Crystal structure of guaiacol and phenol bound to a heme peroxidase

Guaiacol is a universal substrate for all peroxidases, and its use in a simple colorimetric assay has wide applications. However, its exact binding location has never been defined. Here we report the crystal structures of guaiacol bound to cytochrome c peroxidase (CcP). A related structure with phenol bound is also presented. The CcP–guaiacol and CcP–phenol crystal structures show that both guaiacol and phenol bind at sites distinct from the cytochrome c binding site and from the δ‐heme edge, which is known to be the binding site for other substrates. Although neither guaiacol nor phenol is seen bound at the δ‐heme edge in the crystal structures, inhibition data and mutagenesis strongly suggest that the catalytic binding site for aromatic compounds is the δ‐heme edge in CcP. The functional implications of these observations are discussed in terms of our existing understanding of substrate binding in peroxidases [Gumiero A et al. (2010) Arch Biochem Biophys500, 13–20]. Guaiacol is a universal substrate for all peroxidases and its use in a simple colorimetric assay has wide applications. However its exact binding location has never been defined. Here we report the crystal structures of guaiacol bound to cytochrome c peroxidase (CcP). A related structure, with phenol bound, is also presented.