Small Molecules Govern Thiol Redox Switches

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Opinion

Small Molecules Govern Thiol Redox Switches Johannes Knuesting1 and Renate Scheibe1,* Oxygenic photosynthesis gave rise to a regulatory mechanism based on reversible redox-modifications of enzymes. In chloroplasts, such on–off switches separate metabolic pathways to avoid futile cycles. During illumination, the redox interconversions allow for rapidly and finely adjusting activation states of redox-regulated enzymes. Noncovalent effects by metabolites binding to these enzymes, here addressed as ‘small molecules’, affect the rates of reduction and oxidation. The chloroplast enzymes provide an example for a versatile regulatory principle where small molecules govern thiol switches to integrate redox state and metabolism for an appropriate response to environmental challenges. In general, this principle can be transferred to reactive thiols involved in redox signaling, oxidative stress responses, and in disease of all organisms.

Highlights Metabolic control is based on reversible redox-modifications leading to a dynamic steady state ratio of two enzyme forms differing in their activities. Fine-tuning of the activation state of each redox-controlled enzyme is achieved by non-covalently bound ‘small molecules’ which affect the rates of the redox-interconversions. ‘Small molecules’ such as substrates or products of the particular enzyme are able to shift redox potentials of the regulatory thiol groups by influencing the local microenvironment of these cysteine residues.

Redox Switches as the Basis for Cellular Homeostasis Redox chemistry is a central topic concerning all biological systems. In photoautotrophic organisms, during photosynthesis light energy is converted into strong reductants. At the same time, due to the asymmetrical architecture of the thylakoid membrane and vectorial electron flow, a proton motive force is generated which is used for ATP production. Reductants and ATP generated continuously during photosynthetic electron transport (PET) are needed for all cellular activities, in particular for biomass production by the C-, N-, and S-assimilatory processes. Since pool sizes of the involved reductants and energy carriers are extremely small, they need to be kept in a state that is ‘half full and half empty’ to allow for continuous fluxes under changing supply and demand. Therefore, flexible and fast regulation of key enzymes in all the energy-requiring pathways is essential to maintain metabolic and redox homeostasis. Light–dark modulation (see Glossary) of chloroplast enzymes by redox modifications at the various steps of the Calvin-Benson cycle (CBC) has been intensively investigated over the past decades. Originally it was thought to be a plant-specific type of regulation, to avoid a futile cycle with light and dark metabolism going on at the same time. But now, redox-controlled processes become apparent in all organisms. The historical development of the redox field over the past 50 years of research is provided in comprehensive reviews [1,2]. It started with the discovery of light–dark modulation in plants and its fine-tuning by ‘small molecules’. More recently, the redox field expanded also to non-plant research areas, finally leading to very active investigations in medical sciences where disturbance of redox homeostasis is causative for many diseases and aging [3,4]. Reversible thiol modifications are generally mediated by redox-active proteins, among them numerous members of the thioredoxin (Trx) superfamily that can transfer electrons to target proteins. Trx, in turn, mediate reduction and oxidation of redox-regulated target enzymes [5–7]. The chloroplasts of the model plant Arabidopsis thaliana contain a large number of Trx isoforms

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This principle of fine-tuning exemplified here for light-dark-modulated chloroplast enzymes is suggested for other proteins that are also subjected to reversible post-translational modifications.

1

Department of Plant Physiology, Faculty of Biology and Chemistry, Osnabrück University, Barbarastr. 11, 49076 Osnabrück, Germany

*Correspondence: [email protected] (R. Scheibe).

https://doi.org/10.1016/j.tplants.2018.06.007 © 2018 Elsevier Ltd. All rights reserved.

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as well as glutaredoxins (Grx) and peroxiredoxins [8,9]. Electrons for the reduction of the various Trx originate from the PET and are withdrawn at ferredoxin via ferredoxin-thioredoxin reductase (FTR), or from NADPH mediated by NADPH-Trx reductases NTRA (cytosol), NTRB (mitochondria), or NTRC (chloroplasts). The redox-regulated chloroplast enzymes are characterized by a high reactivity towards oxidants due to thermodynamic and kinetic effects that modify the properties of their thiols [10]. Furthermore, alkalization of the stroma of the photosynthesizing chloroplast increases deprotonation of the regulatory cysteines, leading to enhanced thiol reactivity. Therefore, continuous electron flow is required to maintain a certain portion of each enzyme in the reduced state. The chloroplast with the production of both highly reducing components, such as reduced ferredoxin (–420 mV), and oxygen (+816 mV) from photosynthetic water oxidation, appears to be perfectly suited to facilitate continuous thiol/disulfide cycling as a basis for a regulatory mechanism under nonstressed conditions with no increase in reactive oxygen species (ROS). Indeed, photosynthesis provides ample amounts of electrons, mostly in excess of ATP. The cost for regulation can be covered easily from light-generated electron flow, since the adjustment of the required ATP/NADPH ratio is achieved by various poising mechanisms [11,12]. In general, three types of thiol switches for regulation can be distinguished that exhibit different switching frequencies: (i) on–off switches between light and dark metabolism, (ii) continuous redox-cycling of redox-modulated chloroplast enzymes in the light at the expense of additional energy in the form of electrons, and (iii) redox sensors and signal transducers to prevent oxidative stress (Figure 1). In each case, the responsiveness of the switches can be influenced by small molecules that bind noncovalently. In this opinion paper, we present evidence from biochemical and physiological studies for the role of small molecules in rapidly and flexibly adjusting the redox switches. We will summarize the essential features of the chloroplast system, which is unique in some respect but offers general ideas to comprehend other regulatory systems. In the case of chloroplasts, the redox switches are not only operating as on–off switches (type 1) but provide the basis for fine-tuning (type 2). The effect of small molecules, originating from metabolic activities and environmental stress, on the redox interconversions allows for the integration of different types of information. Any disturbance of cellular homeostasis will result in changes of pool sizes of metabolites and inorganic ions. Change of the concentrations of these small molecules acts as indicators for the need to readjust metabolism to maintain homeostasis. Small molecules directly affect the interconversion rates of thiol switches and therewith the steady-state ratios of unmodified and modified protein, each of the two exhibiting different properties. Such a fine-controlled regulatory system is likely to also be realized in other cases where thiol switches have been found. To date, numerous further examples have become apparent, establishing the redox chemistry of thiol groups as the universal basis for controlling cellular homeostasis, sensing, and signal transduction [13–17].

Redox-Modulated Enzymes in Chloroplasts Light–dark-modulated chloroplast enzymes represent well-studied examples for redox regulation (Figure 2). Several enzymes of the CBC are activated by light-dependent reduction, mediated by Trx isoforms (mainly Trx m and f). Three of them catalyze practically irreversible reactions, namely fructose 1,6-bisphosphatase (FBPase; E.C.3.1.3.11), sedoheptulose 1,7bisphosphatase (SBPase; E.C.3.1.3.37), and phosphoribulose kinase (PRK; E.C.2.7.1.19), controlling fluxes through different parts of the branched pathway. The reductive phase of the CBC is catalyzed by a NAD(P)-dependent glyceraldehyde 3-phosphate dehydrogenase 2

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Glossary Calvin-Benson cycle (CBC): the main reductive process in chloroplasts for biomass production in the form of carbohydrates and carbon skeletons for all organic compounds by using light-generated NADPH and ATP. Carbon assimilation comprises three steps: (i) fixation of CO2, (ii) reduction of 3PGA for triose phosphate production, and (iii) regeneration of the CO2 acceptor (see also supplemental information online, Figure S1). Cysteine modifications: oxidation of Cys leads to intra- and intermolecular disulfide bridges, Sglutathionylation, S-nitrosylation, Ssulfhydration or results in sulfenic, sulfinic, or sulfonic acid residues, the latter one being irreversible. Cysteines are most reactive when deprotonated in alkaline (micro) environment. Light–dark modulation: the chloroplast-specific regulatory principle involving thiol-disulfide exchange post-translationally adjusts enzyme activities of metabolism during illumination and in darkness to avoid futile cycling of reductive (CBC) and oxidative pentose-phosphate (OPP) cycle. Malate valve: regeneration of the electron acceptor NADP+ is required to prevent overreduction of the photosynthetic electron transport chain and ROS formation. The key enzyme of the malate valve is NADPMDH, which uses NADPH to form malate that is transported out of the chloroplast, allowing for flexible adjustment of the NADPH/ATP ratio inside the chloroplast. This step needs to be strictly controlled because continuous NADPH supply is required, with the highest priority for 3-PGA reduction in the CBC (see also supplemental information online, Figure S2). Oxidative pentose-phosphate (OPP) pathway: glucose-6phosphate is oxidized generating NADPH, pentose phosphate, and CO2. In the illuminated chloroplast, this reaction would interfere with the CBC, resulting in futile cycling and ATP loss, if it would not be shut off in the light. Redox-active proteins: cysteines in proteins fulfill different roles for

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(A)

(B)

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Dark Light Dark Light

Connuous cycling

Light

(C)

Oxidave stress

Stress

Recovery

red.

PET

O2

PET

O2

PET ROS

NADPH

ox. Figure 1. Thiol switches Involved in Diverse Cellular Functions. Thiol switches operate in the context of light–dark cycles (A), during illumination (B), and upon oxidative stress (C). The turnover of the redox cycle of various thiol switches can be once a day (A), continuously during illumination (B), or exclusively in periods of stress (C). (A) Metabolism in illuminated and in darkened chloroplasts is separated by an on–off switch. Photosynthetic electron transport (PET) provides reductant. When electron flow stops upon darkening, regulatory thiol proteins are oxidized. This diurnal light–dark modulation prevents waste of energy. (B) During illumination, due to the continuous reoxidation of regulatory cysteines that are characterized by their high disposition to become oxidized, photosynthetic electrons are sacrificed to run the redox cycle continuously (see also supplemental information online, Figure S3). These particular thiol switches serve as the basis for fine-tuning activation states individually, depending on the metabolic situation at each regulated step. (C) Oxidative stress provokes disruption of cellular redox homeostasis and formation of reactive oxygen species (ROS). The transiently modified proteins can take over moonlighting functions and are involved in signaling and changing gene transcription. Upon reestablishment of redox homeostasis or for continued ROS scavenging and repair, NADPH reverses these modifications. ox., oxidized; red., reduced.

(NADP-GAPDH; E.C.1.2.1.13). RubisCO is indirectly redox-dependent due to the activity of RubisCO activase, which is a target of Trx. The F-ATP synthase (CF1; E.C.3.6.1.34) and NADPmalate dehydrogenase (NADP-MDH; E.C.1.1.1.82), both involved in poising the ATP-toNADPH ratio, are also activated by reduced Trx. The ATPase uses the proton gradient of the thylakoid membrane to generate ATP. The NADP-MDH of C3 plants as part of the malate valve [18] is involved in the export of surplus, reducing equivalents as malate from the chloroplast into the cytosol. All these enzymes are switched off by oxidation. Interestingly, oxidation of enzymes by Trx can also lead to activation, as is the case for glucose 6-phosphate dehydrogenase (G6PDH; E.C.1.1.1.49), the key enzyme of the oxidative pentose-phosphate (OPP) pathway. Light inactivation of the plastid isoforms of G6PDH ensures that the OPP pathway and CBC do not operate simultaneously, avoiding futile cycling. There are more Trx targets in plastids, such as ADP-glucose pyrophosphorylase (AGPase; E.C.2.7.7.27) to generate the activated substrate ADP-glucose for starch biosynthesis [19], and acetyl-CoA carboxylase (ACCase; E.C.6.4.1.2) for the synthesis of malonyl-CoA as the activated starter molecule to initiate fatty acid synthesis [20]. Furthermore, plastidial transcription and translation, and defense against oxidative stress, involve the other plastid isoforms Trx x, y, and z, as well as Trx-like proteins such as ACHT1 and 4 and CDSP32 in a redoxdependent manner or also redox-independently [21–26]. As another example, tetrapyrrole biosynthesis has been shown to be dependent upon NTRC and Trx at various steps [27]. Processes in other cellular compartments, such as nuclear transcription and cytosolic translation, also respond in many ways to redox-dependent signals [28,29], pointing to a broader

structure and function. On the one hand, they can build disulfide bridges to stabilize protein structures. These permanently oxidized proteins are found in endoplasmic reticulum, Golgi, vacuole, and apoplast. In general, the majority of the intracellular cysteine-containing proteins are permanently reduced. Upon oxidation they are irreversibly damaged. In contrast, some intracellular redox-active proteins undergo a reversible redox change, depending on the presence of reductants or oxidants. Reduced and oxidized proteins are characterized by different properties and the ratio of both forms determines the actual enzyme activity. Small molecules: thiol switches are affected by small molecules acting as positive or negative effectors of reduction or oxidation of the regulatory thiols of redox-active proteins. They comprise metabolites and ions that bind noncovalently to these proteins, thus linking the metabolic state and their redox interconversions. Thioredoxin (Trx): two cysteines in their catalytic center are common to members of this ubiquitous family of small soluble proteins. Each Trx can exist in the reduced dithiol and the oxidized disulfide state. The formation of the energetically favored 14-membered ring upon oxidation at the protruding active site makes Trx a perfect redox mediator. Trx systems: chloroplasts possess two systems for reduction of the Trx, namely ferredoxin-Trx reductase (FTR) and NADPH-Trx reductase (NTRC). Cytosol and mitochondria possess additional Trx and the NADPH-Trx reductases, NTRA and NTRB, respectively. Even the nucleus has been found to contain a Trx system. The plant redox network contains further redoxins, such as glutaredoxins (Grx) and peroxiredoxins (Prx), also mediating redox changes.

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PET NADPH

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Figure 2. Redox-Regulated Chloroplast Enzymes. The activities of the chloroplast enzymes shown in the scheme are regulated by redox interconversions. Some of the enzymes are reductively activated (yellow and orange), others are reductively inactivated (black). These reversible cysteine modifications are specifically suited to control metabolism and redox homeostasis in the daily light–dark cycle and under changing light regimes during the day. The Calvin-Benson cycle (CBC) as a branched, cyclic pathway is controlled at these essential steps in the different parts (yellow). The malate valve enzyme NADP-MDH, as well as the chloroplast ATP synthase, maintain the required ATP/NADPH ratio (orange). Oxidation leads to disulfide formation of the regulatory cysteines of these enzymes, resulting in their inactivation. In contrast, the key enzyme of the oxidative pentose phosphate (OPP) pathway, namely G6PDH (black), is inactive in the light, when reduced, and active in the dark, when its thiols are oxidized. DicT, dicarboxylate translocator; FBP, fructose1,6-bisphosphate; FBPase, fructose 1,6-bisphosphatase; G6PDH, glucose6-phosphate dehydrogenase; NADP-GAPDH, NADP-dependent glyceraldehyde3-phosphate dehydrogenase; NADP-MDH, NADP-malate dehydrogenase; SBPase, sedoheptulose1,7bisphosphatase;PET, photosynthetic electron transport; PGA, phosphoglycerate; PRK,phosphoribulose kinase; TPT, triosephosphate-phosphate translocator.

distribution of redox-controlled steps that are potentially also regulated by additional factors, such as small molecules, for flexible adjustment.

Dynamics of Reduction and Reoxidation as Influenced by Small Molecules Oxidation of the regulatory thiols occurs when light-dependent photosynthetic electron flow is shut down. Addition of oxygen to an anaerobic chloroplast suspension was shown to enhance oxidative inactivation upon darkening [7,30]. Due to the aerobic environment inside the chloroplast, the reduced cysteine residues of the redox-modified enzymes are readily reoxidized also during illumination. This applies even more under high light, when the flux through the CBC should be increased and not decreased in the presence of light-generated ROS, such as H2O2. Continuous re-reduction is required to maintain a certain portion of the enzyme in the reduced form under steady-state conditions in the light. Since specific metabolites act as positive or negative effectors on the reduction and oxidation rates of the interconversion in each case, the steady-state fraction of reduced enzyme is determined by the current metabolic

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Net effect in steady state

NADP-GAPDH S S

HS

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(A2B2)

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Malate + NADP

-

G6PDH + 6PGlu + HS

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Feed-back acvaon of reducve inacvaon by NADPH

Figure 3. Redox-Regulated Chloroplast Enzymes Subjected to Individual Fine-Tuning by ‘Small Molecules’. The redox-regulated chloroplast enzymes shown in the schemes are reduced by the thioredoxin system in the light and continuously reoxidized due to the presence of oxygen. Small molecules (red), which are often substrates or products of the reaction catalyzed by the respective active enzyme (round symbol), modulate the rates of interconversion between (See figure legend on the bottom of the next page.) Trends in Plant Science, Month Year, Vol. xx, No. yy

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situation [31–33] (Figure 3). Additionally, as is true for most metabolic enzymes, catalysis of the active form itself is also influenced by substrates, products, cellular ion concentrations, and pH. The concept of fine-tuning an on–off switch, such as light–dark modulation of chloroplast enzymes, grew from work with the spinach chloroplast NADP-MDH. Interconversion of the active and inactive forms of NADP-MDH is directly influenced by the small molecule NADP+ [34]. Similarly, redox-dependent rates of activation of other chloroplast enzymes are controlled by metabolites [35]. Binding of a specific metabolite in each case was found to shift the oxidation constant (Kox) for the respective regulatory cysteines after equilibration with dithiothreitol (DTT)-redox buffers [36]. Such experiments, as well as kinetic studies, have provided the first evidence that a common principle (redox-cycling) is used to control fluxes via small molecules, specifically at defined metabolic steps, according to the demand [37]. Effectordriven change in the steady-state concentration of the active form of the enzyme allows for rapid adjustment of fluxes. Local metabolite concentrations are direct indicators of the actual carbon flux at each of the redox-regulated steps. Functioning as positive or negative effectors, they immediately can readjust the fluxes specifically upon any imbalance in metabolite pools, in this way maintaining homeostasis. The redox-dependent changes of the kinetic parameters can concern Vmax (NADP-MDH, PRK), Km (NADP-GAPDH, FBPase, G6PDH, and AGPase), or Ki (AGPase, RubisCO activase). The monocascade of the continuous redox cycle between the two enzyme forms enables a hysteretic response within less than a minute to gradually or rapidly alter actual enzyme activities as required (Figure 3). NADP-GAPDH The chloroplast NADP-GAPDH catalyzes the reduction of 3-PGA, which is achieved after its conversion into 1,3-bisphosphoglycerate (1,3bisPGA) by 3-phosphoglycerate kinase (PGK). The active enzyme consists of two different subunits, GapA and GapB, in a heterotetramer (A2B2). The dark form with two regulatory thiols in the oxidized state exists as a hexadecameric complex (A8B8), which is virtually inactive under physiological substrate (1,3bisPGA) concentrations. Only the simultaneous presence of reduced Trx and 1,3bisPGA, acting as an effector, leads to the dissociated tetrameric form with an increased affinity for the substrate 1,3bisPGA [38,39]. For GapA/B, reduction of the disulfide bridge at the C terminal extension of GapB in the hexadecameric complex lowers the activation constant (Ka) for 1,3bisPGA, acting here as a positive effector to facilitate dissociation and activation at physiological 1,3bisPGA concentrations. In addition, another complex consisting of inactive NADP-GAPDH, PRK, and the small chloroplast protein CP12 is even more readily activated by reduction and dissociation in the presence of NADPH already in low light. This leads to activation of a fraction of the total NADP-GAPDH pool and all of the PRK, even in low light [40]. In contrast, the A8B8 complex releases the active enzyme only when the substrate 1,3bisPGA starts to accumulate, indicating oxidized and reduced forms. Redox cycling in the light, when reduction and concomitant reoxidation lead to a steady state of reduced and oxidized forms, provides the basis for individual fine-tuning by metabolites. Small molecules act as effectors (positive or negative) on either reduction or oxidation or both. The equilibrium between active and inactive enzyme can be shifted according to the nature of the effectors, as indicated by the arrows and the + and affecting the rates of the interconversions. The net effect is that a very rapid and efficient adjustment of enzyme activities is achieved according to the actual metabolic demand. This can result in feed-forward activation when substrate pools increase (NADP-GAPDH, FBPase), or feedback inhibition from the increase of product (NADP-MDH or G6PDH). Oxidation in the dark leads to disulfide formation of the regulatory cysteine residues. In most cases (NADP-GAPDH, FBPase, PRK, NADP-MDH), the oxidized enzymes are inactive (square symbols), except for G6PDH which is active upon oxidation. FBP, fructose 1,6bisphosphate; FBPase, fructose 1,6-bisphosphatase; F6P, fructose 6-phosphate; GAP, glyceraldehyde 3-phosphate; G6P, glucose 6-phosphate; G6PDH, glucose 6-phosphate dehydrogenase; NADP-GAPDH, NADP-dependent glyceraldehyde 3-phosphate dehydrogenase; NADP-MDH, NADP-malate dehydrogenase; OAA, oxaloacetate; PGA, 3-phosphoglycerate; PRK, phosphoribulose kinase; 1,3bisPGA, 1,3-bisphosphoglycerate; Ru1,5bisP, ribulose 1,5bisphosphate; Ru5P, ribulose 5-phosphate; 6PGlu, 6-phosphogluconate.

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the need for an increased flux through this step. Interestingly, this results in NADP-GAPDH activities that correspond to the actual flux rate of the whole CBC [41]. In this context, CP12 itself can be seen as a specific redox regulator. Due to its properties as an intrinsically unstructured protein, carrying two disulfide bridges, it assists the oligomerization of a specific NADP-GAPDH/PRK complex upon oxidation and releases both CBC enzymes in their active state upon the onset of illumination [42–44]. PRK PRK is a unique enzyme in chloroplasts for regeneration of the CO2 acceptor in the CBC. It is rapidly activated in the light upon release from the NADP-GAPDH/CP12/PRK complex [40]. Both reductive activation and oxidative inactivation of PRK in vitro, using the purified dimeric form, is slowed down in the presence of ATP [45]. This results in no net effect of ATP on the activation state of PRK, which is fully reduced and active in standard assay medium already in low light [40]. Actual fluxes through this step are determined by metabolic inhibitors (inorganic phosphate, FBP, 6-phosphogluconate) at the level of catalysis of the fully reduced enzyme [46]. FBPase Chloroplast FBPase is reductively activated, but only when its substrate FBP is present as an effector [47]. In addition, the rate of oxidative inactivation is decreased in the presence of FBP [48]. FBP binding impacts thiol sensitivity of the distant vicinal thiol groups by long distance conformational changes, resulting in facilitated reduction and the stabilization of the reduced form of FBPase [49]. This results in an increased activation state of FBPase as soon as substrate starts to accumulate. Reduced FBPase is characterized by increased affinities for FBP and Mg2+, with [Mg2+-FBP]2 being the true substrate of the reaction, and by a pH optimum for catalysis in a more physiological range [50]. These shifts of Km (FBP; Mg2+) and pH optimum, together with the facilitated reduction, result in an increased FBPase activity in illuminated chloroplasts. NADP-MDH Poising mechanisms are required when the ATP demand in the chloroplast exceeds its production and NADPH accumulates. Under this condition, NADPH is reoxidized by NADP-MDH and malate is formed, resulting in indirect export of reducing equivalents as malate. In the cytosol, malate can be reoxidized, leading to the formation of NADH for nitrate reduction, or, in the mitochondria, for ATP production [18]. Reductive activation of NADP-MDH is only enabled when NADPH starts to accumulate in the stroma (e.g., due to the lack of ATP or CO2 for continued CBC activity), and when NADP+ levels decrease correspondingly. In the lag phase of photosynthesis, upon a switch from dark to light, overreduction of the electron transport chain, monitored as fluorescence emission from photosystem II, is counteracted by the fast activation of NADP-MDH and typical oscillations that are dampened in the next few minutes to reach a new steady-state [51]. NADP+ acts as a negative effector for light activation of NADP-MDH, indicating the preferential consumption of reducing equivalents in the CBC or by competing electron acceptors such as nitrite [52,53]. This is an essential feature of the selfcontrolled malate valve. NADPH in contrast, inhibits both activation as well as inactivation, thus decreasing the frequency of the redox cycle, but without any net effect on the ratio between reduced and oxidized form. Upon decrease of light intensity from high to lower light, a short peak of NADP+ accumulation and a transient decrease of O2 evolution coincides with a rapid decrease of NADP-MDH activity and the re-establishment of a new steady-state activity [54]. The oxidized enzyme is characterized by a Vmax of zero, where the oxidized C terminus of NADP-MDH is acting as an autoinhibitory peptide [55]. The C terminus is released from the active site upon reduction only in the absence of the negative effector NADP+ [56].

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G6PDH Reductive inactivation of chloroplast G6PDH rapidly and efficiently occurs when NADPH is sufficiently provided in the light reactions; here NADPH acts as a positive effector of the reductive inactivation. The reduced inactive G6PDH is characterized by its low affinity towards the substrate G6P (Km > 20 mM), rendering it practically inactive at physiological G6P concentrations. It is worth mentioning that this enzyme is active when its regulatory thiols are oxidized by the formation of a disulfide bridge; a unique case for an intracellular enzyme. Here, oxidation results in a shift of the Km value to the physiological range (1–2 mM) of the substrate G6P [57,58]. Other Enzymes Redox state and small molecules appear to act together in other cases also. The carboxylation step in the CBC, catalyzed by RubisCO, is regulated by the ATP-dependent RubisCO activase, which removes incorrect metabolites from the active site [59]. The reduced form of the activase is less affected by the inhibitor ADP. Therefore, the carboxylation activity can be adjusted to the conditions as indicated by the ADP/ATP ratio, particularly in low light [60]. The g-subunit of chloroplast CF0/CF1-ATP synthase undergoes a redox change of two cysteine residues, positioned in a chloroplast-specific amino acid sequence insertion, thus preventing ATP hydrolysis in the dark [61–63]. However, ATP synthesis is not only redox controlled, but also responds to pH gradient, proton motive force, and other metabolic factors. Ambient conditions, such as low light or elevated CO2, can therefore influence the ease of reductive activation and thus the rate of ATP synthesis [64,65]. The reductive activation of AGPase is promoted by the regulatory metabolite trehalose 6-phosphate (T6P) [66]. For glutamine synthase (GS), glutamate synthetase (GOGAT), and acetyl-CoA carboxylase (ACCase), similar mechanisms of an integrated activation have been suggested but remain to be studied in more detail. Indeed, it is experimentally challenging to identify the site of action of the effectors and their role in shifting the equilibrium redox potential, particularly when activation and activity cannot be tested separately. It is also difficult to distinguish and characterize the different mechanisms of regulation, when substrate or products act simultaneously as effectors on the redox interconversion or when both catalysis and activation/inactivation are influenced by the same small molecules. Taken together, small molecules act as positive or negative effectors on reduction and/or oxidation rates of the redox interconversions. This principle allows for the individual adjustment of the redox states and concomitantly the properties of the respective proteins (Figure 4A, Key Figure). As an example, for the CBC enzyme FBPase and the malate-valve enzyme NADPMDH, this can result in a diverging effect concerning their activation state and consequently their actual activities (Figure 4B). The extraordinarily negative redox potential of reduced ferredoxin is capable of reducing these enzymes, however, only in the presence of the positive effector (FBP for FBPase) and the absence of the negative effector (NADP+ for NADP-MDH). Photosynthetic thylakoid energization and redox state of the electron transport chain are thus tightly interconnected with the redox cycles of target enzymes. The control of these monocascades by metabolic intermediates results in a continuous adjustment and maintenance of fluxes under challenging conditions.

Concluding Remarks and Future Perspectives Usually in lab experiments, when huge effects are anticipated for clear-cut results, a large impact is applied to produce visible displacements which provoke system disturbance, so that oscillations and subsequent dampening can be easily observed [51]. In reality, however, infinitely small, immediate changes of the activation states result in a smooth adjustment of 8

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Key Figure

Opposite Effects on Calvin-Benson Cycle (CBC) and Malate Valve Activation Depending on the Metabolic State (A)

Small molecules (posive or negave effector) 2e-+ 2H+

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the steady-state activities and fluxes, which are tightly linked to the metabolic situation. The complexity of the regulatory network that controls energy distribution, biomass production, antioxidant defense, gene transcription, and translation relies on the presence of many redox transmitters. The interplay between redox systems and metabolism is also the basis of genetic and epigenetic mechanisms in the context of adaptive responses. At this level, but also in the classic metabolic cases of redox-based modifications as described in this article, the combinatorial aspect of post-translational modifications and allosteric effects with gradual impact on each other might explain the robustness of plant metabolism [67]. While the plastid Trx are characterized by exceptionally negative redox potentials in the range of 330 to 370 mV (at stromal pH 7.9) [68], cytosolic Trx and all Grx range between 220 and 270 mV (at pH 7) [69]. Redox potentials of the cysteines in the C-X-X-C motif of the Trx-fold can vary dynamically in the range between 95 mV (protein-disulfide isomerase in the endoplasmatic reticulum) and 287 mV (cytTrx) [70], or even be more negative as in chloroplast Trx f and m. As one pH unit changes the redox potential of cysteines by 60 mV [71], values from measurements performed at pH 7, as done in many studies, should be corrected to a more negative value when compared with the values of the plastid isoforms, namely to 280 to 330 mV. It is interesting to note that the midpoint potentials of chloroplast Trx with their rather negative values can only be determined in redox buffers with similar oxidation constants (Kox). In fact, only DTT-redox buffers are suitable for this purpose. Finally, in a steady-state system, red/ ox ratios are not only determined by their equilibrium redox potentials (by redox titrations), but also by the kinetics of reduction and oxidation [72]. It is still a challenge to assign specific targets to each of the many Trx. Some specificity of Trx might be explained by differences in their redox potentials, those of the various plastidial Trx isoforms ranging around 360 mV for Trx m and f [68] and below 310 mV for Trx x, y, and z [73]. However, the main driving force for target recognition appears to lie in the surface properties that facilitate the noncovalent interaction between Trx and the target [74,75]. Genetic studies and in vitro experiments using the recombinant proteins indicate some specificity of plastidial Trx isoforms for different targets [73,76]. Furthermore, mutant plants with a changed expression of the various Trx systems showed deviations in photosynthetic performance, Figure 4. (A) The redox cycle of protein dithiol-disulfides is driven by electron flow from reductants to an oxidant. Such a seemingly futile cycle serves as a basis for fine-tuning. The steady-state concentration of active enzyme can be readily shifted by ‘small molecules’ that either positively or negatively act as effectors on the rates of interconversion. (B) Chloroplast components are arranged according to their midpoint potentials. The disulfide bridges of light–dark-regulated enzymes are reduced by the thioredoxins Trx m and f, which obtain electrons from the strong reductant Fd (ferredoxin). Differential activation of FBPase (fructose 1,6-bisphosphatase) and NADP-MDH (NADP-malate dehydrogenase) are shown as examples for individual fine-tuning. Changing stromal concentrations of FBP (fructose 1,6-phosphate) and NADP+ are visualized by color gradients. In the case of FBPase, a low concentration of the positive effector FBP leads to a very negative redox potential of the regulatory cysteines and hinders enzyme activation by thioredoxin (Trx). FBP (blue oval) at increasing concentrations binds to the active site and, by an allosteric effect (marked by the grey arrow), shifts the redox potential of the regulatory cysteines to a less negative value, so that FBPase can be easily reduced. An increasing FBP concentration represents the carbohydrate pool to be further processed in the chloroplast and opens the gate by facilitating FBPase activation. When the flux through the CBC is high and light-generated NADPH is continuously used for CO2 assimilation, NADP+ (red oval) as a negative effector impedes reductive activation of NADP-MDH and the autoinhibitory C terminus blocks the active site, rendering the enzyme inactive. As a consequence, export of NADPH as malate is impeded when reductants are preferentially used in the CBC, and increased NADP acts as a brake to avoid inappropriate opening of the malate valve by inhibition of reductive activation. In contrast, a low NADP+/(NADP+ + NADPH) ratio (corresponding to high NADPH), renders the C terminal disulfide bridge accessible and shifts the redox potential to a less negative value, thus enabling reductive activation of NADP-MDH. DTT, dithiothreitol; FBP, fructose 1,6-bisphosphate; GSSG/GSH, oxidized and reduced glutathione; ox, oxidized; red, reduced.

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enzyme activation kinetics, and adaptation to fluctuating light conditions, but are also suggesting partial redundancy [77–79]. In an in vitro system, FTR and NTRC reduce the various chloroplast Trx isoforms more or less efficiently [80]. However, the well-studied chloroplast redox systems and their possible targets, particularly their specificities for the targets, are not yet fully understood [81–83]. The availability of crystal structures of redox-regulated targets improves the knowledge of the molecular details of these proteins. Their sequence characteristics and special properties came into existence during the period when oxygenic photosynthesis created conditions that were favorable for using oxidation and re-reduction as a means to post-translationally change enzyme properties [84]. During evolution, upon the emergence of oxygenic photosynthesis, some chloroplast enzymes acquired sequence extensions or inserts carrying the regulatory thiols, while the remaining sequence is highly similar to the counterparts that are not subjected to redox modulation [84,85]. The extra sequences carry two vicinal thiols and act as a redox switch. Upon reversible modification, local restriction or a conformational impact changes the catalytic or regulatory properties of the enzyme (Figure 4B). Some structures of the respective enzymes in their reduced and oxidized forms are already available, like those for FBPase [49,86] and for NADP-MDH [87,88] with NADP+ bound to the oxidized form in such a way that disulfide reduction is impeded [6]. From crystal structures of two very similar Trx h isoforms from barley, the importance of hydrogen bonds between backbones of Trx and target protein has been suggested as a determinant for specific recognition [89,90]. Moreover, the various Trx h isotypes were also shown to possess specific recognition sites for their targets [91]. To explain the specificity among the unusually high number of redox systems and their targets, the in vivo situation with its high protein concentration promoting protein–protein interactions should be taken into account. More specific protein–protein interactions might, therefore, be achieved only in vivo. Steric constraints for disulfide formation can be increased or decreased by noncovalent interactions that are affected by ligand binding or additional post-translational modifications (PTM code) [92–95], thus changing the reductive and oxidative properties, respectively, of each redox mediator protein. In order to predict a change of redox potential, the interplay of bound small molecules, additional modifications, and other factors should be taken into account. All these factors can shift the pKa of the respective thiols. Also, steric constraints can impede disulfide formation, and noncovalent binding of effectors influences the local electrostatic environment and affects thiolate formation [96–100]. Binding of small molecules puts further impact on the reactivity of the thiol(s), as shown for OOC-5, a Torsin-family protein in Caenorhabditis elegans, where ADPbinding shifts the redox potential from 210 to 240 mV. In this way, by integrating the ADP/ ATP ratio and redox state, this protein acts as a sensor for metabolic requirements [101]. The regulatory principle described here for well-studied examples is not only present in chloroplasts of plants growing under challenging environmental conditions, which require the continuous and rapid adjustment of enzyme activities by post-translational modification. Similar redox-regulatory mechanisms also exist in yeast and animals during oxidative stress affecting many cellular functions, including signaling and regulation of nuclear transcription [70,102–105]. Redox changes of intracellular proteins occur in all organisms, particularly in the context of development [106], aging, pathogen attack, and disease [107]. Better knowledge of the ubiquitous redox switches and their appropriate adjustment in complex regulatory Trends in Plant Science, Month Year, Vol. xx, No. yy

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networks will enable advances in all fields, including medical sciences (see Outstanding Questions). Acknowledgments Work performed in the author’s lab was supported with funds from the DFG (SCHE 217; SPP 1710) over the past four decades.

Supplemental Information Supplemental information associated with this article can be found, in the online version, at https://doi.org/10.1016/j. tplants.2018.06.007.

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Outstanding Questions Are there additional thiol-switches in signaling, subcellular localization or gene expression that are controlled by ‘small molecules’? Does local influence on thiolate formation from further yet unknown modifications of neighboring amino acids need to be taken into account to fully understand the dynamic behavior of a highly integrated regulatory network? Is the principle of fine-tuning by ‘small molecules’ also valid in other cases of post-translationally regulated proteins, for example, nitrate reductase, sucrose-P synthase and PEP-carboxylase which are subject to protein phosphorylation/-dephosphorylation? Is specificity in vivo reached by local concentrations of ‘small molecules’ such as protons, inorganic ions, metabolites, or further covalent modifications, or protein-protein interactions? Which other sterical and/or biochemical features determine the observed shifts of the redox potentials of the regulatory cysteines? Can simulations and modeling of the respective transient holo-complexes help to better understand and predict specificities, new targets, and functions?

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