Hydrogen peroxide-independent generation of superoxide by plant peroxidase: hypotheses and supportive data employing ferrous ion as a model stimulus

Abstract

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.

Salicylic acid-induced superoxide generation catalyzed by plant peroxidase in hydrogen peroxide-independent manner

“…19 We can expect that SA may interact with native form of plant peroxidases. In fact, such preliminary data on the SA-dependent stimulation of native HRP 21 and SBP 32 leading to generation of O 2 ¡ have been shown in our previous articles. However, when we have reported such data, it was early for us to clarify our working hypothesis on the mode of SA action in the H 2 O 2 -independent peroxidase action leading to generation of ROS.…”

“…However, when we have reported such data, it was early for us to clarify our working hypothesis on the mode of SA action in the H 2 O 2 -independent peroxidase action leading to generation of ROS. After publication of recent work by Kimura et al, 21 we examined and confirmed that SA actually induces the generation of O 2 ¡ in the presence of model enzymes such as HRP and SBP, even in the absence of H 2 O 2 supplementation. Here, typical data obtained with SBP are shown (Fig.…”

“…Among three potential inducers of O 2 ¡ (IAA, SA, Fe 2C ), only Fe 2C induced an intense and long-lasting peak of CLA-CL in both HRP 21 and SBP (unpublished results). These data might be reflecting the facts that both IAA and SA present at excess level behave as suicide substrates by targeting Compound III (Fig.…”

“…These theoretical graph models are based on our previous bio-computational approach for algebraically expressing the cyclic behavior (redox cycling) of HRP among native enzyme and its 2 electron-oxidized and single electron-oxidized intermediates (Compounds I and II) as a cyclic additive group Z 3 D {C 0 , C 2 , C 1 } D {C 0 , 1C 2 , 2C 2 } D {0, 2, 1}, and a cyclic multiplicative group Z 3 * D {C 1 , C 2 } D {C 1 , C 2 1 } D {1, 2}, with C 2 as the common generator (generalized inputs of e-donating and e-accepting substrates); by viewing that the system is simply consisted of "states" and "transitions." 31 Each of small graphs in Figure 1B 21 and NO to soybean peroxidase (SBP), 32 reportedly result in arrest of the enzymes at Compound II suggesting that these enzymes enter the conventional peroxidase cycle after oxygenation cycle. Upon addition of peroxidase substrates such as phenolics or amines, the peroxidase cycle is completed and native enzyme was shown to be regenerated.…”

Hydrogen peroxide-independent generation of superoxide catalyzed by soybean peroxidase in response to ferrous ion

“…We have recently reported a likely mechanism involving free ferrous ion (Fe 2C ) in reduction of ferric native enzyme of purified HRP into ferrous enzyme intermediate which readily produces O 2 ¡ via formation of Compound III, especially under alkaline condition, thus, possibly contributing to the plant defense mechanism associated with pH changes. 18 Here, we report on the spectroscopic and CL evidences in support of the model for soybean POX (SBP)-catalyzed robust production of O 2 ¡ to be stimulated in the absence of conventional POX substrates but in the presence of free Fe 2C .…”