Ec 1111

The E.C. 1.11.1 subclass contains the peroxidases, which use hydrogen peroxide (H2O2) as electron acceptor to oxidize the donor, thereby forming the oxidized donor and water. Members include horseradish peroxidase (E.C. 1.11.1.7; also known as guaiacol peroxidase and scopoletin peroxidase), manganese peroxidase (E.C. 1.11.1.13) and diarylpropane peroxidase (E.C. 1.11.1.14). All three classes are hemoproteins. Horseradish peroxidase and related peroxidases are involved in the oxidative coupling of lignans, lignin, and tannins. Mechanistically, hydrogen peroxide oxidizes the active site of the peroxidase enzyme, and upon binding of the substrate in the active site, the substrate becomes oxidized and the enzyme returns to its reduced state.

Peroxidases are encoded by large multi-gene families, which has complicated the study of individual peroxidase enzymes (cf. Christensen et al., 1998). Manganese and diarylpropane peroxidases are used by white rot fungi (basiodiomycetes) to degrade lignin via oxidation.

The origin of the H2O2 that peroxidases use is not entirely clear. The best studied peroxidases are the ones involved in cell wall lignification, and several mechanisms that describe the generation of H2O2 have been identified in different plant species, as will be discussed below.

Ogawa et al. (1997) investigated the formation of H2O2 and the superoxide radical (O2 ) in spinach (Spinacia oleracea) hypocotyls with the use of histochemical stains. Nitroblue tetrazolium (NBT) is used to detect O2.- radicals. The colored reaction product formazan was only detected in the vascular tissue of developing spinach hypocotyls if CuZn-superoxide dismutase (CuZn-SOD; E.C. 1.15.1.1) was inhibited by DDC

(^N-diethyldithiocarbamate), suggesting that CuZn-SOD effectively catalyzes the elimination of the O2- radicals. The mechanism for the elimination of these radicals is through dismutation of superoxide into oxygen (O2) via oxidation, and H2O2 via reduction. Imidazole and DPI (diphenyleneiodonium), inhibitors of NAD(P)H-oxidase (E.C. 1.6.99.6), were shown to suppress the formation of formazan, indicating the involvement of NAD(P)H-oxidase in superoxide radical formation. NADPH is nicotinamide dinucleotide phosphate, a compound that is used as an electron donor throughout cellular metabolism. Based on these results they proposed a mechanism for the generation of H2O2, shown below in Figure 2-9, that involves the concerted action of a membrane-bound NADPH oxidase and a CuZn-superoxide dismutase The H2O2 that is generated by the combined action of these two enzymes is then used to activate a peroxidase that oxidizes monolignols.

NADPH-oxidase NAD(P)H +2 O2 -► NAD(P)+ + 2 O2"

CuZn SOD

Figure 2-9. Formation of H2O2 through the concerted action of NAD(P)H oxidase and CuZn-superoxide dismutase, as proposed by Ogawa et al. (1997).

There are cases where the peroxidase is not activated by H2O2, but by a reaction product instead. Ferrer et al. (1990) described the oxidation of the auxin indole-3-acetic acid (IAA; 2.49) and molecular oxygen by a cell wall peroxidase that was able to oxidize coniferyl alcohol (2.48) in the absence of H2O2.

Auxins are plant hormones involved in a number of developmental processes in plants, including embryo development, leaf formation, and apical dominance (reviewed by Leyser (2005), and Woodward and Bartel (2005)). IAA is transported through the extracellular space, and would thus be readily available as a reductor to cell-wall bound peroxidases. The cellwall bound peroxidases in this study were isolated from lupin (Lupinus alba), and the oxidation of coniferyl alcohol at the expense of IAA was monitored spectrophotometrically in the UV range of the spectrum. The IAA was converted to oxindoles. Previous studies showed that 3-methylene 2-oxindole (2.54) is the predominant oxindole formed (Ricard and Job, 1974). Ferrer et al. (1990) showed that the oxidation of coniferyl alcohol was dependent on the concentration of IAA, whereby high concentrations inhibited the reaction. This makes sense physiologically, since lignification is associated with a terminal developmental process, whereas high levels of auxin are correlated with growth and differentiation. Folkes et al. (2002) proposed a reaction mechanism for the peroxidase-mediated oxidation of IAA without the involvement of H2O2, which is shown in Figure 2-10.

Oxidation of IAA (2.49) results in cation 2.50, which undergoes decarboxylation and results in the skatolyl radical (2.51). This compound reacts with molecular oxygen to form peroxyl radical 2.52. With IAA or another cellular reductor, the hydroperoxide 2.53 is formed. It is this compound that activates the peroxidase, and thus allows the oxidation of other substrates, such as coniferyl alcohol. Among the degradation products of 2.53, 3-methylene 2-oxindole (2.54) is the most abundant.

Figure 2-10. Oxidation of IAA.

Figure 2-10. Oxidation of IAA.

An alternative mechanism for the generation of H2O2 was described by Caliskan and Cuming (1998), who studied the wheat protein germin. This protein is synthesized de novo when wheat embryos germinate. It was shown to be highly resistant to proteolytic degradation, and to have oxalate oxidase (E.C.. 1.2.3.4) activity. This enzyme catalyzes the oxidation of oxalate (2.55), and the formation of H2O2 as shown in Figure 2-11. Given that in 9-day old seedlings both the oxalate oxidase mRNA and protein were localized to the vascular tissue, the authors speculated that wheat germin plays a role in providing H2O2 in those tissues of the seedling where the cell wall needs to be cross-linked to restrict cell growth.

Figure 2-11. Formation of H2O2 via oxidation of oxalate by oxalate oxidase.

A fourth possibility is the generation of H2O2 via oxidation of putrescine (butane-1,4-diamine; 2.56). This reaction is catalyzed by copper amine oxidase (E.C. 1.4.3.6). Copper amine oxidases are homodimers in which each unit contains a copper ion and a 1,3,5-trihydroxyphenylalanine quinine co-factor. In plants copper amine oxidases generally oxidize putrescine to 4-aminobutanal (2.57). This latter compound undergoes spontaneous cyclization to A1 pyrroline (2.58), ammonia, and H2O2, as shown in Figure 2-12 (Medda et al., 1995).

Figure 2-12. Formation of H2O2 via oxidation of putrescine by copper amine oxidase.

Figure 2-12. Formation of H2O2 via oxidation of putrescine by copper amine oxidase.

The copper amine oxidase in the model plant Arabidopsis thaliana is encoded by the ATAO1 gene (Moller and McPherson, 1999). In situ hybridizations and analyses of transgenic plants expressing a reporter gene under control of the ATAO1 promoter revealed expression of the ATAO1 gene in the root cap and the vascular tissue. This would make it feasible that H2O2 generated via this mechanism could be used by a peroxidase involved in lignification and cross-linking of cell wall proteins in Arabidopsis.

Ros-Barcelo et al. (2002) analyzed lignifying xylem of Zinnia elegans. Based on the inhibition of H2O2 production as a result of treatment with imidazole, an involvement of NADPH-oxidase (E.C. 1.6.3.1) was hypothesized. This enzyme catalyzes the formation of H2O2 from the oxidation of NADPH with molecular oxygen, as shown in Figure 2-13.

Figure 2-13. Formation of H2O2 via oxidation of NADPH by NADPH oxidase.

Onnerud et al. (2004) recently proposed a mechanism involving a redox shuttle for the oxidative coupling of coniferyl alcohol (2.48), as it may occur during lignification. Given that lignin is very compact, the authors speculated that enzymes such as peroxidases may be too large to be effective. They investigated whether manganese (II) oxalate (2.55; see Figure 2-11) could function as a redox shuttle. Figure 2-14 depicts how Mn(II) oxalate is reduced to Mn(III) oxalate by a membrane or cell-wall bound manganese peroxidase (E.C. 1.11.1.13). The Mn(III) diffuses into the cell wall, oxidizes monolignols and the lignin polymer, and returns to the manganese peroxidase to get oxidized again.

h2o2

Mn peroxidase

Mn3+

Mn2+ monolignol radical oxidized lignin residue

Mn3+

lignifying cell wall monolignol lignin

Figure 2-14. Oxidation of monolignols via a manganese oxalate redox shuttle as proposed by Onnerud et al. (2004).

Based on this overview, it is clear that there are many different mechanisms by which H2O2 can be generated, and it is possible that even more mechanisms exist. Further research is needed to determine to what extent these mechanisms are unique to particular plant species, tissues, metabolic processes, or developmental stages. Given that the availability and concentration of enzyme substrates is likely to fluctuate as a function of both the developmental stage and the environmental conditions, the availability of multiple mechanisms to generate H2O2 offers a high degree of flexibility.

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