Heme Redox Cycling in Soybean Peroxidase: Hypothetical Model and Supportive Data (original) (raw)
In plants, the peroxidase family is involved in a great deal of oxidation reactions essential for living cells, using H2O2 as an electron acceptor and a variety of substrates as electron donors. Nowadays, plant peroxidases are widely used as biological sensing materials in various areas such as medical diagnosis, biosensors, and nanotechnology. Among plant peroxidases, horseradish peroxidase (HRP) and soybean peroxidase (SBP) are widely used as model enzymes applicable to various purposes. For the purpose of providing a basis for future innovation by using SBP as a sensing material, we focus on the behavior of purified SBP in responses to known plant peroxidase substrates reportedly involved in superoxide-generating reactions. A chemiluminescence study showed that SBP can catalyze the generation of superoxide in the presence of salicylic acid and indole-3-acetic acid used as the tester substrates for examining the involvement of the conventional peroxidase cycle (involving native form, Compounds I and II) and oxygenase cycle (involving native form, ferrous form, and Compound III), respectively. Enzyme intermediates in the above two redox cycles were spectroscopically determined by recording the typical spectra reflecting the presence of Compound II (peroxidise cycle) and Compound III (oxygenase cycle). Interestingly, addition of nitric oxide in the absence of H2O2 resulted in the formation of an intermediate resembling Compound II, the catalytically active form in the peroxidase cycle; thus, the novel gaseous regulation of SBP-mediated reaction was proposed.
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It is well documented that extracellular alkalization occurs in plants under the challenges by pathogenic microbes. This may eventually induce the pH-dependent extracellular peroxidase-mediated oxidative burst at the site of microbial challenges. By employing the purified proteins of horseradish peroxidase as a model, we have recently proposed a likely role for free Fe2+ in reduction of ferric enzyme of plant peroxidases into ferrous intermediate and oxygen-bound form of enzyme known as Compound III which may eventually releases superoxide anion radical (O2-), especially under alkaline condition, possibly contributing to the plant defense mechanism. In the present study, we employed the purified protein of soybean peroxidase (SBP) as an additional model, and examined the changes in the redox status of enzyme accompanying the generation of O2- in response to Fe2+ under alkaline condition.
When plants are threaten by microbial attacks or treated with elicitors, alkalization of extracellular space is often induced and thus pH-dependent extracellular peroxidase-mediated oxidative burst reportedly takes place, especially at the site of microbial challenge. However, direct stimulus involved in activation of peroxidase-catalyzed oxidative burst has not been identified to date. Here, we would like to propose a likely role for free ferrous ion in reduction of ferric native peroxidase into ferrous enzyme intermediate which readily produces superoxide anion via mechanism involving Compound III, especially under alkaline condition, thus, possibly contributing to the plant defense mechanism. Through spectroscopic and chemiluminescence (CL) analyses of reactions catalyzed by horseradish peroxidase (HRP), the present study proposed that plant peroxidase-catalyzed production of superoxide anion can be stimulated in the absence of conventional peroxidase substrates but in the presence of free ferrous ion.
Reactions of soybean peroxidase and hydrogen peroxide pH 2.4-12.0, and veratryl alcohol at pH 2.4
Biochimica et biophysica acta, 2001
Peroxidase from soybean seed coat (SBP) has properties that makes it particularly suited for practical applications. Therefore, it is essential to know its fundamental enzymatic properties. Stopped-flow techniques were used to investigate the pH dependence of the reaction of SBP and hydrogen peroxide. The reaction is linearly dependent on hydrogen peroxide concentration at acidic and neutral pH with the second order rate constant k(1)=2.0x10(7) M(-1) s(-1), pH 4-8. From pH 9.3 to 10.2 the reaction is biphasic, a novel observation for a peroxidase at alkaline pH. A fast reaction has the characteristics of the reaction at neutral pH, and a slow reaction shows hyperbolic dependence on hydrogen peroxide concentration. At pH >10.5 only the slow reaction is seen. The shift in mechanism is coincident with the change in haem iron co-ordination to a six-coordinate low spin hydroxy ligated alkaline form. The pK(a) value for the alkaline transition was observed at 9.7+/-0.1, 9.6+/-0.1 and 9...
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