Abstract
| - Fourier transform infrared (FTIR) difference spectroscopy allows the study of molecular changesoccurring at active sites in proteins with high sensitivity. Reactions are triggered by light, potential, ortemperature steps and more recently by the diffusion of buffers containing effectors above membraneproteins deposited as films on ATR crystals. We have adapted a microdialysis system to an ATR, tostudy metal sites in soluble proteins. In this study, we identified a Cd2+- or Zn2+-binding site in cytochromec with dissociation constants of 17 and 42 μM, respectively, which affects the oxidation rate offerrocytochrome c by hydrogen peroxide. Using the microdialysis ATR−FTIR setup, we determined thata histidine and the carboxylate group of a glutamate are involved in Zn2+ binding. The implication of His33 and Glu 104 in the binding site was deduced from the comparison of FTIR data recorded with horseheart and the variant tuna cytochrome c lacking these two amino acids. A two-dimensional NMR analysisof the Zn2+-binding site in horse heart cytochrome c confirmed that His 33 and residues close to the Cterminus are sensitive to Zn2+ binding. This study demonstrates that the microdialysis ATR−FTIR setupis promising for the analysis of metal sites in proteins. From H2O/2H2O exchange experiments, we concludedthat the impact of Zn2+ and Cd2+ binding on the oxidation kinetics of ferrocytochrome c by H2O2 isassociated to the perturbation of a hydrogen-bonding network involving His 33 that is sensitive to theredox state of cytochrome c.
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