Identification of a Cd(2+)- and Zn(2+)-Binding Site in Cytochrome c Using FTIR Coupled to an ATR Microdialysis Setup and NMR Spectroscopy - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue Biochemistry Année : 2005

Identification of a Cd(2+)- and Zn(2+)-Binding Site in Cytochrome c Using FTIR Coupled to an ATR Microdialysis Setup and NMR Spectroscopy

Résumé

Fourier transform infrared (FTIR) difference spectroscopy allows the study of molecular changes occurring at active sites in proteins with high sensitivity. Reactions are triggered by light, potential, or temperature steps and more recently by the diffusion of buffers containing effectors above membrane proteins deposited as films on ATR crystals. We have adapted a microdialysis system to an ATR, to study metal sites in soluble proteins. In this study, we identified a Cd(2+)- or Zn(2+)-binding site in cytochrome c with dissociation constants of 17 and 42 microM, respectively, which affects the oxidation rate of ferrocytochrome c by hydrogen peroxide. Using the microdialysis ATR-FTIR setup, we determined that a histidine and the carboxylate group of a glutamate are involved in Zn(2+) binding. The implication of His 33 and Glu 104 in the binding site was deduced from the comparison of FTIR data recorded with horse heart and the variant tuna cytochrome c lacking these two amino acids. A two-dimensional NMR analysis of the Zn(2+)-binding site in horse heart cytochrome c confirmed that His 33 and residues close to the C terminus are sensitive to Zn(2+) binding. This study demonstrates that the microdialysis ATR-FTIR setup is promising for the analysis of metal sites in proteins. From H(2)O/(2)H(2)O exchange experiments, we concluded that the impact of Zn(2+) and Cd(2+) binding on the oxidation kinetics of ferrocytochrome c by H(2)O(2) is associated to the perturbation of a hydrogen-bonding network involving His 33 that is sensitive to the redox state of cytochrome c

Dates et versions

hal-00112817 , version 1 (09-11-2006)

Identifiants

Citer

S. Gourion-Arsiquaud, Soizic Chevance, P. Bouyer, L. Garnier, J.-L. Montillet, et al.. Identification of a Cd(2+)- and Zn(2+)-Binding Site in Cytochrome c Using FTIR Coupled to an ATR Microdialysis Setup and NMR Spectroscopy. Biochemistry, 2005, 44 (24), pp.8652-63. ⟨10.1021/bi050322l⟩. ⟨hal-00112817⟩
113 Consultations
0 Téléchargements

Altmetric

Partager

Gmail Facebook X LinkedIn More