Proximal Cavity, Distal Histidine, and Substrate Hydrogen-Bonding Mutations Modulate the Activity of Amphitrite ornata Dehaloperoxidase

Abstract

Dehaloperoxidase (DHP) from Amphitrite ornata is the first globin that has peroxidase activity that approaches that of heme peroxidases. The substrates 2,4,6-tribromophenol (TBP) and 2,4,6-trichlorophenol are oxidatively dehalogenated by DHP to form 2,6-dibromo-1,4-benzoquinone and 2,6-dichloro-1,4-benzoquinone, respectively. There is a well-defined internal substrate-binding site above the heme, a feature not observed in other globins or peroxidases. Given that other known heme peroxidases act on the substrate at the heme edge there is great interest in understanding the possible modes of substrate binding in DHP. Stopped-flow studies (Belyea, J., Gilvey, L. B., Davis, M. F., Godek, M., Sit, T. L., Lommel, S. A., and Franzen, S. (2005) Biochemistry 44, 15637-15644) show that substrate binding must precede the addition of H2O2. This observation suggests that the mechanism of DHP relies on H2O2 activation steps unlike those of other known peroxidases. In this study, the roles of the distal histidine (H55) and proximal histidine (H89) were probed by the creation of site-specific mutations H55R, H55V, H55V/V59H, and H89G. Of these mutants, only H55R shows significant enzymatic activity. H55R is 1 order of magnitude less active than wild-type DHP and has comparable activity to sperm whale myoglobin. The role of tyrosine 38 (Y38), which hydrogen bonds to the hydroxyl group of the substrate, was probed by the mutation Y38F. Surprisingly, abolishing this hydrogen bond increases the activity of the enzyme for the substrate TBP. However, it may open a pathway for the escape of the one-electron product, the phenoxy radical leading to polymeric products.

Caenorhabditis elegans and Human Dual Oxidase 1 (DUOX1) “Peroxidase” Domains

“…However, when peroxidase activity is measured with two standard peroxidase substrates, ABTS and a tyrosine derivative, the peroxidase activity of CeDUOX1 1-589 is found to be much lower than that of LPO. This lower activity is consistent with the absence of a distal histidine residue that is critical for the high activity of most peroxidases (42)(43)(44)(45)(46)). However, a low level of activity for the CeDUOX1 peroxidase domain may be desirable in the context of its function in the intact organism.…”

“…It has been shown through site-specific mutagenesis that the distal catalytic histidine in peroxidases is crucial to their activity. When mutated in such enzymes as horseradish peroxidase, cytochrome c peroxidase, and dehaloperoxidase, a significant loss of activity is observed (42)(43)(44)(45)(46). This fact has led researchers to postulate that the N-terminal region of DUOX1 may not act as a classical peroxidase but rather as a SOD (8,12).…”

The Mechanism of Oxidative Halophenol Dehalogenation by Amphitrite ornata Dehaloperoxidase Is Initiated by H2O2 Binding and Involves Two Consecutive One-Electron Steps: Role of Ferryl Intermediates

“…Furthermore, the mechanism with two consecutive one-electron transfers is supported by rapid scan stopped-flow techniques (31 (33). 2 The formation and proposed reactivity of Cpd ES are inconsistent with previously proposed mechanisms for DHP-catalyzed reactions (25)(26)(27). Mb, best known for O 2 storage, catalyzes the same reaction under conditions of oxidative stress (13).…”

Caldariomyces fumago Chloroperoxidase Catalyzes the Oxidative Dehalogenation of Chlorophenols by a Mechanism Involving Two One-Electron Steps

“…[8][9][10][11] Mutagenesis and pH versus activity studies with DHP have led to a proposal that the mechanism of DHP-catalyzed oxidative dehalogenation involves a net twoelectron oxidation of bound substrate, which cannot be activated starting from the compound II state. 12,13 In contrast, peroxidases such as CCPO typically oxidize organic substrates, especially phenols, by two consecutive one-electron steps. The ferric enzyme reacts with H 2 O 2 to form the high-valent ferryl/porphyrin radical cation, compound I (CCPO-I), which is reduced back to the ferric state in two one-electron steps with concomitant substrate oxidation via a second ferryl species, compound II (CCPO-II).…”