Identification of skatolyl hydroperoxide and its role in the peroxidase-catalysed oxidation of indol-3-yl acetic acid.

Indol-3-yl acetic acid (IAA, auxin) is a plant hormone whose degradation is a key determinant of plant growth and development. The first evidence for skatolyl hydroperoxide formation during the plant peroxidase-catalysed degradation of IAA has been obtained by electrospray MS. Skatolyl hydroperoxide degrades predominantly non-enzymically to oxindol-3-yl carbinol but in part enzymically into indol-3-yl methanol via a peroxidase cycle in which IAA acts as an electron donor. Skatolyl hydroperoxide is degradable by catalase. Horseradish peroxidase isoenzyme C (HRP-C) and anionic tobacco peroxidase (TOP) exhibit differences in their mechanisms of reaction. The insensitivity of the HRP-C-catalysed reaction to catalase is ascribed to the formation of HRP-C Compound III at the initiation step and its subsequent role in radical propagation. This is in contrast with the TOP-catalysed process in which skatolyl hydroperoxide has a key role. Indol-3-yl aldehyde is produced not via the peroxidase cycle but by catalysis involving ferrous peroxidase. Because indol-3-yl aldehyde is one of the main IAA-derived products identified in planta, we conclude that ferrous peroxidases participate in IAA catalytic transformations in vivo. A general scheme for peroxidase-catalysed IAA oxidation is presented.

Anaerobic stopped-flow studies of indole-3-acetic acid oxidation by dioxygen catalysed by horseradish C and anionic tobacco peroxidase at neutral pH: catalase effect.

The effect of order of reagent mixing in the absence and in the presence of catalase on the transient kinetics of indole-3-acetic acid (IAA) oxidation by dioxygen catalysed by horseradish peroxidase C and anionic tobacco peroxidase at neutral pH has been studied. The data suggest that haem-containing plant peroxidases are able to catalyse the reaction in the absence of exogenous hydroperoxide. The initiation proceeds via the formation of the ternary complex enzyme-->IAA-->oxygen responsible for IAA primary radical generation. The horseradish peroxidase-catalysed reaction is independent of catalase indicating a significant contribution of free radical processes into the overall mechanism. This is in contrast to the tobacco peroxidase-catalysed reaction where the peroxidase cycle plays an important role. The transient kinetics of IAA oxidation catalysed by tobacco peroxidase exhibits a biphasic character with the first phase affected by catalase. The first phase is therefore associated with the common peroxidase cycle while the second is ascribed to native enzyme interaction with skatole peroxy radicals yielding directly Compound II.

Anionic tobacco peroxidase is active at extremely low pH: veratryl alcohol oxidation with a pH optimum of 1.8.

Tobacco peroxidase (36 kDa, pI 3.5) exhibits unique catalytic and spectral properties that are modulated by pH, calcium and magnesium ions. It catalyses the oxidation of veratryl alcohol by hydrogen peroxide over a wide pH range (1.5-5.0) in the presence of these metal ions with a pH optimum of 1.8. This is the only example of a holoperoxidase described so far that is active and comparatively stable at such a low pH. The enhancement of tobacco peroxidase activity by magnesium ions is to our knowledge the first example of a magnesium-induced peroxidase activation. UV/visible spectra of tobacco peroxidase showed that the Soret band shifted and its absorption coefficient increased upon the addition of calcium or magnesium ions and on lowering the pH. The tobacco peroxidase spectrum at pH 1.85, in the presence of calcium chloride (> 50 mM), is similar to that of lignin peroxidase at pH 6.0, with the Soret band shifting from 403 to 409 nm and the molar absorption coefficient increasing from 108,000 to 148,000 +/- 2000 M-1.cm-1 (results given +/- S.E.M.; n = 3). The data provide evidence for a low-affinity site for bivalent metal ion binding in addition to the two constitutive calcium sites that are present in all plant peroxidases. The presence of a glutamic acid residue (Glu-141) at the entrance to the haem-binding pocket, analogous to Glu-146 in lignin peroxidase and not present in other plant peroxidases, may account for these novel properties.

Wild-type and mutant forms of recombinant horseradish peroxidase C expressed in Escherichia coli. Substrate specificity and stability under irradiation.

Two horseradish peroxidase C (HRPC) mutants with substitutions in the active center, i.e., Phe41-->His and Phe143-->Glu, were compared to the wild-type recombinant enzyme expressed in Escherichia coli in terms of the enzymatic activity and stability under irradiation. Both mutations caused a significant decrease in activity, but it was still possible to follow the effect of mutations on the key steps of the reaction mechanism. Phe41 can be considered a nonpolar barrier separating histidine residues in the active center and providing a firm noncovalent binding with the highly hydrophobic porphyrin ring. The replacement of Phe41 with the ionizable His residue destabilizes the enzyme. The Phe143-->Glu replacement creates a negative charge at the entrance of the heme-binding pocket, and protects the latter from both donor substrates and free radicals. The radiolytic inactivation of the wild-type and mutant forms of recombinant HRP suggested different binding sites for iodide, 2,2'-bis(3-ethylbenzothiasoline-6-sulfonate (ABTS), guaiacol, and O-phenylene diamine. The study of kinetics and inactivation is in agreement with the direct binding of iodide to the heme porphyrin ring. The results also suggest that the ABTS binding site is less accessible than that for O-phenylene diamine.

Expression of fungal Mn peroxidase in E. coli and refolding to yield active enzyme.

The cDNA encoding Mn peroxidase isozyme H4 from Phanerochaete chrysosporium was expressed in Escherichia coli. The portion of the cDNA encoding the enzyme's signal peptide, not found in the processed holoenzyme, was deleted from the cDNA. The polypeptide was produced as inactive inclusion bodies that could be solubilized in 8 M urea and the reducing agent dithiothreitol. Reconstitution of activity was accomplished by diluting the urea concentration to 2M in the presence of hemin, calcium, and oxidized glutathione. All of the additives were required for recovery of activity. The activity of the recombinant enzyme was dependent on both Mn2+ and H2O2.

[Production and catalytic properties of point mutants Phe41--->His and Phe143--->Glu of horseradish peroxidase, expressed in Escherichia coli]. / Poluchenie i kataliticheskie svoistva tochechnykh mutantov Phe41--->His i Phe143--->Glu peroksidazy khrena, ékspressiruemykh v Escherichia coli.

Recombinant horseradish peroxidase and its single-point mutants, F4IH and H143E, have been reactivated from E. coli inclusion bodies with a 25% yield. Both mutations affect heme entrapment as well as enzyme stability and activity. A more than 40-fold decrease in the specific activity towards ABTS is associated with different steps of peroxidase catalysis. F41H replacement results in a drop of both rate constants by two and one orders or magnitude for hydrogen peroxide and ABTS, respectively. The mechanism of iodide oxidation by the F41H mutant fits into a ternary interaction. The F143E replacement mainly affects the steps of ABTS oxidation and product dissociation. It is suggested that the role of replaced phenylalanine residues consists in the formation of a highly hydrophobic pocket allowing for strong non-covalent binding of the heme porphyrin ring.