Role of histidine 42 in ascorbate peroxidase

Abstract

To examine the role of the distal His42 residue in the catalytic mechanism of pea cytosolic ascorbate peroxidase, two site-directed variants were prepared in which His42 was replaced with alanine (H42A) or glutamic acid (H42E). Electronic spectra of the ferric derivatives of H42A and H42E (pH 7.0, l ¼ 0.10 M, 25.0°C) revealed wavelength maxima [k max (nm): 397, 509, % 540 sh , 644 (H42A); 404, 516, % 538 sh , 639 (H42E)] consistent with a predominantly fiveco-ordinate high-spin iron. The specific activity of H42E for oxidation of L-ascorbate (8.2 ± 0.3 UAEmg )1 ) was % 30-fold lower than that of the recombinant wild-type enzyme (rAPX); the H42A variant was essentially inactive but activity could be partially recovered by addition of exogenous imidazoles. The plant peroxidase superfamily has been classified [1] into three major categories: class I contains the enzymes of prokaryotic origin, class II contains the fungal enzymes (e.g. manganese peroxidase, lignin peroxidase) and class III contains the classical secretory peroxidases [e.g. horseradish peroxidase (HRP)]. The most notable member of the class I peroxidase subgroup is cytochrome c peroxidase (CcP), which was first identified in 1940 [2]. In spite of the fact that CcP has some rather unusual features, most notably the existence of a stable tryptophan radical during catalysis [3][4][5][6] and the utilization of a large macromolecular substrate (cytochrome c), it has been the subject of such intense mechanistic, structural and spectroscopic scrutiny that it has become the benchmark against which all other peroxidases are measured.More recently, it has been possible to isolate and purify in good yields a second member of the class I peroxidase subgroup, ascorbate peroxidase (APX) [7,8]. Ascorbatedependent peroxidase activity was first reported in 1979 [9,10] and the enzyme catalyses the reduction of potentially damaging H 2 O 2 in plants and algae using ascorbate as a source of reducing equivalents [11,12]. APX was known from sequence comparisons [13] to contain the same activesite Trp residue (Trp179) as is used by CcP (Trp191) during catalysis. With high-resolution structural information available for the recombinant pea cytosolic enzyme (rAPX) [14] ( Fig. 1), APX has provided a new opportunity to reassess the functional properties of CcP and to determine whether it is indeed representative of class I peroxidases. As detailed functional information has emerged, however, it seems that APX has several rather curious features of its own, and, in some ways, more questions have been raised than answered. (In fact, even the current classification of APX as a class I enzyme has been recently questioned [15].) For example, Trp179 in APX is not a necessary requirement for oxidation of ascorbate [16] and there is general agreement from kinetic [17][18][19] and EPR data [20] that the initial product (Compound I) of the reaction of APX with H 2 O 2 is a porphyrin p-cation intermediate and not a protein-based trytophan radical. Equally intriguing is the existence of a

Resonance Raman spectroscopy of oxoiron(IV) porphyrin π-cation radical and oxoiron(IV) hemes in peroxidase intermediates

“…9). Nevertheless, the importance of the distal histidine, such as His-42 in HRP isozyme C [149,229] and other peroxidases [72,[242][243][244] continues to be reaffirmed. For CO bound HRP the guanidinium group of Arg-38 may change conformation when His-42 is protonated [245].…”

Anticancer and Antimicrobial Activities of Some Antioxidant-Rich Cameroonian Medicinal Plants

“…Hemoglobin ascorbate peroxidase activity has previously been described in detail [57], [58]. Stock solutions of met-hemoglobin, hydrogen peroxide and ascorbate of 1.4 mM (in PBS, pH 7.4), 50 mM (in water) and 70 mM (in water), respectively, were used.…”

The role of Ala134 in controlling substrate binding and reactivity in ascorbate peroxidase

“…The residue equivalent to Ala134 in APX is also present in CcP (Ala147) but is replaced with a proline in other class II and III peroxidases [18,19], Figure 1C. To examine the role of Ala134 in controlling peroxidase reactivity at the -heme edge, we prepared a variant of APX in which Ala134 has been replaced with a proline and have examined the effect of this mutation on the Binding constants for binding of anionic ligands to the heme were determined according to previous protocols [24].…”

“…Pre-steady state (pH 7.0, 5.0 o C) kinetic experiments were carried out according to published protocols [24]. Compound I and Compound II spectra were obtained by addition of a 1:1 and 10:1 ratio of hydrogen peroxide to wild-type APX respectively.…”

“…Compound I and Compound II spectra were obtained by addition of a 1:1 and 10:1 ratio of hydrogen peroxide to wild-type APX respectively. Second order rate constants for Compound I, k1, and Compound II, k2, formation were obtained as previously [24]; Compound II reduction was measured at 420 nm (Compound I/Compound II isosbestic point) after pre-formation of Compound I.…”