The O27H2O2 System

In biological organisms, the four-step reaction sequence of ROS as shown in Figure 53 is tamed and energetically tuned at transition metal centers, which are encapsulated in specifically functionalized protein matrices. This mode of catalysis of the "hot water redox chemistry" avoids the uncontrolled formation of ROS. In photosynthesis, the highly endergonic oxidative water splitting (ДС° = +237.13 kJ/mol, see Atkins, 2014) is catalyzed by a unique MmOsCa cluster of the water-oxidizing complex (WOC) of photosystem II and energetically driven by the strongly oxidizing cation radical P680+* (Klimov et al., 1978; Rappaport et al., 2002) formed via light-induced charge separation (for review, see Renger, 2012).

Correspondingly, the highly exergonic process in the reverse direction is catalyzed by a binuclear heme iron-copper center of the cytochrome oxidase (COX), and the free energy is transformed into a transmembrane electrochemical potential difference for protons (for a review, see Renger and Ludwig, 2011), which provides the driving force for ATP synthesis (for a review, see Junge, 2008). In spite of the highly controlled reaction sequences in photosynthetic WOC and respiratory COX, the formation of ROS in living cells cannot be completely avoided.

The general so called “water-water cycle” as shown in Figure 53 is at most responsible for the subsequent formation of O 2 or HO , H2O2 and HO *. Among all ROS, the O 2 /H2O2 system is one of the key elements in cell signaling and other plant functions. O 2 and H2O2 are assumed to initiate reaction cascades for the generation of “secondary” ROS as necessary for long-distance signaling from the chloroplasts to or between other cell organelles (Baier and Dietz, 2005; Sharma et al., 2012; Bhattacharjee, 2012).

The initial step in formation of redox intermediates of the H2O2/O2 system in all cells is the one-electron reduction of O2 to O 2 (see Figure 53). O 2 and H2O2 are mainly formed in chloroplasts, peroxisomes, mitochondria and cell walls (Corpas et al., 2012; Bhattacharjee, 2012). Enzymatic sources of O 2 / H2O2 generation have been identified such as cell wall-bound peroxidases, aminooxidases, flavin-containing oxidases, oxalate and plasma membrane NADPH oxidases (Bolwell et al., 2002; Mori and Schroeder, 2004; Svedruzic at al., 2005). In particular, sources of ROS in the apoplast are oxidases bound to the cell wall, peroxidases, and polyamino oxidases (Minibayeva et al., 1998, 2009). Recent studies have brought strong evidence that O2 by direct reduction by plastohydroquinone is possible (Khorobrykh et al., 2015). As the direct two electron reduction by 1AgO2 delivers a stoichiometric amount of H2O2 it is assumed without great doubt in the literature that O 2 is the source for formation of H2O2 (see Figure 53) (Khorobrykh et al., 2015).

It was proposed for a long time that the major source of O 2 /H2O2 production in chloroplasts is the acceptor side of photosystem I (PS I) (Asada, 1999, 2006). So the exact mechanism of O2 reduction might still remain a matter of discussion. It was assumed that O2 mainly is reduced by transfer of electrons from reduced ferredoxin (Fd) to O2 via ferre- doxin-thioredoxin reductase (Gechev et al., 2006) although this assumption was challenged since a long time (Asada et al., 1974; Golbeck and Radmer, 1984). New findings showed that reduced Fd was only capable of low rates of O2 reduction in the presence of NADP+ with contribution to the total O2 reduction not exceeding 10% (Kozuleva and Ivanov, 2010; Kozuleva et al., 2014). NADPH oxidase (NOX) is considered to be involved into ROS production both in animal and plant cells (Sagi and Fluhr, 2006) according to the reaction NADPH + 2О2 A NADPH + 2O + Н+.

Under conditions of limited NADPH consumption due to impaired CO2 fixation rates via the Benson-Bassham-Calvin cycle in photosynthetic organisms, some components of the electron transport chain (ETC) will stay reduced and can perform 32 g O2 reduction to O 2. It is suggested that H2O2 formation takes place in the plastoquinone (PQ) pool, but with a low rate (Ivanov et al., 2007), studies on mutants of Synechocystis sp. PCC 6803 lacking phylloquinone (menB mutant) show the involvement of phylloquinone in O2 reduction (Kozuleva et al., 2014).

Very recent studies revise the assumption that PS I is a predominant source of O 2 and at all challenge the assumption of a one electron transfer from the PS I acceptor side (Prasad et al., 2015). Prasad et al. (2015) showed with a novel catalytic amperometric biosensor that formation of superoxide can occur via reduction of molecular oxygen by semiplastoquinone. In fact (Prasad et al., 2015) determined the reduction by Q B as the major source of O 2 challenging the opinion that also PS I is involved. EPR spin-trapping data obtained using the urea-type herbicide DCMU showed the involvement of plastosemiquinone in O 2 production. From these findings and concomitant measurements of H2O2 in accordance with the reaction scheme shown in Figure 53 the authors (Prasad et al., 2015) also conclude that H2O2 in PSII membrane is exclusively formed by the dismutation of O 2.

Recent literature suggests very short lifetimes for O 2 radicals in the gs regime (1 gs half-life is published in (Sharma et al., 2012), while 2-4 gs are found in (Gechev et al., 2006) - which is about one order of magnitude longer than that of :AgO2 (vide supra). O 2 radicals are rapidly transformed into H2O2 via the one-electron steps of the dismutation reaction catalyzed by the membrane-bound Cu/Zn-superoxide dismutase (SOD) (see Figure 53) (Asada, 1999, 2006). Three forms of SODs exist in plants containing different metal centers, such as manganese (Mn-SOD), iron (Fe-SOD), and copper-zinc (Cu/Zn-SOD) (Bowler et al., 1992; Alscher et al., 2002), from which Cu/Zn-SOD is the dominant form. The non-enzymatic O 2 dismutation reaction is very slow (Foyer and Noctor, 2009; Foyer and Shigeoka, 2011). Earlier literature suggested generally a low reactivity of O 2 radicals indicating that the exact mechanisms of the O 2 reaction pathways in living cells might need further elucidation (see Halliwell and Gutteridge, 1985 and references therein). In earlier studies, Halliwell (1977) pointed out that O 2 is a moderately reactive nucleophilic reactant with both oxidizing and reducing properties. The negative charge of the O 2 radical leads to an inhibition of its electrophilic properties in presence of molecules with many electrons, while molecules with a low electron number might be oxidized. O 2 oxidizes enzymes containing [4Fe-4S] clusters (Imlay, 2003), while cytochrome c is reduced (McCord et al., 1977). Among the amino acids, mainly histidine, methionine, and tryptophan can be oxidized by O 2 (Dat et al.,

2000). These radicals interact quickly with other radicals due to the spin selection rules. For example, superoxide interacts with radicals like nitric oxide and with transition metals or with other superoxide radicals (dismutation). As an example, Fe(III) is reduced by O 2, then H2O2

interacts with Fe2+ (Fenton reaction), in effect forming HO *, which is the most reactive species among all ROS (see also Figure 53). This reaction is particularly mentioned due to its importance for the generation of highly

reactive HO* from long-lived H2O2 which might act as long distance messenger. Further information about various reaction rate constants of O 2 at different conditions, concentrations and pH are found in (Rigo et al., 1977; Fridovich, 1983; Loffler et al., 2007)

H2O2 might be the most prominent cell toxic scavenged by a broad series of enzymes including catalases, peroxidases and peroxiredoxins. Within the chloroplasts, H2O2 is reduced to H2O by ascorbate (Asc) via a reaction catalyzed by soluble stromal ascorbate peroxidase (APX)

(Asada, 2006; Noctor et al., 1998) or APX bound to the thylakoid membrane (t-APX). As shown in Figure 54, the Asc oxidized to the monodehydroascorbate radical (MDHA) is regenerated by reduction of MDHA either directly by Fd or by NAD(P)H catalyzed by MDHA reductase (MDHAR). The MDHA radical always decays partially into dehydroascorbate (DHA), which is reduced by DHA reductase (DHAR). In that step, reduced glutathione (GSH) is oxidized to glutathione disulfide (GSSG). The reduction of GSSG to GSH occurs from NAD(P)H by glutathione reductase (GR) (Noctor and Foyer, 1998; Asada, 2006).

Scheme of pseudocyclic “HO-HO” electron transport according to (Schmitt et al., 2014a). Image reproduced with permission

Figure 54. Scheme of pseudocyclic “H2O-H2O” electron transport according to (Schmitt et al., 2014a). Image reproduced with permission.

Assuming O2 reduction to O ' at the acceptor side of PS I, followed by dismutation of O by SOD, and the reduction of H2O2 by t-APX one O2 molecule is finally reduced to two H2O molecules. This four-electron reduction process counterbalances the oxidation of two H2O molecules to one O2 molecule at the donor side of PS II so that no net change in the overall turnover of O2 is obtained, as is schematically illustrated in Figure 54. The exact role of PS I and PS II might be a matter of discussion. However, this “water-water cycle” is referred to as pseudocyclic electron transport (for details, see Foyer and Shigeoka, 2011; Asada, 1999, 2006) driven by H2O2. It has to be kept in mind that this pseudocyclic electron transport can be coupled to the formation of a transmembrane pH difference, ApH.

Figure 53 and Figure 54 indicate that H2O2 could also be generated by oxidation of two H2O molecules. Formation of H2O2 has long been reported to take place at a disturbed water-oxidizing complex (WOC) under special circumstances (Ananyev et al., 1992; Klimov et al., 1993; Pospisil, 2009). However, under physiological conditions, this process is negligible if taking place at all. Accordingly, H2O2 production at PS II should occur via the reductive pathway at the acceptor side under conditions where the PQ pool is over-reduced as proposed by (Ivanov et al., 2007; Prasad et al., 2015). Detailed reaction schemes for the reactions of H2O2 are found in (Mattila et al., 2015).

H2O2 is the ROS with the longest lifetime, which is in the order of 1 ms (Henzler and Steudle, 2000; Gechev and Hille, 2005). This is mostly supported as the molecule is neutral and therefore can pass lipophilic regions of the cell especially membranes including water channels like aquaporins (Bienert et al., 2007). Therefore, it can travel over large distances and play a central role in signaling of stress (see chapter 4) or undertakes even the role of a secondary photochemical electron transporter from PS II to PS I as shown in Figure 54.

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