The high affinity metal binding site of isolated F1-ATPase from beef heart mitochondria was studied by High Field CWEPR and Pulsed EPR spectroscopy, using Mn(II) as a paramagnetic probe. The protein F1 was fully depleted of endogenous Mg(II) and nucleotides (stripped F1 or MF1(0,0)) and loaded with stoichiometric Mn(II) and stoichiometric or excess amounts of ADP or AMPPNP. Mn(II) and nucleotides were added to MF1(0,0) both subsequentially and together as preformed complexes. Metal-ADP inhibition kinetics analysis was performed showing that Mn(II) enters one catalytic site on a b subunit. From the HF-EPR spectra the 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 on the protein/metal/nucleotide molar ratios. ESEEM technique was used to obtain information on the interaction between Mn(II) and the 31P nuclei of the metal coordinating nucleotide. In the case of the 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 of the remarkably differences present among their HFEPR spectra. This fact has been attributed to changes in the metal site coordination symmetry due to ligands not involving phosphate groups. In this investigation we showed that the divalent metal triggers the high affinity conformation of the catalytic site and ADP binds the preformed Mn(II) – MF1 (0,0) complex to give the Mg(II)-ADP inhibited form upon tri-site catalytic activity. By studying protein samples prepared in different ways, we were able to prove for MF1 what was previously demonstrated for E. Coli F1 and CF1: the active role of the divalent cation in creating a competent catalytic site upon binding.

The high affinity metal binding site in beef heart mitochondrial F-1 ATPase: an EPR spectroscopy study

DABBENI SALA, FEDERICA;ZOLEO, ALFONSO;MANIERO, ANNA LISA
2004

Abstract

The high affinity metal binding site of isolated F1-ATPase from beef heart mitochondria was studied by High Field CWEPR and Pulsed EPR spectroscopy, using Mn(II) as a paramagnetic probe. The protein F1 was fully depleted of endogenous Mg(II) and nucleotides (stripped F1 or MF1(0,0)) and loaded with stoichiometric Mn(II) and stoichiometric or excess amounts of ADP or AMPPNP. Mn(II) and nucleotides were added to MF1(0,0) both subsequentially and together as preformed complexes. Metal-ADP inhibition kinetics analysis was performed showing that Mn(II) enters one catalytic site on a b subunit. From the HF-EPR spectra the 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 on the protein/metal/nucleotide molar ratios. ESEEM technique was used to obtain information on the interaction between Mn(II) and the 31P nuclei of the metal coordinating nucleotide. In the case of the 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 of the remarkably differences present among their HFEPR spectra. This fact has been attributed to changes in the metal site coordination symmetry due to ligands not involving phosphate groups. In this investigation we showed that the divalent metal triggers the high affinity conformation of the catalytic site and ADP binds the preformed Mn(II) – MF1 (0,0) complex to give the Mg(II)-ADP inhibited form upon tri-site catalytic activity. By studying protein samples prepared in different ways, we were able to prove for MF1 what was previously demonstrated for E. Coli F1 and CF1: the active role of the divalent cation in creating a competent catalytic site upon binding.
File in questo prodotto:
Non ci sono file associati a questo prodotto.
Pubblicazioni consigliate

Caricamento pubblicazioni consigliate

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11577/156251
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? 0
social impact