Protein engineering of cytochrome c by semisynthesis: substitutions at Glutamic acid 66

We have used protein semisynthesis to prepare four analoguesof horse cytochrome c, in which the glutamic acid residue atposition 66 has been removed and replaced by norvaline, glutamine,lysine and, as a methodological control, glutamic acid. Thisresidue is quite strongly conserved in mitochondrial cytochromec, and forms part of a cluster of acidic residues that occursin all cytochromes c but whose function is obscure. Comparativestudies of the physical and biochemical properties of the analogueshave now disclosed two specific roles for Glu⁶⁶ in the protein.It contributes significantly to the stabilization of the activeconformation of the protein, probably by salt bridge formation,and it appears to participate in the redox-state-dependent ATP-bindingsite of cytochrome c. Our results also support two general viewsof the role of surface charged residues in cytochrome c, namelythat their disposition influences both redox potential, throughthe electrostatic field felt at the redox centre, and the kineticsof electron transfer, through the dipole moment they generate.