Semisynthesis of cytochrome c analogues. The effect of modifying the conserved residues 38 and 39

Abstract

We have used chemical and enzymic protein engineering techniques to create analogues of the semisynthetic two-fragment complex (1-37).(38-104) of mitochondrial cytochrome c. This complex, unlike the natural product of specific tryptic cleavage, (1-38).(39-104), from which it is prepared, quite closely resembles the parent protein in functional characteristics and is thus a suitable substrate for modifications designed to study structure-function relations. We have replaced the invariant Arg-38 and the conserved Lys-39 with a range of alternative amino acids and have studied the effects on the principal functional parameters. The hydrogen-bonding capacity of Arg-38 is crucial to the stabilization of the bottom omega-loop, while the positive charge of Lys-39 helps maintain the high redox potential by electrostatic effects at the haem iron.

Role of arginine-38 in regulation of the cytochrome c oxidation-reduction equilibrium

“…However, even with this latter system, interpretation of functional differences is not straightforward. A recent report by Proudfoot and Wallace (1987) illustrates this point. These workers present studies of complexes in which the peptide bond between residues 37 and 38 was hydrolyzed and in which residue 38 was replaced by either Lys or Gin.…”