The Electron Transfer Complex between Cytochrome c552 and the CuA Domain of the Thermus thermophilus ba3 Oxidase

Abstract

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 [ 15 N, 1 H]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.

Alternative ground states enable pathway switching in biological electron transfer

“…Previous pathways calculations considering only the σ u * ground state weighted by metalligand covalencies suggested that the Cys ligands act as the electron access point (26). However, our pathways analysis in the ET complex between cytochrome c 552 (Cyt c 552 ) and the Cu A domain of T. thermophilus (13) reveals that the axial Met160 ligand provides the favored electron entry point to the Cu A site (SI Appendix, Tables S6 and S7). Even though the metal-ligand covalency is very low for the axial Cu-S Met bond in the σ u * ground state (Fig.…”

“…Moreover, transient binding of redox protein partners is known to modify the reduction potential of donor and acceptor sites (28) and to induce geometrical distortions of copper redox centers (29,30). In fact, the study of the Cyt 552 -Cu A complex reveals subtle structural perturbations at the Cu A site in a redox state-dependent fashion (13). In addition, the prediction that moderate electric fields alter the barrier for σ u *-π u interchange suggests that the local membrane potential could exert regulation on the electroprotonic energy transduction, as previously proposed based on different evidence (31,32).…”

“…These steps involve two long, nearly perpendicular pathways through the protein milieu. Despite the low driving forces, ET takes place with high rates along these two pathways (12)(13)(14). This efficiency has been attributed to the unique coordination features of the Cu A site (Fig.…”

Electron transfer kinetics of soluble fragments indicate a direct interaction between complex III and the caa3 oxidase in Thermus thermophilus

“…Electron transfer in T. thermophilus between complex III and the terminal oxidases had previously been analyzed by a variety of structural and kinetic approaches (5,7,9,10,14,22). Using soluble fragments of redox protein complexes to determine ET interactions has proven a successful approach (5, 6, 9, 23 -26): it offers the advantages of a simplified experimental handling, as no detergents are required, and avoids kinetic interference from subsequent internal ET or energy transducing steps, allowing the following of only the single ET reaction of interest.…”

“…Electron transfer (ET) between the bc-complex and the terminal oxidases via cytochrome c 552 has been studied by presteady state (5,6,9) and steady-state (3, 10, 13) kinetics as well as NMR and computational approaches (14), revealing fast and efficient ET reactions (bimolecular rate constants of ca.…”