A RATIONALE FOR THE ABSOLUTE CONSERVATION OF ASN(70) AND PRO(71) IN MITOCHONDRIAL CYTOCHROMES-C SUGGESTED BY PROTEIN ENGINEERING

The absolutely conserved residues Asn(70) and pro(71) of mitochondrial cytochrome c have been targeted for protein engineering by semisynthe sis. 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 am ino acids prepared by solid-phase methods. High yields of the purified analogs, homoserine(70) and norvaline(71) cytochromes c, were obtaine d. Functional tests revealed minor destabilization of the Hse(70)-cont aining structure, with little adverse effect in in vitro assays, but [ Nva(71)] cytochrome c was essentially devoid of activity in these syst ems. This appeared to be a consequence of a shift, more pronounced tha n any yet reported, in the conformational equilibrium between the acti ve state III conformer and the inactive, 'alkaline' state IV. The resu lts support our view that this dipeptide is optimal for, and rigidifie s, the right-angle bend between two alpha-helices, thus determining th e conformation of the 70s loop that terminates in the sixth ligand Met (80), and 'forcing' the coordination of iron by thioether sulfur in th e presence of the adjacent more avid amine ligands of state IV. Not on ly is [Nva(71)] cytochrome c inactive at pH 7, but it also proves to b e an extremely potent inhibitor of electron transfer by native state I II, thus providing the rationale for the evolutionary conservation of a high pK for the ligand exchange reaction.