title: The role of the Mg2+ cation in ATPsynthase studied by electron paramagnetic resonance using VO2+ and Mn2+ paramagnetic probes.
abstract: 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.
authors.affiliation: CEA/Saclay, Département de Biologie Cellulaire et Moléculaire Gif-sur-Yvette, France. jean-luc.zimmermann@cea.fr
authors.forename: J L
authors.initials: JL
authors.lastname: Zimmermann
authors.forename: B
authors.initials: B
authors.lastname: Schneider
authors.forename: S
authors.initials: S
authors.lastname: Morlet
authors.forename: T
authors.initials: T
authors.lastname: Amano
authors.forename: C
authors.initials: C
authors.lastname: Sigalat
mesh_terms: D002736:Chloroplasts / Q000201:enzymology; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D008274:Magnesium / Q000737:chemistry*; D008345:Manganese; D006180:Proton-Translocating ATPases / Q000737:chemistry*; D013113:Spin Labels; D018724:Spinacia oleracea / Q000201:enzymology; D014638:Vanadates
chemical_list: D013113:Spin Labels; D014638:Vanadates; D008345:Manganese; D006180:Proton-Translocating ATPases; D008274:Magnesium