Mutation of Distal Residues of Horseradish Peroxidase:  Influence on Substrate Binding and Cavity Properties

Abstract

The manner in which the distal heme pocket residues of peroxidases control the reaction mechanism and ligand binding has been investigated further by analysis of the electronic absorption and resonance Raman (RR) spectra of distal site mutants of recombinant horseradish peroxidase (HRP-C*). The roles of the conserved distal histidine and arginine residues, particularly in the context of the catalytic mechanism originally proposed for cytochrome c peroxidase (CCP), have been evaluated by studying the His42 --> Leu, His42 --> Arg, Arg38 --> Gly, and Arg38 --> Leu variants of HRP-C*. Spectra of the ferric forms, their complexes with benzohydroxamic acid (BHA), and the ferrous forms have been recorded at neutral pH. In addition, the ferric forms have been studied at alkaline pH. The relative populations of the three heme spin states characteristic of HRP-C* and its mutants were found to vary markedly from mutant to mutant. This diversity of heme spin state populations among the various mutants has allowed a well-defined set of RR frequencies to be compiled for the three heme spin states. These frequencies support the analysis of wild-type HRP-C* in terms of two heme states, five- (5cHS#) and six-coordinate high-spin (6cHS#), which exhibit anomalous RR frequencies compared to those of model heme systems. The third heme spin state is identified as being six-coordinate high-spin, displaying typical RR frequencies (6cHS). The 6cHS# and the 6cHS heme states are characterized by H bonding between the iron-bound water molecule and the Arg38 residue or the His42 residue, respectively. The proportion of six-coordinate high-spin heme states is at a minimum in the Arg38Leu mutant, indicating that the occupancy of the distal water molecule site is reduced in this mutant. The His42Arg mutant is distinguished from the other mutants by the unexpected presence of an iron-bound hydroxyl group at neutral pH. The spectral changes induced upon complexation with BHA indicate that both the distal histidine and arginine are involved in BHA binding; however, the arginine residue appears to play a more critical role. Measurements at pH 12 suggest there is a concerted involvement of both distal residues in mediating the alkaline transition of HRP-C*. Arg38 appears to be essential for stabilization of the OH- ligand, while His42 acts as a H bond acceptor. A striking similarity between the roles of these residues in the reaction of H2O2 with the enzyme and the alkaline transition is noted. By comparison with the results from corresponding mutants of CCP, it appears that although the hydrogen-bonding network linking the distal and proximal sides of the heme is conserved the distal cavity in HRP-C differs significantly from that of CCP. However, some similarities in the local environment of the distal arginine are suggested.

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).…”

On the Relationship of Coral Allene Oxide Synthase to Catalase

“…Whereas a water-ligated 6c/HS structure is a major species in the T66V mutant similar to WT cAOS, a 5c/HS species was observed as a minor component in the T66V mutant. The fact that the water coordination to the heme iron was stabilized by a hydrogen bond and/or a negatively charged group in other heme proteins (41)(42)(43) suggests that the mutation of Thr-66 possibly affects the distal environment. Furthermore, the CN affinity was increased to 8-fold of that of WT cAOS by the mutation, which also provides evidence for the structural perturbation at the heme distal site upon the mutation.…”

Predicting the functionally distinct residues in the heme, cation, and substrate-binding sites of peroxidase from stress-tolerant mangrove specie, Avicennia marina

“…7b). The distance between heme (Fe) and catalytic water molecule is 2.65 Å which is the same as in case of HRP and is enough to control the electronic structure of heme system (Howes et al 1997). Crystallographic study has revealed that BHA was found at the peroxidase catalytic site, whereas hydrophobic residues are present near the aromatic portion of the substrate (Henriksen et al 1998b).…”