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Unusual kinetic properties of anionic tobacco peroxidase related to the mechanism of oxidation of indole-3-acetic acid

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Abstract

Anionic tobacco peroxidase (TOP) (mol wt 36 kDa, pI 3.5) was purified from transgenic tobacco plants with the yield of 60 mg/1 kg leaves. The enzyme exhibits unusual properties, i.e., Compound I is less reactive than Compound II. The enzyme was investigated in oxidation of indole-3-acetic acid (IAA) oxidation by oxygen in the air. The aerobic steady-state spectral studies reveal that Compound II is the key intermediate of the reaction mechanism. This was confirmed in the anaerobic stopped-flow experiments. No reaction between the enzyme and IAA is observed under anaerobic conditions. The data obtained are interpreted in terms of a ternary complex formation (ferric enzyme-IAA-oxygen) at the initiation step resulting in production of IAA radicals. The latter interacts with the ferric enzyme and oxygen producing Compound II. The oxidative cycle involves the ferric enzyme and Compound II, and is independent from the peroxidative one.

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Abbreviations

TOP:

tobacco anionic peroxidase

HRP:

horseradish peroxidase

E:

ferric enzyme, native form of peroxidase with Fe (III)

ferrous enzyme:

reduced Fe (II) state of native peroxidase

Compound I:

two-electron oxidized form of ferric peroxidase, the socalled oxyferryl-heme

Compound II:

one-electron oxidized form of the ferric enzyme

Compound III:

four-electron oxidized state of ferric enzyme

IAA:

indole-3-acetic acid (I)

IAA•+ :

indole-3-acetate cationradical (II)

IAA•:

indolyl radical (III)

In-CH2•:

indole-3-methyl (skatole) radical (IV)

In-CH2O2•:

skatole peroxyl radical

In-CH2OOH:

skatole hydroperoxide

In-CH2O:

indole-3-epoxide (V); 3-methylene-in-dolenine (VI)

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Authors and Affiliations

  1. Department of Chemical Enzymology, Chemical Faculty, Moscow State University, 119899, Moscow, GSP, Russia

    I. G. Gazarian

  2. Nitrogen Fixation Laboratory, AFRC Institute of Plant Science Research, University of Sussex, Brighton, UK

    G. A. Ashby & R. N. F. Thorneley

  3. Department of Horticulture and Crop Science, Ohio State University, Columbus, OH

    L. M. Lagrimini

Authors
  1. I. G. Gazarian
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  2. G. A. Ashby
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  3. R. N. F. Thorneley
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  4. L. M. Lagrimini
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Gazarian, I.G., Ashby, G.A., Thorneley, R.N.F. et al. Unusual kinetic properties of anionic tobacco peroxidase related to the mechanism of oxidation of indole-3-acetic acid. Appl Biochem Biotechnol 61, 1–12 (1996). https://doi.org/10.1007/BF02785683

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