Different interaction modes of two cytochrome-c oxidase soluble CuA fragments with their substrates.
Cytochrome-c oxidase is the terminal enzyme in the respiratory chains of mitochondria and many bacteria and catalyzes the formation of water by reduction of dioxygen. The first step in the cytochrome oxidase reaction is the bimolecular electron transfer from cytochrome c to the homobinuclear mixed-valence CuA center of subunit II. In Thermus thermophilus a soluble cytochrome c552 acts as the electron donor to ba3 cytochrome-c oxidase, an interaction believed to be mainly hydrophobic. In Paracoccus denitrificans, electrostatic interactions appear to play a major role in the electron transfer process from the membrane-spanning cytochrome c552. In the present study, soluble fragments of the CuA domains and their respective cytochrome c electron donors were analyzed by stopped-flow spectroscopy to further characterize the interaction modes. The forward and the reverse electron transfer reactions were studied as a function of ionic strength and temperature, in all cases yielding monoexponential time-dependent reaction profiles in either direction. From the apparent second-order rate constants, equilibrium constants were calculated, with values of 4.8 and of 0.19, for the T. thermophilus and P. denitrificans c552 and CuA couples, respectively. Ionic strength strongly affects the electron transfer reaction in P. denitrificans indicating that about five charges on the protein interfaces control the interaction, when analyzed according to the Brønsted equation, whereas in the T. thermophilus only 0.5 charges are involved. Overall the results indicate that the soluble CuA domains are excellent models for the initial electron transfer processes in cytochrome-c oxidases.
Maneg
Oliver
O
Molekulare Genetik, Biozentrum, J. W. Goethe-Universität, Marie-Curie-Strasse 9, Frankfurt D-60439, Germany.
Ludwig
Bernd
B
Malatesta
Francesco
F
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D003300:Copper; D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D009994:Osmolar Concentration; D010231:Paracoccus denitrificans / Q000201:enzymology; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D016963:Thermus thermophilus / Q000201:enzymology
D001426:Bacterial Proteins; D003574:Cytochrome c Group; D021122:Protein Subunits; C083951:cytochrome c553; D003300:Copper; C022302:cytochrome C-552; D003576:Electron Transport Complex IV