Resonance Raman study of the interactions between cytochrome c variants and cytochrome c oxidase

• Published in: Biochemistry (Easton) Vol. 32; no. 40; pp. 10912 - 10922
• Main Authors: Hildebrandt, Peter, Vanhecke, Franck, Buse, Gerhard, Soulimane, Tewfik, Mauk, A. Grant
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 12-10-1993
• Subjects: Alanine; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Binding Sites; Biological and medical sciences; Cattle; Cytochrome c Group - chemistry; Cytochrome c Group - metabolism; Cytochromes c; Electron Transport Complex IV - chemistry; Electron Transport Complex IV - metabolism; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Heme - metabolism; Horses; Interactions. Associations; Intermolecular phenomena; Models, Molecular; Molecular biophysics; Mutagenesis, Site-Directed; Oxidoreductases; Protein Conformation; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins; Spectrum Analysis, Raman - methods; Yeast; Bovine; Enzyme; Cytochrome-c oxidase; Electrostatic interaction; Electron transfer; In vitro; Structure function relationship; Cytochrome c; Intermolecular interaction; Vertebrata; Mammalia; Artiodactyla; Oxidoreductases; Ungulata; Conformational transition
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00091a047

The structural changes in oxidized yeast iso-1-cytochrome c and fully oxidized bovine cytochrome c oxidase that are induced upon complex formation have been analyzed by resonance Raman spectroscopy. The main spectral changes could be ascribed to cytochrome c, which in the case of the wild-type protein are essentially the same as previously observed in the complex of horse heart cytochrome c and bovine cytochrome c oxidase [Hildebrandt et al. (1990) Biochemistry 29, 1661-1668]. These spectral changes are attributed to the formation of the conformational state II (approximately 45%) which exhibits an open heme pocket structure. The structural changes are assumed to be induced by the electrostatic interactions between the negatively charged binding domain on cytochrome c oxidase and the positively charged lysine residues on the front surface of cytochrome c. Substituting one of these lysine residues (i.e., Lys-72) by an alanine significantly lowers the state II content (< 15%), implying that this lysine is essential for controlling the conformational equilibrium of the bound protein. On the other hand, the replacement of lysine-79 by alanine only slightly lowers the state II content (approximately 35%). However, the analysis of the spectra suggests that lysine-79 may be involved in controlling conformational details within the heme pocket of the bound cytochrome c. Due to the underlying structural changes and the lowered redox potential, formation of state II may be of functional importance for the physiological electron-transfer process by lowering the reorganization energy and increasing the driving force. The spectral changes caused by complex formation that are attributable to cytochrome c oxidase indicate structural changes of the vinyl and formyl substituents while the ground-state conformations of the porphyrin macrocycles are preserved. This finding implies that the conformational changes in the heme pockets of cytochrome c oxidase are much smaller than those in cytochrome c. These changes refer not only to heme a but also to heme a3, located remote from the cytochrome c binding site, pointing to a long-range structural communication between the binding domain and the oxygen reduction site. The possible functional implications of these structural changes are discussed.