A Copper Protein and a Cytochrome Bind at the Same Site on Bacterial Cytochrome c Peroxidase
| Autor: | Neil Errington, Stephen E. Harding, Alan Cooper, Margaret Nutley, Celia F. Goodhew, Françoise Guerlesquin, Graham W. Pettigrew, José J. G. Moura, Sofia R. Pauleta, Isabel Moura |
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| Rok vydání: | 2004 |
| Předmět: |
Models
Molecular Cytochrome Macromolecular Substances Copper protein Stereochemistry Centrifugation Cytochrome c Group Plasma protein binding Calorimetry Binding Competitive Biochemistry chemistry.chemical_compound Bacterial Proteins Azurin Metalloproteins Centrifugation Density Gradient Computer Simulation Nuclear Magnetic Resonance Biomolecular Heme chemistry.chemical_classification biology Cytochrome c peroxidase Cytochrome-c Peroxidase Enzyme chemistry Paracoccus pantotrophus biology.protein Proton NMR Thermodynamics Copper Protein Binding Peroxidase |
| Zdroj: | Biochemistry. 43:14566-14576 |
| ISSN: | 1520-4995 0006-2960 |
| Popis: | Pseudoazurin binds at a single site on cytochrome c peroxidase from Paracoccus pantotrophus with a K(d) of 16.4 microM at 25 degrees C, pH 6.0, in an endothermic reaction that is driven by a large entropy change. Sedimentation velocity experiments confirmed the presence of a single site, although results at higher pseudoazurin concentrations are complicated by the dimerization of the protein. Microcalorimetry, ultracentrifugation, and (1)H NMR spectroscopy studies in which cytochrome c550, pseudoazurin, and cytochrome c peroxidase were all present could be modeled using a competitive binding algorithm. Molecular docking simulation of the binding of pseudoazurin to the peroxidase in combination with the chemical shift perturbation pattern for pseudoazurin in the presence of the peroxidase revealed a group of solutions that were situated close to the electron-transferring heme with Cu-Fe distances of about 14 A. This is consistent with the results of (1)H NMR spectroscopy, which showed that pseudoazurin binds closely enough to the electron-transferring heme of the peroxidase to perturb its set of heme methyl resonances. We conclude that cytochrome c550 and pseudoazurin bind at the same site on the cytochrome c peroxidase and that the pair of electrons required to restore the enzyme to its active state after turnover are delivered one-by-one to the electron-transferring heme. |
| Databáze: | OpenAIRE |
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