A copper protein and a cytochrome bind at the same site on bacterial cytochrome c peroxidase.
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.
Pauleta
Sofia R
SR
Veterinary Biomedical Sciences, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Summerhall, UK.
Cooper
Alan
A
Nutley
Margaret
M
Errington
Neil
N
Harding
Stephen
S
Guerlesquin
Francoise
F
Goodhew
Celia F
CF
Moura
Isabel
I
Moura
Jose J G
JJ
Pettigrew
Graham W
GW
D001400:Azurin / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001667:Binding, Competitive; D002151:Calorimetry; D002498:Centrifugation; D002499:Centrifugation, Density Gradient; D003198:Computer Simulation; D003300:Copper / Q000737:chemistry*; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism; D003578:Cytochrome-c Peroxidase / Q000737:chemistry* / Q000378:metabolism; D046911:Macromolecular Substances / Q000737:chemistry / Q000378:metabolism; D008667:Metalloproteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D042084:Paracoccus pantotrophus / Q000201:enzymology* / Q000378:metabolism; D011485:Protein Binding; D013816:Thermodynamics
D001426:Bacterial Proteins; D003574:Cytochrome c Group; D046911:Macromolecular Substances; D008667:Metalloproteins; C047163:pseudoazurin; D001400:Azurin; D003300:Copper; C031732:cytochrome C-550; D003578:Cytochrome-c Peroxidase