High-affinity metal-binding site in beef heart mitochondrial F1ATPase:: An EPR spectroscopy study

The high-affinity metal-binding site of isolated F-1-ATPase from beef heart mitochondria was studied by high-field (HF) continuous wave electron paramagnetic resonance (CW-EPR) and pulsed EPR spectroscopy, using Mn-II as a paramagnetic probe. The protein F, was fully depleted of endogenous Mg-II and nucleotides [stripped F-1 or MF1(0,0)] and loaded with stoichiometric Mn-II and stoichiometric or excess amounts of ADP or adenosine 5'-(beta,gamma-imido)-triphosphate (AMPPNP). Mn-II and nucleotides were added to MF1(0,0) either subsequently or together as preformed complexes. Metal-ADP inhibition kinetics analysis was performed showing that in all samples Mn-II enters one catalytic site on a beta subunit. From the HF-EPR spectra, the zero-field splitting (ZFS) parameters of the various samples were obtained, showing that different metal-protein coordination symmetry is induced depending on the metal nucleotide addition order and the protein/metal/nucleotide molar ratios. The electron spin-echo envelope modulation (ESEEM) technique was used to obtain information on the interaction between Mn-II and the P-31 nuclei of the metal-coordinated nucleotide. In the case of samples containing ADP, the measured P-31 hyperfine couplings clearly indicated coordination changes related to the metal nucleotide addition order and the protein/ metal/nucleotide ratios. On the contrary, the samples with AMPPNP showed very similar ESEEM patterns, despite the remarkable differences present among their HF-EPR spectra. This fact has been attributed to changes in the metal-site coordination symmetry because of ligands not involving phosphate groups. The kinetic data showed that the divalent metal always induces in the catalytic site the high-affinity conformation, while EPR experiments in frozen solutions supported the occurrence of different precatalytic states when the metal and ADP are added to the protein sequentially or together as a preformed complex. The different states evolve to the same conformation, the metal(II)-ADP inhibited form, upon induction of the trisite catalytic activity. All our spectroscopic and kinetic data point to the active role of the divalent cation in creating a competent catalytic site upon binding to MF1, in accordance with previous evidence obtained for Escherichia coli and chloroplast F-1.