| Abstract
| - The high-affinity metal-binding site of isolated F1-ATPase from beef heart mitochondria wasstudied by high-field (HF) continuous wave electron paramagnetic resonance (CW-EPR) and pulsed EPRspectroscopy, using MnII as a paramagnetic probe. The protein F1 was fully depleted of endogenous MgIIand nucleotides [stripped F1 or MF1(0,0)] and loaded with stoichiometric MnII and stoichiometric orexcess amounts of ADP or adenosine 5‘-(β,γ-imido)-triphosphate (AMPPNP). MnII and nucleotides wereadded to MF1(0,0) either subsequently or together as preformed complexes. Metal-ADP inhibition kineticsanalysis was performed showing that in all samples MnII enters one catalytic site on a β subunit. Fromthe HF-EPR spectra, the zero-field splitting (ZFS) parameters of the various samples were obtained, showingthat different metal-protein coordination symmetry is induced depending on the metal nucleotide additionorder and the protein/metal/nucleotide molar ratios. The electron spin-echo envelope modulation (ESEEM)technique was used to obtain information on the interaction between MnII and the 31P nuclei of the metal-coordinated nucleotide. In the case of samples containing ADP, the measured 31P hyperfine couplingsclearly 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 tochanges in the metal-site coordination symmetry because of ligands not involving phosphate groups. Thekinetic 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 whenthe metal and ADP are added to the protein sequentially or together as a preformed complex. The differentstates evolve to the same conformation, the metalII−ADP inhibited form, upon induction of the trisitecatalytic activity. All our spectroscopic and kinetic data point to the active role of the divalent cation increating a competent catalytic site upon binding to MF1, in accordance with previous evidence obtainedfor Escherichia coli and chloroplast F1.
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