Cu(II)-Binding properties of a cytochrome c with a synthetic metal-binding site: His-X_3-His in an α-helix

A metal‐binding site consisting of two histidines positioned His‐X_3‐His in an α‐helix has been engineered into the surface of Saccharomyces cerevisiae iso‐1‐cytochrome c. The synthetic metal‐binding cytochrome c retains its biological activity in vivo. Its ability to bind chelated Cu(II) has been characterized by partitioning in aqueous two‐phase polymer systems containing a polymer‐metal complex, Cu(II)IDA‐PEG, and by metal‐affinity chromatography. The stability constant for the complex formed between Cu(II)IDA‐PEG and the cytochrome c His‐X3‐His site is 5.3 × 104^ M^(−1), which corresponds to a chelate effect that contributes 1.5 kcal mol^(−1) to the binding energy. Incorporation of the His‐X_3‐His site yields a synthetic metal‐binding protein whose metal affinity is sensitive to environmental conditions that alter helix structure or flexibility