The metal binding sites of isolated F1 ATPase from spinach chloroplasts (CF1) and from the thermophilic bacterium Bacillus PS3 (TF1) have been studied by EPR and pulsed EPR spectroscopy using Mn(II) as a paramagnetic probe. After dialysis in the presence of EDTA, purified CF1 retains 0.14 +/- 0.07 Mg(II) and approximately 0.75 +/- 0.25 ADP. TF1 retains 0.31 +/- 0.03 Mg(II) and 0.08 +/- 0.01 nucleotide (ADP + ATP) after the same treatment. Supplementing known quantities of Mn(II) to metal-depleted CF1 allowed a spectroscopic characterization of the bound Mn(II) cations, for which the EPR spectra at X- and Q-band are reported. The zero field splitting parameters of Mn(II) are derived from the simulation of the EPR signal recorded at Q-band for a sample supplemented with 0.3 Mn/CF1. The values, magnitude of D approximately 200 x 10(-4) cm-1 and magnitude of E approximately 40 x 10(-4) cm-1 suggest that the Mn(II) binds to CF1 in a slightly distorted environment. The ESEEM spectra of complexes of Mn(II) with CF1 were also recorded for different Mn/CF1 ratios. For a complex with 0.8 Mn/CF1, the ESEEM spectrum shows two frequencies at 3.7 and 8.6 MHz that are attributed to the magnetic coupling with 31P with a hyperfine coupling constant of magnitude of A approximately 5.3 MHz, reflecting the interaction with a phosphate group from the endogenous ADP molecule. This demonstrates close proximity of the strong affinity metal site M1 and the endogenous ADP binding site N1, and binding of the ADP beta-phosphate to the divalent metal cation. For Mn(II) complexes with higher Mn/CF1 ratios, new frequency components below approximately 5 MHz are resolved in the spectra in addition to the peaks from 31P. From a comparison of the CF1 spectra and their magnetic field dependence across the Mn(II) EPR line shape with those of Mn(II) complexes with imidazole, glycine, poly-L-lysine, and nucleotide ligands, it is concluded that additional metal binding sites are filled at higher Mn contents and that these involve 14N donors. It is suggested that the most probable set of ligands of the divalent metal(s) for these additional metal sites in CF1 includes a lysine residue, in line with a previous proposal [Houseman, A. L. P., Morgan, L., LoBrutto, R., & Frasch, W. D. (1994) Biochemistry 33, 4910-4917]. Similar experiments for a Mn(II) complex with TF1 (0.4 Mn/TF1) showed no interaction with 31P; instead modulations are detected in the ESEEM below approximately 5 MHz that are attributed to a 14N ligand. This is tentatively attributed to the deprotonated amine of Lys-162 from a beta subunit, on the basis of the structural data available for the mitochondrial F1 complex. Addition of the substrate ATP to this Mn.TF1 complex leads to the formation of a ternary Mn.TF1.ATP complex with coordination of the Mn(II) by a phosphate group from the ATP as judged from the ESEEM results (magnitude of A(31P) approximately 4.5 MHz). An increase in the hyperfine coupling constant of 31P of the phosphate bound to Mn(II) to magnitude of A(31P) approximately 5....

The conformations of the phytotoxic cyclic tetrapeptide tentoxin [cyclo-(L-MeAla1-L-Leu2-MePhe[(Z) delta]3-Gly4)] have been studied in aqueous solution by two-dimensional proton nmr at various temperatures. Contrary to what is observed in chloroform, tentoxin exhibits multiple exchanging conformations in water. Aggregation phenomena were also observed. Four conformations with different proportions (51, 37, 8, and 4%) were observed at -5 degrees C. Models were constructed from nmr parameters and restrained molecular dynamics simulations. All the models exhibit cis-trans-cis-trans conformation of the amide bond sequence. The conversion from one form to another is accomplished by a conformational peptide flip consisting of a 180 degree rotation of a nonmethylated peptide bond.

Optimal conditions for the reconstitution of bacteriorhodopsin and H+-transporting ATP synthase from therinophilic Bacillus PS3 (TF,,F,) were determined. Phosphalidylcholine/phosphatidic acid liposonies prepared by reverse-phase evaporation were treated with various amounts of Triton X-100, octyl glucoside, octaethylcne glycol n-dodecylether, sodium cholate or sodium deoxycholate and the incorporation of proteins by these detergents was studied at each step crf the solubilization process. After removal of detergent by means of SM-2 Bio-Beads, the light-driven ATP synthase activities of the resulting proteoliposomes were analyzed at 40°C. The nature of the detergent used for reconstilution wits important for determining the mechanism of protein insertions. The most efficient reconstitutions were obtained with octyl glucoside or Triton X-I00 by insertion of the proteins into detergent-saturated liposomes.The conditions for reconstitution were further optimized with regard to functional coupling between bacteriorhodopsin and TF,,F,. It was demonstrated that one of the main factors limiting the production of efficient reconstituted proteoliposomes was related to activation of the highly stable TF,F,. Activation was accomplished by total solubilization of phospholipids and proteins in a Triton X-lOO/octyl glucoside mixture containing 20 mM octyl glucoside, leading to a threefold slii1iulation of the ATP synthase activily. Final ATP synthasc activities depended greatly on the lipidlbactcriorhodopsin and the lipid/TF,,F, ratios as well as on the phospholipid used. In particular, light-driven ATP synthesis depcnded upon the presence of negatively charged phospholipids. Cholesterol was found to induce a fourfold increase in ATP synthase activity with a concomitant 65 % decrease in the K,,, for ADP, suggesting that sterols can modulate catalytic events mediated by F,.Preparations obtained by this step-by-step reconstitution procedure displayed activities up Io 20-fold higher (500-800 nmol ATP . min-' 1 mg TF,,F,-l in the presencc of cholesterol) than the maximal values reported in the literature for light-driven ATP synthesis by TF,,F, measured under similar conditions. This study also allowed riitionalization of the different parameters involved in reconstitution experiments and the present simple method is shown to be of general use for preparation of efficient proteoliposomes containing bacteriorhodopsin and choloroplast or mitochondria1 F,,F,-type ATP synthases.Keywords: F,,F, ATP synthasc; bacteriorhodopsin: reconstitution; ATP synthesis; proteoliposomes.The F,,F,-type H -transporting ATP synthases, which are found in similar forms in the membranes of bacteria, mitochondria and chloroplasts, couple H+ translocation to ATP synthesis or hydrolysis. During the past decade a number of different approaches have led to information about the stoichiometi k composition and the function of the different subunits of these complicated enzymes and to the location and function of their nuclcotide-binding domains and the role of c...