THE HOMOLOGOUS TRYPTOPHAN CRITICAL FOR CYTOCHROME-C PEROXIDASE FUNCTION IS NOT ESSENTIAL FOR ASCORBATE PEROXIDASE-ACTIVITY

The crystal structures of ascorbate peroxidase (APX) and cytochrome c peroxidase (CCP) show that the active site structures are nearly ident ical. Both enzymes contain a His-Asp-Trp catalytic triad in the proxim al pocket. The proximal Asp residue hydrogen bonds with both the His p roximal heme ligand and the indole ring nitrogen of the proximal Trp. The Trp is stacked parallel to and in contact with the proximal His li gand. This Trp is known to be the site of free radical formation in CC P compound I and also is essential for activity. However, APX forms a porphyrin radical and not a Trp-centered radical, even though the His- Asp-Trp triad structure is the same in both peroxidases. We found that conversion of the proximal Trp to Phe has no effect on APX enzyme act ivity and that the mutant crystal structure shows that changes in the structure are confined to the site of mutation. This indicates that th e paths of electron transfer in CCP and APX are distinctly different. The Trp-to-Phe mutant does alter the stability of the APX compound I p orphyrin radical, by a factor of two. Electrostatic calculations and m odeling studies show that a potassium cation located about 8 Angstrom from the proximal Trp in APX, but absent in CCP, makes a significant c ontribution to the stability of a cation Trp radical. This underscores the importance of long-range electrostatic effects in enzyme catalyze d reactions.