RESONANCE RAMAN-STUDY OF THE INTERACTIONS BETWEEN CYTOCHROME-C VARIANTS AND CYTOCHROME-C-OXIDASE

The structural changes in oxidized yeast iso-1-cytochrome c and fully oxidized bovine cytochrome c oxidase that are induced upon complex for mation have been analyzed by resonance Raman spectroscopy. The main sp ectral 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 ox idase [Hildebrandt et al. (1990) Biochemistry 29, 1661-1668]. These sp ectral changes are attributed to the formation of the conformational s tate II (approximately 45%) which exhibits an open heme pocket structu re. The structural changes are assumed to be induced by the electrosta tic interactions between the negatively charged binding domain on cyto chrome c oxidase and the positively charged lysine residues on the fro nt 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 co nformational 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 sugg ests that lysine-79 may be involved in controlling conformational deta ils within the heme pocket of the bound cytochrome c. Due to the under lying structural changes and the lowered redox potential, formation of state II may be of functional importance for the physiological electr on-transfer process by lowering the reorganization energy and increasi ng the driving force. The spectral changes caused by complex formation that are attributable to cytochrome c oxidase indicate structural cha nges of the vinyl and formyl substituents while the ground-state confo rmations of the porphyrin macrocycles are preserved. This finding impl ies that the conformational changes in the heme pockets of cytochrome c oxidase are much smaller than those in cytochrome c. These changes r efer not only to heme a but also to heme a3, located remote from the c ytochrome c binding site, pointing to a long-range structural communic ation between the binding domain and the oxygen reduction site. The po ssible functional implications of these structural changes are discuss ed.