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 modu lation (ESEEM) and hyperfine sublevel correlation (HYSCORE) spectra of Mg2-depleted chloroplast F1-ATPase substituted with stoichiometric VO2+ are re ported. The ESEEM and HYSCORE spectra of the complex are dominated by the h yperfine and quadrupole interactions between the VO2+ paramagnet and two di fferent nitrogen ligands with isotropic hyperfine couplings \A(1)\ = 4.11 M Hz and \A(2)\ = 6.46 MHz and nuclear quadrupole couplings e(2)qQ(1), approx imate to 3.89-4.39 MHz and e(2)qQ(2) approximate to 1.91-2.20 MHz, respecti vely. 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, ver y characteristic correlations are detected in the HYSCORE spectra between t he N-14 Delta M-1 = 2 transitions in the negative quadrant, and also betwee n some of the Delta M-I = 1 transitions in the positive quadrant. The inter action 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.ADP and Mn.ATP with the whole enzyme show di stinct and specific hyperfine couplings with the P-31 atoms of the bonding phosphates in the HYSCORE (ADP, A(P-31(beta)) = 5.20 MHz; ATP, A(P-31(beta) ) = 4.60 MHz and A((31)p(gamma)) = 5.90 MHz) demonstrating the role of the enzyme active site in positioning the di- or triphosphate chain of the nucl eotide for efficient catalysis. When the complexes are formed with the isol ated alpha or beta subunits of the enzyme, the HYSCORE spectra are substant ially modified, suggesting that in these cases the nucleotide binding site is only partially structured. (C) 2000 Elsevier Science B.V. All rights res erved.