATPase as determined by pulsed EPR spectroscopy.

The conformation of di- and triphosphate nucleosides in the active site of ATPsynthase (H(+)-ATPase) from thermophilic Bacillus PS3 (TF1) and their interaction with Mg(2+)/Mn(2+) cations have been investigated using EPR, ESEEM, and HYSCORE spectroscopies. For a ternary complex formed by a stoichiometric mixture of TF1, Mn(2+), and ADP, the ESEEM and HYSCORE data reveal a (31)P hyperfine interaction with Mn(2+) (|A((31)P)| approximately 5.20 MHz), significantly larger than that measured for the complex formed by Mn(2+) and ADP in solution (|A((31)P)| approximately 4.50 MHz). The Q-band EPR spectrum of the Mn.TF1.ADP complex indicates that the Mn(2+) binds in a slightly distorted environment with |D| approximately 180 x 10(-4) cm(-1) and |E| approximately 50 x 10(-4) cm(-1). The increased hyperfine coupling with (31)P in the presence of TF1 reflects the specific interaction between the central Mn(2+) and the ADP beta-phosphate, illustrating the role of the enzyme active site in positioning the phosphate chain of the substrate for efficient catalysis. Results with the ternary Mn.TF1.ATP and Mn.TF1.AMP-PNP complexes are interpreted in a similar way with two hyperfine couplings being resolved for each complex (|A((31)P(beta))| approximately 4.60 MHz and |A((31)P(gamma))| approximately 5.90 MHz with ATP, and |A((31)P(beta))| approximately 4.20 MHz and |A((31)P(gamma))| approximately 5.40 MHz with AMP-PNP). In these complexes, the increased hyperfine coupling with (31)P(gamma) compared with (31)P(beta) reflects the smaller Mn.P distance with the gamma-phosphate compared with the beta-phosphate as found in the crystal structure of the analogous enzyme from mitochondria [3.53 vs 3.70 A (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628)] and the different binding modes of the two phosphate groups. The ESEEM and HYSCORE data of a complex formed with Mn(2+), ATP, and the isolated beta subunit show that the (31)P hyperfine coupling is close to that measured in the absence of the protein, indicating a poorly structured nucleotide site in the isolated beta subunit in the presence of ATP. The inhibition data obtained for TF1 incubated in the presence of Mg(2+), ADP, Al(NO(3))(3), and NaF indicate the formation of the inhibited complex with the transition state analogue namely Mg.TF1.ADP.AlF(x) with the equilibrium dissociation constant K(D) = 350 microM and rate constant k = 0.02 min(-1). The ESEEM and HYSCORE data obtained for an inhibited TF1 sample, Mn.TF1.ADP.AlF(x), confirm the formation of the transition state analogue with distinct spectroscopic footprints that can be assigned to Mn.(19)F and Mn.(27)Al hyperfine interactions. The (31)P(beta) hyperfine coupling that is measured in the inhibited complex with the transition state analogue (|A((31)P(beta))| approximately 5.10 MHz) is intermediate between those measured in the presence of ADP and ATP and suggests an increase in the bond between Mn and the P(beta) from ADP upon formation of the transition state.

Mechanism of activation of the chloroplast ATP synthase. A kinetic study of the thiol modulation of isolated ATPase and membrane-bound ATP synthase from spinach by Eschericia coli thioredoxin.

The mechanism of thiol modulation of the chloroplast ATP synthase by Escherichia coli thioredoxin was investigated in the isolated ATPase subcomplex and in the ATP synthase complex reconstituted in bacteriorhodopsin proteoliposomes. Thiol modulation was resolved kinetically by continuously monitoring ATP hydrolysis by the isolated subcomplex and ATP synthesis by proteoliposomes. The binding rate constant of reduced thioredoxin to the oxidized ATPase subcomplex devoid of its epsilon subunit could be determined. It did not depend on the catalytic turnover. Reciprocically, the catalytic turnover did not seem to depend on thioredoxin binding. Thiol modulation by Trx of the epsilon-bearing ATPase subcomplex was slow and favored the release of epsilon. The rate constant of thioredoxin binding to the membrane-bound ATP synthase increased with the protonmotive force. It was lower in the presence of ADP than in its absence, revealing a specific effect of the ATP synthase turnover on thioredoxin-gamma subunit interaction. These findings, and more especially the comparisons between the isolated ATPase subcomplex and the ATP synthase complex, can be interpreted in the frame of the rotational catalysis hypothesis. Finally, thiol modulation changed the catalytic properties of the ATP synthase, the kinetics of which became non-Michaelian. This questions the common view about the nature of changes induced by ATP synthase thiol modulation.

The role of the Mg2+ cation in ATPsynthase studied by electron paramagnetic resonance using VO2+ and Mn2+ paramagnetic probes.

The electron paramagnetic resonance (EPR), electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation (HYSCORE) spectra of Mg2+-depleted chloroplast F1-ATPase substituted with stoichiometric VO2+ are reported. The ESEEM and HYSCORE spectra of the complex are dominated by the hyperfine and quadrupole interactions between the VO2+ paramagnet and two different nitrogen ligands with isotropic hyperfine couplings /A1/ = 4.11 MHz and /A2/ = 6.46 MHz and nuclear quadrupole couplings e2qQ1 approximately 3.89-4.49 MHz and e2qQ2 approximately 1.91-2.20 MHz, respectively. Aminoacid functional groups compatible with these magnetic couplings include a histidine imidazole, the epsilon-NH2 of a lysine residue, and the guanidinium group of an arginine. Consistent with this interpretation, very characteristic correlations are detected in the HYSCORE spectra between the 14N deltaM1 = 2 transitions in the negative quadrant, and also between some of the deltaM1 = 1 transitions in the positive quadrant. The interaction of the substrate and product ADP and ATP nucleotides with the enzyme has been studied in protein complexes where Mg2+ is substituted for Mn2+. Stoichiometric complexes of Mn x ADP and Mn x ATP with the whole enzyme show distinct and specific hyperfine couplings with the 31P atoms of the bonding phosphates in the HYSCORE (ADP, A(31Pbeta) = 5.20 MHz: ATP, A(31Pbeta) = 4.60 MHz and A(31Pgamma) = 5.90 MHz) demonstrating the role of the enzyme active site in positioning the di- or triphosphate chain of the nucleotide for efficient catalysis. When the complexes are formed with the isolated alpha or beta subunits of the enzyme, the HYSCORE spectra are substantially modified, suggesting that in these cases the nucleotide binding site is only partially structured.

Identification and characterization of Mg2+ binding sites in isolated alpha and beta subunits of H(+)-ATPase from Bacillus PS3.

The properties of the nucleotide binding sites in the isolated beta and alpha subunits of H(+)-ATPase from Bacillus PS3 (TF1) have been examined by studying the EPR properties of bound VO(2+), which is a paramagnetic probe for the native Mg2+ cation cofactor. The amino acid ligands of the VO2+ complexes with the isolated beta subunit, with the isolated alpha subunit, with different mixtures of both alpha and beta subunits, and with the catalytic alpha 3 beta 3 gamma subcomplex have been characterized by a combination of EPR, ESEEM, and HYSCORE spectroscopies. The EPR spectrum of the isolated beta subunit with bound VO2+ (1 VO2+/beta) is characterized by (51)V hyperfine coupling parameters (A( parallel) = 168 x 10(-)(4) cm(-)(1) and A( perpendicular) = 60 x 10(-)(4) cm(-)(1)) that suggest that VO2+ binds to the isolated beta subunit with at least one nitrogen ligand. Results obtained for the analogous VO2+ complex with the isolated alpha subunit are virtually identical. ESEEM and HYSCORE spectra are also reported and are similar for both complexes, indicating a very similar coordination scheme for VO2+ bound to isolated alpha and beta subunits. In the isolated beta (or alpha) subunit, the bound VO2+ cation is coordinated by one nitrogen ligand with hyperfine coupling parameters A( parallel)((14)N) = 4.44 MHz, and A( perpendicular)((14)N) = 4.3 MHz and quadrupole coupling parameters e(2)()qQ approximately 3.18 MHz and eta approximately 1. These are typical for amine-type nitrogen ligands equatorial to the VO2+ cation; amino acid residues in the TF1 beta and alpha subunits with nitrogen donors that may bind VO2+ are reviewed. VO2+ bound to a mixture of alpha and beta subunits in the presence of 200 mM Na2SO4 to promote the formation of the alpha 3 beta 3 hexamer has a second nitrogen ligand with magnetic properties similar to those of a histidine imidazole. This situation is analogous to that in the alpha 3 beta 3 gamma subcomplex and in the whole TF1 enzyme [Buy, C., Matsui, T., Andrianambinintsoa, S., Sigalat, C., Girault, G., and Zimmermann, J.-L. (1996) Biochemistry 35, 14281-14293]. These data are interpreted in terms of only partially structured nucleotide binding sites in the isolated beta and alpha subunits as compared to fully structured nucleotide binding sites in the alpha 3 beta 3 heterohexamer, the alpha 3 beta 3 gamma subcomplex, and the whole TF1 ATPase.

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.

Interrelation between high and low affinity tentoxin binding sites in chloroplast F1-ATPase revealed by synthetic analogues.

Eight synthetic analogues of tentoxin (cyclo-(L-N-MeGlu1-L-Leu2-N-MeDeltaZPhe3-Gly4)) modified in residues 1, 2, and 3 were checked for their ability to inhibit and reactivate the ATPase activity of the activated soluble part of chloroplast ATP synthase. The data were consistent with a model involving two binding sites of different affinities for the toxins. The occupancy of the high affinity site (or tight site) gave rise to an inactive complex, whereas filling both sites (tight + loose) gave rise to a complex of variable activity, dependent on the toxin analogue. Competition experiments between tentoxin and nonreactivating analogues allowed discrimination between the absence of binding and a nonproductive binding to the site of lower affinity (or loose site). The affinity for the loose site was not affected significantly by the modifications of the tentoxin molecule, whereas the affinity for the tight site was found notably changed. Increasing the size of side chain 1 or 2 and introducing a net electrical charge both resulted in a decrease of affinity for the tight site, but the second change dominated the first one. The activity of different ternary complexes enzyme-tentoxin-analogue depended on the nature of the toxin bound on each site and not only on that bound on the loose site. This demonstrates that the reactivation process results from an interaction, direct or not, between these two binding sites. Possible molecular mechanisms are discussed.

Binding sites for Mg(II) in H(+)-ATPase from Bacillus PS3 and in the alpha 3 beta 3 gamma subcomplex studied by one-dimensional ESEEM and two-dimensional HYSCORE spectroscopy of oxovanadium(IV) complexes: a possible role for beta-His-324.

The binding sites for Mg2+ in wild type F1 ATPase (TF1) and in the alpha 3 beta 3 gamma subcomplex from the thermophilic bacterium Bacillus PS3 have been studied by EPR and by ESEEM and HYSCORE spectroscopy of complexes with the oxovanadium cation VO2+. Complexes of metal-depleted TF1 and substoichiometric amounts of VO2+ display low-temperature EPR signals with spectral parameters g parallel = 1.947 and g perpendicular = 1.980, and hyperfine couplings with 51V, A parallel = 169 x 10(-4) cm-1 and A perpendicular = 61 x 10(-4) cm-1, that are indicative of a binding site for VO2+ with nitrogen ligands from the protein. This binding site is probably identical with the metal binding site with strong affinity M1 that has been characterized using Mn2+ in a previous study [Buy, C., Girault, G., & Zimmermann, J. L. (1996) Biochemistry 35, 9880-9891]. The three-pulse ESEEM spectrum of the VO2+ complex with TF1 shows a frequency pattern with spectral properties that are evidence for two nitrogen ligands to the VO2+ with hyperfine couplings A1 = 4.75 MHz and A2 = 6.5 MHz and nuclear quadrupole parameters e2Qq1 = 2.8-3.2 MHz and e2Qq2 = 2.0-2.3 MHz. The ligands are identified as a lysine terminal amine and a histidine imidazole, which are proposed as Lys-164 and His-324 from a beta subunit. The HYSCORE data obtained for the VO.TF1 complex show correlations within each pair of the ESEEM nu dq peaks from the 14N nuclei, confirming the interpretation of the one-dimensional spectra. Evidence for the formation of a ternary complex by addition of VO2+ and ATP to metal-depleted TF1 is shown in the EPR and ESEEM spectra and in the contour plots of the HYSCORE data. Two pairs of correlation patterns are resolved in addition to the peaks from the two 14N ligands, which are interpreted as hyperfine couplings with 31P beta and 31P gamma of the ATP that binds the VO2+ cation. The assignment of the two hyperfine couplings to the specific phosphates, A(31P beta) = 15.5 MHz and A(31P gamma) = 8.7 MHz, in the VO.TF1.ATP complex is proposed by comparison with those measured for VO2+ in solution with ATP at pH 6.3 and 2.3. These results are discussed in light of the previous data with the analogous Mn.TF1 complex, and a model is proposed in which the native Mg2+ in the M1 site is coordinated by the side chain of beta-Lys-164 and is in close proximity to a histidine residue (probably beta-His-324) that may have a critical role. Additional coordination by two phosphates from ATP (probably the beta- and gamma-phosphates) is observed in the ternary complex VO.TF1.ATP. ESEEM and HYSCORE data are also obtained for the analogous complexes VO. alpha 3 beta 3 gamma and VO. alpha 3 beta 3 gamma .ATP that show very similar properties in terms of coordination of the divalent metal cation, except for the lysine ligand that is found to be lost in the ternary complex with ATP. It is suggested that this observation may reflect changes in the metal and nucleotide active sites that are associated with the absence of the delta and epsilon subunits in the subcomplex.

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.

Proton coupling is preserved in membrane-bound chloroplast ATPase activated by high concentrations of tentoxin.

The effect of tentoxin at high concentrations was investigated in thylakoids and proteoliposomes containing bacteriorhodopsin and CF0CF1. Venturicidin-sensitive ATP hydrolysis, ATP-generated delta pH and ATP synthesis were practically 100% inhibited at 2 microM tentoxin, and restored to various extents beyond 50 microM. With respect to the native enzyme, tentoxin-reactivated ATPase had the following properties: (i) a higher delta pH requirement to synthetise ATP; (ii) a decreased futile proton flow through CF0CF1 (without ADP), which remains 100% blocked by ADP. It is concluded that despite its altered kinetic performances, tentoxin-modified CF0CF1 preserves its mechanism and remains a tightly coupled proton pump.

Flow-force relationships in lettuce thylakoids. 1. Strict control of electron flow by internal pH.

The regulation by the proton gradient of the electron flow from water to ferricyanide was investigated in thylakoids extracted from lettuce leaves. When the transmembrane proton current was varied by an uncoupler or by the ATP synthase activity, a unique relationship was found between the rate of ferricyanide reduction and the proton gradient, restricted here to its delta pH component. This behavior was conserved in CF1-depleted thylakoids where the passive proton flow was varied by the concentration of an Fo inhibitor or by the concentration of an uncoupler after 100% inhibition of Fo. This shows that under our experimental conditions no direct proton transfer exists in steady state between the site of regulation of the redox chain and the ATPase. Studies at two different pH's indicate that the internal pH, and not the transmembrane pH difference, controls the electron transfer between PS2 and PS1. Modeling the data suggests that a single deprotonation step is kinetically limiting.

Flow-force relationships in lettuce thylakoids. 2. Effect of the uncoupler FCCP on local proton resistances at the ATPase level.

The relationship between the steady-state proton gradient (delta pH) and the rate of phosphorylation was investigated in thylakoids under various conditions. Under partial uncoupling by carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), the rate of ATP synthesis was reduced by less than expected from the decrease of delta pH. This was observed in the case of the pyocyanine-mediated cyclic electron flow around photosystem 1, but not with the H2O-->photosystem 2-->cytochrome b6f-->photosystem 1-->methyl viologen system. In state 4, a unique relation was found between delta pH and the "phosphate potential", delta Gp, regardless of whether the energy level was controlled by light input or FCCP. The anomalous effect of FCCP on the rate of ATP synthesis disappeared when the ATPase was partially blocked by the reversible inhibitor venturicidin, but not in the presence of tentoxin, an irreversible inhibitor. These results are consistent with the existence of a small kinetic barrier for protons, limiting their access to the ATPase. This resistance would be collapsed by FCCP.