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(1) 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 (31)P nuclei of the metal-coordinated nucleotide. In the case of samples containing ADP, the measured (31)P 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).
Zoleo
Alfonso
A
Department of Chemistry, University of Padova, via Marzolo 1, I-35131 Padova, Italy.
Contessi
Stefania
S
Lippe
Giovanna
G
Pinato
Luca
L
Brustolon
Marina
M
Brunel
Louis Claude
LC
Dabbeni-Sala
Federica
F
Maniero
Anna Lisa
AL
D000244:Adenosine Diphosphate / Q000737:chemistry; D000266:Adenylyl Imidodiphosphate / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D002413:Cations, Divalent / Q000737:chemistry; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D046911:Macromolecular Substances; D008345:Manganese / Q000737:chemistry* / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000201:enzymology*; D025261:Mitochondrial Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*
D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D046911:Macromolecular Substances; D000266:Adenylyl Imidodiphosphate; D008345:Manganese; D000244:Adenosine Diphosphate; D025261:Mitochondrial Proton-Translocating ATPases