Interaction of Cytochrome c with Cytochrome c Oxidase: An NMR Study on Two Soluble Fragments Derived from Paracoccus denitrificans ² Hans Wienk, ‡,⊥,| Oliver Maneg, §,| Christian Lu ¨cke, ‡,# Primoz ˇ Pristovs ˇek, ¶ Frank Lo ¨hr, ‡ Bernd Ludwig, § and Heinz Ru ¨terjans* ,‡ Institute of Biophysical Chemistry, J.W. Goethe-UniVersity, and Molecular Genetics, Institute of Biochemistry, J.W. Goethe-UniVersity, Marie-Curie-Strasse 9, D-60439 Frankfurt am Main, Germany, and National Institute of Chemistry, HajdrihoVa 19, SI-1000 Ljubljana, SloVenia ReceiVed NoVember 19, 2002; ReVised Manuscript ReceiVed March 17, 2003 ABSTRACT: 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 tran- sient 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 be- tween 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 [ 15 N, 1 H]-TROSY spectra of the [ 15 N]-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. In the soil bacterium Paracoccus denitrificans, the elec- tron transfer from complex III (cytochrome bc 1 ) to complex IV (heme aa 3 -type cytochrome c oxidase) is mediated by the 18-kDa membrane-bound cytochrome c 552 (1, 2). In the second half of this shuttle pathway, the reduced cyto- chrome c 552 interacts with the hydrophilic domain of sub- unit II of the oxidase. Positively charged lysine residues on the cytochrome c 552 surface, clustered around its exposed heme edge, and patches of opposite charge above the binuclear Cu A center in subunit II provide the basis for long- range electrostatic attractions that define the encounter complex (3). This is thought to be followed by a phase of molecular rearrangement that involves hydrophobic resi- dues on the surface of both molecules, enabling the extra electron in the reduced heme of cytochrome c 552 to be transferred to the Cu A center, the first acceptor in the oxi- dase. This heme-to-copper electron transfer is mediated by tryptophan 121 at the contact surface of the oxidase subunit II (4). The study of transient electron-transfer complexes in atomic detail is not straightforward. In vivo, protein-protein interactions during electron transfer are optimized to support high turnover numbers, implying that only transient com- plexes between the redox partners are formed. Moreover, due to fast redox equilibration in bimolecular complexes, the redox state of the individual components (electron donor and acceptor) is likely to be inhomogeneous under physi- ological conditions. For practical reasons, however, in vitro studies usually employ nonfunctional protein derivatives or use an excess of redox reagent to produce a homogeneous redox state. Only a limited number of experimental techniques yield structural information on an atomic level. High-resolution NMR 1 spectroscopy has been established as a powerful method to obtain detailed three-dimensional structures of biomolecules. In addition, it is particularly useful for the topological analysis of transient complexes (e.g., refs 5 and 6) and for the study of different redox states, since the chemical shifts observed in NMR spectra provide information on the electronic shielding of the observed nuclei. ² This work was supported by the European Union (grant no. QLG2- CT-1999-01003) and by the Deutsche Forschungsgemeinschaft (SFB 472). H.W. received a fellowship from the Alexander-von-Humboldt Foundation. P.P. received financial support from the Ministry of Education, Science and Sport of Slovenia. * To whom correspondence should be addressed. E-mail: hruet@ bpc.uni-frankfurt.de. ‡ Institute of Biophysical Chemistry, J.W. Goethe-University. § Molecular Genetics, Institute of Biochemistry, J.W. Goethe- University. ¶ National Institute of Chemistry, Slovenia. ⊥ Present address: Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. # Present address: Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, D-06120 Halle, Germany. | These authors contributed equally to the experiments reported in this study. 1 Abbreviations: CuA, water-soluble CuA fragment of subunit II from P. denitrificans cytochrome c oxidase; cyt c552, water-soluble domain of P. denitrificans cytochrome c552; NMR, nuclear magnetic resonance; TROSY, transverse relaxation optimized spectroscopy. 6005 Biochemistry 2003, 42, 6005-6012 10.1021/bi027198f CCC: $25.00 © 2003 American Chemical Society Published on Web 04/29/2003