Cu(II)‐Binding properties of a cytochrome c with a synthetic metal‐binding site: His‐X3‐His in an α‐helix

Abstract

A metal-binding site consisting of two histidines positioned His-X₃-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-X₃-His site yields a synthetic metal-binding protein whose metal affinity is sensitive to environmental conditions that alter helix structure or flexibility.