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≈3.89-4.49 MHz and e²qQ₂≈1.91-2.20 MHz, respectively. Aminoacid functional groups compatible with these magnetic couplings include a histidine imidazole, the ε-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 ΔM_I = 2 transitions in the negative quadrant, and also between some of the ΔM_I = 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·ADP and Mn·ATP with the whole enzyme show distinct and specific hyperfine couplings with the ³¹P atoms of the bonding phosphates in the HYSCORE (ADP, A(³¹P_β) = 5.20 MHz; ATP, A(³¹P_β) = 4.60 MHz and A(³¹P_γ) = 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 α or β subunits of the enzyme, the HYSCORE spectra are substantially modified, suggesting that in these cases the nucleotide binding site is only partially structured.