Structure-activity relation of horseradish peroxidases as studied with mutations at heme distal and proximal sites

Abstract

The distal His-Asn-Glu hydrogen bond network in the heme distal site is highly conserved among various fungal and plant peroxidases. To gain an insight into the functional roles of this hydrogen network in peroxidase activity, we have mutated the Asn70 to Val (N70V) or Asp (N70D), the Glu64 to Pro (E64P), or Gly (E64G) or Ser (E64S) in horseradish peroxidase. All the mutants disrupted the distal His-Asn hydrogen bond with maintaining the heme electronic structures as revealed by 1H NMR spectra and exhibited substantial depression of the peroxidase activity. The depression of the activity was ascribed to the decreased basicity and dislocation of the distal His induced by these mutations. The replacement of the distal His by Glu (H42E) to mimic the heme distal site of chloroperoxidase impaired the peroxidase activity. We have also studied F221W HRP mutant in which the proximal Phe is replaced with Trp to mimic the heme proximal structure of cytochrome c peroxidase and shown that the HRP-type compound I was formed, followed by transformation to the CcP-type Trp radical.