A copper protein and a cytochrome bind at the same site on bacterial cytochrome c peroxidase

Sofia R. Pauleta, Alan Cooper, Margaret Nutley, Neil Errington, Stephen Harding, Francoise Guerlesquin, Celia F. Goodhew, Isabel Moura, José J. G. Moura, Graham W. Pettigrew

Research output: Contribution to journalArticlepeer-review

27 Citations (Scopus)


Pseudoazurin binds at a single site on cytochrome c peroxidase from Paracoccus pantotrophus with a Kd of 16.4 μM at 25°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 1H 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 Å. This is consistent with the results of 1H 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.

Original languageEnglish
Pages (from-to)14566-14576
Number of pages11
Issue number46
Publication statusPublished - 23 Nov 2004


Dive into the research topics of 'A copper protein and a cytochrome bind at the same site on bacterial cytochrome c peroxidase'. Together they form a unique fingerprint.

Cite this