A rationale for the absolute conservation of Asn70 and Pro71 in mitochondrial cytochromes c suggested by protein...

The absolutely conserved residues Asn70 and Pro71 of mitochondrial cytochrome c have been targeted for protein engineering by semisynthesis. Neither residue has even been implicated in mechanistic schemes, and we reasoned that the conservation of this dipeptide was to fulfill a crucial structural role. Semisynthesis was through condensation by autocatalytic fragment religation of natural fragment 1-65 (H) of the horse protein and synthetic 39-residue peptides containing noncoded amino acids prepared by solid-phase methods. High yields of the purified analogs, homoserine70 and norvaline71 cytochromes c, were obtained. Functional tests revealed minor destabilization of the Hse70-containing structure, with little adverse effect in in vitro assays, but [Nva71] cytochrome c was essentially devoid of activity in these systems. This appeared to be a consequence of a shift, more pronounced than any yet reported, in the conformational equilibrium between the active state III conformer and the inactive, 'alkaline' state IV. The results support our view that this dipeptide is optimal for, and rigidifies, the right-angle bend between two alpha-helices, thus determining the conformation of the 70s loop that terminates in the sixth ligand Met80, and 'forcing' the coordination of iron by thioether sulfur in the presence of the adjacent more avid amine ligands of state IV. Not only is [Nva71] cytochrome c inactive at pH 7, but it also proves to be an extremely potent inhibitor of electron transfer by native state III, thus providing the rationale for the evolutionary conservation of a high pK for the ligand exchange reaction.