The structural and functional role of lysine residues in the binding domain of cytochrome c in the electron transfer to cytochrome c oxidase

The interactions of yeast iso-1 cytochrome c with bovine cytochrome c oxida se were studied using cytochrome c variants in which lysines of the binding domain were substituted by alanines. Resonance Raman spectra of the fully oxidized complexes of both proteins reveal structural changes of both the h eme c and the hemes a and a(3). The structural changes in cytochrome c are the same as those observed upon binding to phospholipid vesicles where the bound protein exists in two conformers, B1 and B2. Whereas the structure of B1 is the same as that of the unbound cytochrome c, the formation of B2 is associated with substantial alterations of the heme pocket. In cytochrome c oxidase, the structural changes in both hemes refer to more subtle pertur bations of the immediate protein environment and may be a result of a confo rmational equilibrium involving two states. These changes are qualitatively different to these observed for cytochrome c oxidase upon poly-L-lysine bi nding. The resonance Raman spectra of the various cytochrome c/cytochrome c oxidase complexes were analyzed quantitatively. The spectroscopic studies were paralleled by steady-state kinetic measurements of the same protein co mbinations. The results of the spectra analysis and the kinetic studies wer e used to determine the stability of the complexes and the conformational e quilibria B2/B1 for all cytochrome c variants. The complex stability decrea ses in the order: wild-type WT > J72K > K79A > K73A > K87A > J72A > K86A > K73A/K79A (where J is the natural trimethyl lysine). This order is not exhi bited by the conformational equilibria. The electrostatic control of state B2 formation does not depend on individual intermolecular salt bridges, but on the charge distribution in a specific region of the front surface of cy tochrome c that is defined by the lysyl residues at positions 72, 73 and 79 . On the other hand, the conformational changes in cytochrome c oxidase wer e found to be independent of the identity of the bound cytochrome c variant . The maximum rate constants determined from steady-state kinetic measureme nts could be related to the conformational equilibria of the bound cytochro me c using a simple model that assumes that the conformational transitions are faster than product formation. Within this model, the data analysis lea ds to the conclusion that the interprotein electron transfer rate constant is around two times higher in state B2 than in B1. These results can be int erpreted in terms of an increase of the driving force in state B2 as a resu lt of the large negative shift of the reduction potential.