High-Affinity Metal-Binding Site in Beef Heart Mitochondrial F1ATPase:  An EPR Spectroscopy Study

• Published in: Biochemistry (Easton) 2004-10, Vol.43 (41), p.13214-13224
• Main Authors: Zoleo, Alfonso, Contessi, Stefania, Lippe, Giovanna, Pinato, Luca, Brustolon, Marina, Brunel, Louis Claude, Dabbeni-Sala, Federica, Maniero, Anna Lisa
• Format: Article
• Language: English
• Subjects: Adenosine Diphosphate - chemistry; Adenylyl Imidodiphosphate - chemistry; Animals; Binding Sites; Catalytic Domain; Cations, Divalent - chemistry; Cattle; Electron Spin Resonance Spectroscopy - methods; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - metabolism; Macromolecular Substances; Manganese - chemistry; Manganese - metabolism; Mitochondria, Heart - enzymology; Mitochondrial Proton-Translocating ATPases - antagonists & inhibitors; Mitochondrial Proton-Translocating ATPases - chemistry; Mitochondrial Proton-Translocating ATPases - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi049525k

The high-affinity metal-binding site of isolated F1-ATPase from beef heart mitochondria was studied by high-field (HF) continuous wave electron paramagnetic resonance (CW-EPR) and pulsed EPR spectroscopy, using MnII as a paramagnetic probe. The protein F1 was fully depleted of endogenous MgII and nucleotides [stripped F1 or MF1(0,0)] and loaded with stoichiometric MnII and stoichiometric or excess amounts of ADP or adenosine 5‘-(β,γ-imido)-triphosphate (AMPPNP). MnII 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 MnII enters one catalytic site on a β 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 MnII and the 31P nuclei of the metal-coordinated nucleotide. In the case of samples containing ADP, the measured 31P 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 metalII−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 F1.