The Electron Transfer Complex between Cytochrome c552 and the CuA Domain of the Thermus thermophilus ba3 Oxidase A COMBINED NMR AND COMPUTATIONAL APPROACH

The structural analysis of the redox complex between the soluble cytochrome c(552) and the membrane-integral cytochrome ba(3) oxidase of Thermus thermophilus is complicated by the transient nature of this protein-protein interaction. Using NMR-based chemical shift perturbation mapping, however, we identified the contact regions between cytochrome c(552) and the Cu-A domain, the fully functional water-soluble fragment of subunit II of the ba(3) oxidase. First we determined the complete backbone resonance assignments of both proteins for each redox state. Subsequently, two-dimensional [N-15, H-1]TROSY spectra recorded for each redox partner both in free and complexed state indicated those surface residues affected by complex formation between the two proteins. This chemical shift analysis performed for both redox states provided a topological description of the contact surface on each partner molecule. Remarkably, very pronounced indirect effects, which were observed on the back side of the heme cleft only in the reduced state, suggested that alterations of the electron distribution in the porphyrin ring due to formation of the protein-protein complex are apparently sensed even beyond the heme propionate groups. The contact residues of each redox partner, as derived from the chemical shift perturbation mapping, were employed for a protein-protein docking calculation that provided a structure ensemble of 10 closely related conformers representing the complex between cytochrome c(552) and the Cu-A domain. Based on these structures, the electron transfer pathway from the heme of cytochrome c(552) to the Cu-A center of the ba(3) oxidase has been predicted.