The pH-dependent conformational equilibria of iso-1-ferricytochrome c that occur between pH 7 and pH 12 have been studied by resonance Raman (RR) spectroscopy. Detailed analysis of the RR spectra provides the number and the spectra of the conformational species that occur over this range of pH as well as their relative concentrations at each pH. Between pH 7 and pH 12, the wild-type protein exhibits five conformers which differ with respect to the axial ligands of the heme iron. In the medium alkaline pH range, the neutral form undergoes two parallel conformational transitions with similar pK as (8.7 and 8.9) to those of the states IVa and IVb. Comparative studies with protein variants in which either Lys79 or Lys73 or both lysyl residues were replaced by alanine(s) confirm previous conclusions (Rosell et al., preceding paper in this issue) that Lys73 and Lys79 are the axial ligands replacing Met80 in the states IVa and IVb, respectively. On the basis of isotopic shifts caused by H/D exchange, the Fe−N(Lys) stretching vibrations of these species were identified at 385 cm-1. In the high alkaline pH range, the wild-type protein forms two further conformers with pK as of 10.5 and 11. In these states, Va and Vb, which are also detected in the Lys73Ala, Lys79Ala, and the Lys73Ala/Lys79Ala variant proteins, another strong-field ligand, presumably a hydroxide, occupies the sixth axial coordination site. Comparison of the RR spectra demonstrates far-reaching similarities between state Va and the conformational state B2 that cytochrome c forms in electrostatically stabilized complexes at neutral pH where, inter alia, Lys73 and Lys79 are involved in intermolecular interactions. This finding as well as the dramatically lowered pK as (<10) for formation of Va and Vb in the double variant (Lys73Ala/Lys79Ala) suggest that the residues 73 and 79 are part of a switching mechanism by which conformational changes at the heme pocket are induced to regulate the function of the protein in biological electron-transfer reactions.

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The interactions of yeast iso-1 cytochrome c with bovine cytochrome c oxidase 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 heme 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 perturbations of the immediate protein environment and may be a result of a conformational equilibrium involving two states. These changes are qualitatively different to those observed for cytochrome c oxidase upon poly-l-lysine binding. 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 combinations. The results of the spectra analysis and the kinetic studies were used to determine the stability of the complexes and the conformational equilibria B2/B1 for all cytochrome c variants. The complex stability decreases 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 exhibited 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 cytochrome 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 were found to be independent of the identity of the bound cytochrome c variant. The maximum rate constants determined from steady-state kinetic measurements could be related to the conformational equilibria of the bound cytochrome c using a simple model that assumes that the conformational transitions are faster than product formation. Within this model, the data analysis leads 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 interpreted in terms of an increase of the driving force in state B2 as a result of the large negative shift of the reduction potential.