Interaction of cytochrome c with cytochrome c oxidase:: An NMR study on two soluble fragments derived from Paracoccus denitrificans

The functional interactions between the various components of the respiratory chain are relatively short-lived, thus allowing high turnover numbers but at the same time complicating the structural analysis of the complexes. Chemical shift mapping by NMR spectroscopy is a useful tool to investigate such transient contacts, since it can monitor changes in the electron-shielding properties of a protein as the result of temporary contacts with a reaction partner. In this study, we investigated the molecular interaction between two components of the electron-transfer chain from Paracoccus denitrificans: the engineered, water-soluble fragment of cytochrome C-552 and the Cu-A domain from the cytochrome c oxidase. Comparison of [N-15,H-1]-TROSY spectra of the [N-15]-labeled cytochrome c(552) fragment in the absence and in the presence of the Cu-A fragment showed chemical shift changes for the backbone amide groups of several, mostly uncharged residues located around the exposed heme edge in cytochrome C-552. The detected contact areas on the cytochrome C-552 surface were comparable under both fully reduced and fully oxidized conditions, suggesting that the respective chemical shift changes represent biologically relevant protein-protein interactions.