title: Protein-protein interactions studied by EPR relaxation measurements: cytochrome c and cytochrome c oxidase.
abstract: The complex formed between cytochrome c oxidase from Paracoccus denitrificans and its electron-transfer partner cytochrome c has been studied by multi-frequency pulse electron paramagnetic resonance spectroscopy. The dipolar relaxation of a fast-relaxing paramagnetic center induced on a more slowly relaxing center can be used to measure their distance in the range of 1-4 nm. This method has been used here for the first time to study transient protein-protein complex formation, employing soluble fragments for both interacting species. We observed significantly enhanced transversal relaxation of the CuA center in cytochrome c oxidase due to the fast-relaxing iron of cytochrome c upon complex formation. The possibility to measure cytochrome c oxidase in the presence and absence of cytochrome c permitted us to separate the dipolar relaxation from other relaxation contributions. This allowed a quantitative simulation and interpretation of the relaxation data. The specific temperature dependence of the dipolar relaxation together with the high orientational selectivity achieved at high magnetic field values may provide detailed information on distance and relative orientation of the two proteins with respect to each other in the complex. Our experimental results cannot be explained by any single well-defined structure of the complex of cytochrome c oxidase with cytochrome c, but rather suggest that a broad distribution in distances and relative orientations between the two proteins exist within this complex.
authors: Institute of Physical and Theoretical Chemistry, Institute of Biochemistry, Center for Biomolecular Magnetic Resonance, and Center of Excellence "Macromolecular Complexes", Johann Wolfgang Goethe University, Frankfurt am Main, Germany.
authors: Sevdalina
authors: S
authors: Lyubenova
authors: M Khalid
authors: MK
authors: Siddiqui
authors: Marloes J M Penning de
authors: MJ
authors: Vries
authors: Bernd
authors: B
authors: Ludwig
authors: Thomas F
authors: TF
authors: Prisner
mesh_terms: D045304:Cytochromes c / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D003576:Electron Transport Complex IV / Q000737:chemistry*; D011485:Protein Binding; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors
chemical_list: D045304:Cytochromes c; D003576:Electron Transport Complex IV