Structurally engineered cytochromes with unusual ligand-binding properties: Expression of Saccharomyces cerevisiae Met-80 --> Ala iso-1-cytochrome c

Abstract

A strategy has been developed to express and purify a recombinant, nonfunctional axial-ligand mutant of iso-1-cytochrome c (Met-80 --> Ala) in Saccharomyces cerevisiae in quantities necessary for extensive biophysical characterization. It involves coexpressing in the same plasmid (YEp213) the nonfunctional gene with a functional gene copy for complementation in a selective medium. The functional gene encodes a product with an engineered metal-chelating dihistidine site (His-39 and Leu-58 --> His) that enables efficient separation of the two isoforms by immobilized metal-affinity chromatography. The purified Met-80 --> Ala protein possesses a binding site for dioxygen and other exogenous ligands. Absorption spectra of several derivatives of this mutant show striking similarities to those of corresponding derivatives of horse-radish peroxidase, myoglobin, and cytochrome P450. The use of a dual-gene vector for cytochrome c expression together with metal-affinity separation opens the way for the engineering of variants with dramatically altered structural and catalytic properties.