The role of electrostatics in macromolecular associations: The cytochrome b(5)-cytochrome c complex

Determination of the degree of specificity in the recognition of electron transfer proteins is essential in order to gain further insights into electron transfer between biomolecules. Electrostatic interactions are generally implicated in these protein-protein interactions, including the association between cytochrome b$\sb5$ and cytochrome c. The dependence of stability of the association between cytochrome b$\sb5$ and cytochrome c on ionic strength and pH conditions is indicative of the charge-charge interactions in the interfacial domain.Selected negatively charged residues of cytochrome b$\sb5$ were replaced with neutral amino acids using site-directed mutagenesis to ascertain the specificity of electrostatic interactions in complex formation with cytochrome c. Information as to contributions to the protein-protein interaction were gained through determination of thermodynamic parameters for complex formation, including the free energy of association obtained from electronic difference spectroscopy and volume changes upon complex formation measured by high pressure techniques. Increases in pressure results in the dissociation of the cytochrome b$\sb5$-cytochrome c complex, yielding a decreased volume of the system, due to the solvation of charged residues that had been previously sequestered in the interface. Combined with site-directed mutagenesis the specific charge groups participating in the association with cytochrome c can be identified, since the neutral amino acids that have replaced the negative groups will not lead to large volume changes upon dissociation. In general, several acidic residues in the region of the exposed heme edge were found to be involved in the association with cytochrome c. It was concluded that a high degree of specificity existed in the interaction between cytochrome b$\sb5$ and cytochrome c. Thus, site-directed mutagenesis combined with high pressure techniques is a feasible method in mapping macromolecular association sites. In addition, site-directed mutagenesis coupled with the measurement of reduction potentials yielded insights into the electrostatic interactions between charged surface residues and the heme iron of cytochrome b$\sb5$.U of I OnlyETDs are only available to UIUC Users without author permissio