Probing the Aromatic‐Donor‐Binding Site of Horseradish Peroxidase Using Site‐Directed Mutagenesis and the Suicide Substrate Phenylhydrazine

Abstract

The haem groups from two classes of site-directed mutants of horseradish peroxidase isoenzyme C (HRP-C) (distal haem pocket mutants, [H42L]HRP-C* and [R38K]-HRP-C* and peripheral-haem-accesschannel mutants, [F142A]HRP-C* and [F143A]HRP-C*) were extracted and analysed by reverse-phase HPLC after phenylhydrazine-induced suicide inactivation. The relative abundance of the two covalently modified haems, C20-phenyl (8-meso phenyl) and C1 8-hydroxymethyl haem, provided a sensitive topological probe for changes induced in the protein architecture in the vicinity of the haem active site and substrate-access channel. Although differing considerably in their efficiency as peroxidases ([H42L]HRP-C* exhibited only approximately 0.03 % of the peroxidase activity of wild type), the variants studied gave rise to a modification pattern typical of an exposed haem edge thereby strengthening the argument that it is the overall protein topology rather than the intrinsic catalytic activity of the active site that determines the sites of covalent haem modification. Mutants which showed impaired ability to bind the aromatic donor benzhydroxamic acid were less readily modified by the phenyl radical at the haem C18-methyl position although the level of arylation at the haem C20 position remained remarkably constant. Our findings suggest that the overall efficacy of haem modification catalysed by HRP-C during turnover with phenylhydrazine and its vulnerability towards inactivation are related to its general ability to bind aromatic donor molecules. Results from phenylhydrazine treatment of HRP-C wild-type and mutant variants were compared with those obtained for Coprinus cinereus peroxidase, an enzyme which from its structure is known to have a remarkably open access channel to the haem edge. We show evidence that C. cinereus peroxidase is able to bind benzhydroxamic acid, albeit with a relatively high Kd (Kd 3.7 mM), a probe for aromatic-donor binding. We suggest reasons why phenylhydrazine-treated C. cinereus peroxidase was more resistant to haem modification and phenyl-radical-based inactivation than HRP-C.Keywords: horseradish peroxidase; suicide inactivation; phenylhydrazine; site-directed mutagenesis.Classical peroxidases, including horseradish peroxidase (HRP), have been shown to catalyse the transfer of electrons in two distinct one-electron steps from a wide range of aromatic donor molecules [l]. The reaction, initiated by hydrogen peroxide, provides two oxidising equivalents to the resting haem prosthetic group and yields compound I, an oxy-ferryl-[Fe(IV) = 01-based porphyrin radical cation. Although compounds I and 11, the high oxidation state intermediates formed during the catalytic cycle, have been well described [2], continued efforts to understand structure/function relationships in HRP have been hampered by the lack of a high-resolution structure of a class 111 (higher plant) peroxidase. Despite the wealth of biocheniical data for this archetypal peroxidase it has been necessary to rely

The Inactivation and Catalytic Pathways of Horseradish Peroxidase with m-Chloroperoxybenzoic Acid

“…In addition the UV-visible spectrum of the kinetically competent, common intermediate on the catalytic and inactivation pathways has been obtained. On the other hand, the topological similarity between mCPBA and commonly used suicide substrates such as phenylhydrazines (44,45) and inhibitors like benzhydroxamic acid (24 -26) make it a useful probe not only of aromatic substrate binding site(s) but also the heme access channel architecture and distal cavity, since it reacts with ferric enzyme to give compound I. Clearly, the characterization of mutants of HRP-C and naturally occurring isoenzymes such as HRP-A2 using mCPBA will provide not only additional mechanistic information but also valuable insights into the factors that determine the functional stability of peroxidases with commercial potential.…”

Effects of phthalic anhydride modification on horseradish peroxidase stability and activity

“…Both HRPs displayed apparent Michaelis-Menten kinetics with ABTS as substrate (Table II Gilfoyle et al (1996) and Smith et al (1992). PA-HRP showed a marginally increased turnover (≈30%) for ABTS but little change in K m .…”

“…Chance and others used spectroscopic methods from the 1940s to explore its catalytic properties (reviewed by Dunford, 1999). Additional biophysical studies using NMR (Veitch et al, 1995(Veitch et al, , 1997, resonance Raman spectroscopy (Smulevitch et al, 1994), and the availability of recombinant mutants (e.g., Gilfoyle et al, 1996;Rodriguez-Lopez et al, 1996a,b) have already yielded new insights into its structure-function relationships; see Veitch and Smith (2001) for a recent review. Many protein folding and stability studies (e.g., Tsaprailis et al, 1998) have used HRP, which has many biotechnological applications.…”

Rescue of His-42 → Ala Horseradish Peroxidase by a Phe-41 → His Mutation

“…2) (10 -13). Evidence that Phe-41 of HRP occupies a position very similar to that of the corresponding residues in peroxidase crystal structures is provided by studies of the reactions of the F41A, F41V, H42A, and H42L mutants with phenyldiazene (8,26).…”