Thiol-dependent redox regulation controls central processes in plant cells including photosynthesis. Thioredoxins reductively activate, for example, Calvin-Benson cycle enzymes. However, the mechanism of oxidative inactivation is unknown despite its importance for efficient regulation. Here, the abundant 2-cysteine peroxiredoxin (2-CysPrx), but not its site-directed variants, mediates rapid inactivation of reductively activated fructose-1,6-bisphosphatase and NADPH-dependent malate dehydrogenase (MDH) in the presence of the proper thioredoxins. Deactivation of phosphoribulokinase (PRK) and MDH was compromised in 2cysprxAB mutant plants upon light/dark transition compared to wildtype. The decisive role of 2-CysPrx in regulating photosynthesis was evident from reoxidation kinetics of ferredoxin upon darkening of intact leaves since its half time decreased 3.5-times in 2cysprxAB. The disadvantage of inefficient deactivation turned into an advantage in fluctuating light. Physiological parameters like MDH and PRK inactivation, photosynthetic kinetics and response to fluctuating light fully recovered in 2cysprxAB mutants complemented with 2-CysPrxA underlining the significance of 2-CysPrx. The results show that the 2-CysPrx serves as electron sink in the thiol network important to oxidize reductively activated proteins and represents the missing link in the reversal of thioredoxin-dependent regulation.
31Thiol-dependent redox regulation controls central processes in plant cells including photosynthesis. 32Thioredoxins reductively activate e.g. Calvin-Benson cycle enzymes. However the mechanism of 33 oxidative inactivation is unknown despite its importance for efficient regulation. Here, the abundant 34 2-cysteine peroxiredoxin (2-CysPrx), but not its site-directed variants, mediates rapid inactivation of 35 reductively activated fructose-1,6-bisphosphatase and NADPH-dependent malate dehydrogenase 36 (MDH)
38Cells contain a thiol redox regulatory network to coordinate metabolic and developmental 39 activities with exogenous and endogenous cues. This network controls the redox state and 40 activity of many target proteins. Electrons are fed into the network from metabolism and reach 41 the target proteins via redox transmitters such as thioredoxin (TRX) and NADPH-dependent 42 thioredoxin reductases (NTR). Electrons are drained from the network by reactive oxygen 43 species (ROS) through thiol peroxidases, e.g., peroxiredoxins (PRX). Mathematical modeling 44 promises access to quantitative understanding of the network function and was implemented for 45 the photosynthesizing chloroplast by using published kinetic parameters combined with fitting to 46 known biochemical data. Two networks were assembled, namely the ferredoxin (FDX), FDX-47 dependent TRX reductase (FTR), TRX, fructose-1,6-bisphosphatase pathway with 2-cysteine 48 PRX/ROS as oxidant, and separately the FDX, FDX-dependent NADP reductase (FNR), 49NADPH, NTRC-pathway for 2-CysPRX reduction. Combining both modules allowed drawing 50 several important conclusions of network performance. The resting H 2 O 2 concentration was 51 estimated to be about 30 nM in the chloroplast stroma. The electron flow to metabolism exceeds 52 that into thiol regulation of FBPase more than 7000-fold under physiological conditions. The 53 electron flow from NTRC to 2-CysPRX is about 5.46-times more efficient than that from TRX-54 f1 to 2-CysPRX. Under severe stress (30 µM H 2 O 2 ) the ratio of electron flow to the thiol 55 network relative to metabolism sinks to 1:251 whereas the ratio of electron flow from NTRC to 56 2-CysPRX and TRX-f1 to 2-CysPRX rises up to 1:80. Thus, the simulation provides clues on 57 experimentally inaccessible parameters and describes the functional state of the chloroplast thiol 58 regulatory network.59 60 Keywords: Calvin-Benson cycle, fructose-1,6-bisphosphatase, peroxiredoxin, reactive 61 oxygen species, redox regulation, thioredoxin 3 62Authors summary 63 The state of the thiol redox regulatory network is a fundamental feature of all cells and 64 determines metabolic and developmental processes. However, only some parameters are 65 quantifiable in experiments. This paper establishes partial mathematical models which enable 66 simulation of electron flows through the regulatory system. This in turn allows for estimating 67 rates and states of components of the network and to tentatively address previously unknown 68 parameters such as the resting hydrogen peroxide levels or the expenditure of reductive power 69 for regulation relative to metabolism. The establishment of such models for simulating the 70 performance and dynamics of the redox regulatory network is of significance not only for 71 photosynthesis but also, e.g., in bacterial and animal cells exposed to environmental stress or 72 pathological disorders. 73 4 74 75 Reduction-oxidation reactions drive life. In aerobic metabolism, electrons from reduced 76 compounds pass on to oxygen t...
Cells contain a thiol redox regulatory network to coordinate metabolic and developmental activities with exogenous and endogenous cues. This network controls the redox state and activity of many target proteins. Electrons are fed into the network from metabolism and reach the target proteins via redox transmitters such as thioredoxin (TRX) and NADPHdependent thioredoxin reductases (NTR). Electrons are drained from the network by reactive oxygen species (ROS) through thiol peroxidases, e.g., peroxiredoxins (PRX). Mathematical modeling promises access to quantitative understanding of the network function and was implemented by using published kinetic parameters combined with fitting to known biochemical data. Two networks were assembled, namely the ferredoxin (FDX), FDX-dependent TRX reductase (FTR), TRX, fructose-1,6-bisphosphatase (FBPase) pathway with 2cysteine PRX/ROS as oxidant, and separately the FDX, FDX-dependent NADP reductase (FNR), NADPH, NTRC-pathway for 2-CysPRX reduction. Combining both modules allowed drawing several important conclusions of network performance. The resting H 2 O 2 concentration was estimated to be about 30 nM in the chloroplast stroma. The electron flow to metabolism exceeds that into thiol regulation of FBPase more than 7000-fold under physiological conditions. The electron flow from NTRC to 2-CysPRX is about 5.32-times more efficient than that from TRX-f1 to 2-CysPRX. Under severe stress (30 μM H 2 O 2) the ratio of electron flow to the thiol network relative to metabolism sinks to 1:251 whereas the ratio of eflow from NTRC to 2-CysPRX and TRX-f1 to 2-CysPRX rises up to 1:67. Thus, the simulation provides clues on experimentally inaccessible parameters and describes the functional state of the chloroplast thiol regulatory network.
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