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

YEp vector with a target nonfunctional variant and a functional cytochrome c gene facilitates the expression and resolution of a recombinant and nonfunctional axial-ligand mutant of iso-1-cytochrome c, Met-80 linked to Ala, in Saccharomyces cerevisiae. The functional cytochrome c gene codes for a gene with an altered metal-cheating dishistidine site, His-39 and Len-58 linked to His, and facilitates the resolution of two isoforms using static metal-attracted chromatography. Cytochromes and variants can be constructed with engineered structural and catalytic features using a double-gene vector for the expressing cytochrome c and a metal-mediates extraction process.