Cytochrome c peroxidase (CCP) from Saccharomyces cerevisiae was subjected to directed mol. evolution to generate mutants with increased activity against the classical peroxidase substrate, guaiacol, thus changing the substrate specificity of CCP from the protein, cytochrome c, to a small org. mol. After 3 rounds of DNA shuffling and screening, mutants were isolated which possessed a 300-fold increased activity against guaiacol and an up to 1000-fold increased specificity for this substrate relative to that for the natural substrate. In all of the selected mutants, the distal Arg-48 residue, which is fully conserved in the superfamily of peroxidases, was mutated to His, showing that this mutation plays a key role in the significant increase in activity against phenolic substrates. The results suggest that, in addn. to stabilizing the reactive intermediate compd. I, the distal Arg residue plays an important role as a gatekeeper in the active site of CCP, controlling the access to the ferryl O atom and the distal His residue. Other isolated mutations increased the general reactivity of CCP, or increased the intracellular concn. of the active holo form, allowing their selection under the employed screening conditions. The results illustrated the ability of directed mol. evolution technologies to deliver solns. to biochem. problems that would not be readily predicted by rational design. [on SciFinder (R)]