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 CcPguaiacol and CcPphenol crystal structures show that both guaiacol and phenol bind at sites distinct from the cytochrome c binding site and from the d-heme edge, which is known to be the binding site for other substrates. Although neither guaiacol nor phenol is seen bound at the d-heme edge in the crystal structures, inhibition data and mutagenesis strongly suggest that the catalytic binding site for aromatic compounds is the d-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 similar to al. (2010) Arch Biochem Biophys500, 1320].