J.-P. Jacquot

Ferredoxin-thioredoxin reductase, an iron-sulfur enzyme linking light to enzyme regulation in oxygenic photosynthesis: Purification and properties of the enzyme from C3, C4, and cyanobacterial species

Ferredoxin-thioredoxin reductase (FTR), an enzyme involved in the light regulation of chloroplast enzymes, was purified to homogeneity from leaves of spinach (a C3 plant) and corn (a C4 plant) and from cells of a cyanobacterium (Nostoc muscorum). The enzyme is a yellowish brown iron-sulfur protein, containing four nonheme iron and labile sulfide groups, that catalyzes the activation of NADP-malate dehydrogenase and fructose 1,6-bisphosphatase in the presence of ferredoxin and of thioredoxin m and f, respectively. FTR is synonymous with the protein earlier called ferralterin. FTR showed an Mr of about 30,000 (determined by sedimentation equilibrium ultracentrifugation, amino acid composition, gel filtration, and gradient gel electrophoresis) and was composed of two dissimilar subunits (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). One of the FTR subunits from each source was similar both in Mr (about 13,000) and immunological properties, while the other subunit (of variable molecular weight) was characteristic of a particular organism. The similar subunit contained a disulfide group that was rapidly reduced by a dithiol (dithiothreitol) but not by monothiols (2-mercaptoethanol or reduced glutathione). Homogeneous FTR formed a tight noncovalent complex with ferredoxin on affinity columns. The basis for the structural variation in the different FTR enzymes remains to be determined.

Ferredoxin-thioredoxin reductase: a catalytically active dithiol group links photoreduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation

The mechanism by which the ferredoxin-thioredoxin system activates the target enzyme, NADP-malate dehydrogenase, was investigated by analyzing the sulfhydryl status of individual protein components with [14C]iodoacetate and monobromobimane. The data indicate that ferredoxin-thioredoxin reductase (FTR)--an iron-sulfur enzyme present in oxygenic photosynthetic organisms--is the first member of a thiol chain that links light to enzyme regulation. FTR possesses a catalytically active dithiol group localized on the 13 kDa (similar) subunit, that occurs in all species investigated and accepts reducing equivalents from photoreduced ferredoxin and transfers them stoichiometrically to the disulfide form of thioredoxin m. The reduced thioredoxin m, in turn, reduces NADP-malate dehydrogenase, thereby converting it from an inactive (S-S) to an active (SH) form. The means by which FTR is able to combine electrons (from photoreduced ferredoxin) with protons (from the medium) to reduce its active disulfide group remains to be determined.

Localization of NADP-malate dehydrogenase of corn leaves by immunological methods

Abstract An antibody raised against the inactive (non-activated) form of NADP-linked malate dehydrogenase (MDH) from corn leaves precipitated the enzyme and inhibited its catalytic activity. The antibody had no significant effect on the low levels of NAD-linked activity in the preparation. When applied in enzyme-linked immunosorption assay (ELISA) and immunofluorescence procedures, the antibody revealed that NADP-MDH is located exclusively in chloroplasts of mesophyll cells. The evidence also indicated that NADP-MDH undergoes a conformational change when activated. The results demonstrate that immunological methods can be used to determine the cellular location of C4 enzymes both in vitro and in situ.

Regulation of corn leaf NADP-malate dehydrogenase light-activation by the photosynthetic electron flow. Effect of photoinhibition studied in a reconstituted system.

Abstract The light-activation of the chloroplastic enzyme NADP-malate dehydrogenase is totally inhibited when chloroplasts are preilluminated with high light. The mechanism of this photoinhibition has been studied in a reconstituted chloroplast system composed of isolated thylakoids and the purified proteins of the ferredoxin-thioredoxin light activation system, by examinating the reduction state of the disulfide bridges located on the different proteins of the system and considered to be involved in the light activation process. The results indicate that, when the reduction of S-S groups on thioredoxin and on NADP-MDH is only partially decreased, NADP-MDH is totally inactive. A reduction vs. activity curve shows that the activity of this last enzyme strongly depends on its reduction state, more than 50% reduction being required for activity to appear. The physiological significance of these observations is discussed.

Energetic aspects of the light activation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase.

The light energy requirements for photoactivation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase were studied in a reconstituted chloroplast system. This system comprised isolated pea thylakoids, ferredoxin (Fd), ferredoxin-thioredoxin reductase (FTR) thioredoxinm and f (Tdm, Tdf) and the photoactivatable enzyme. Light-saturation curves of the photoactivation process were established with once washed thylakoids which did not require the addition of Td for light activation. They exhibited a plateau at 10 W·m−2 under nitrogen and 50 W·m−2 under air, while NADP photoreduction was saturated at 240 W·m−2. Cyclic and pseudocyclic phosphorylations saturated at identical levels as enzyme photoactivations. All these observations suggested that the shift of the light saturation plateau towards higher values under air was due to competing oxygen-dependent reactions. With twice washed thylakoids, which required Td for enzyme light-activation, photophosphorylation was stimulated under N2 by the addition of the components of the photoactivation system. Its rate increased with increasing Td concentrations, just as did the enzyme photoactivation rate, while varying the target enzyme concentration had only a weak effect. Considering that Td concentrations were in a large excess over target enzyme concentrations, it may be assumed that the observed ATP synthesis was essentially dependent on the rate of Td reduction.Under air, Fd-dependent pseudo-cyclic photophosphorylation was not stimulated by the addition of the other enzyme photoactivation components, suggesting that an important site of action of O2 was located at the level of Fd.

Frozen thylakoids: an improvement for reconstituted chloroplast enzyme light-activation systems

Abstract This paper describes a new method for improving both the stability and reproducibility of chloroplast enzyme light-activation systems. Usually, the most labile components of these systems are the thylakoids, the preparation of which must be repeated daily. Freezing the thylakoids in small aliquots in liquid nitrogen and storing them at −90°C in a buffer containing 50% glycerol results in preparations whichare completely stable over an 18-month-period. Enzyme light-activation rates were essentially identical with either frozen or fresh thylakoids. Freezing, however, resulted in a slow decline of NADP-protoreduction rates and also in a gradual uncoupling of non-cyclic photophosphorylation.

Bundle-sheath thylakoids from NADP-malic enzyme-type C4 plants require an exogenous electron donor for enzyme light activation

Light activation of either NADP-malate dehydrogenase (EC 1.1.1.82) or fructose-1,6-bisphosphate phosphatase (EC 3.1.3.11) was assayed in a reconstituted chloroplastic, system comprising the isolated proteins of the ferredoxin-thioredoxin light-activation system and thylakoids from either mesophyll or bundle-sheath tissues of different C4 plants. While C4-plant thylakoids functionned almost equally well with C3-or C4-plant proteins, the photosyntem-II-deficient bundle-sheath thylakoids from the NADP-malic enzyme type, were unable to perform enzyme photoactivation unless supplemented with an electron donor to photosystem I. Bundle-sheath thylakoids isolated from plants showing no photosystem-II deficiency did not require such an addition. The results are discussed with respect to a possible requirement for a physiological reductant of ferredoxin for enzyme light activation in bundle-sheath, tissues.

Effect of high light intensities on oxygen evolution and the light activation of NADP-malate dehydrogenase in intact spinach chloroplasts

The factors limiting the photosynthetic carbon metabolism of intact spinach (Spinacia oleracea L.) chloroplasts after a high-light pretreatment have been studied. Photosynthetic CO2 fixation was decreased and became more sensitive to the inhibitory effect of the cyclic-electron-flow inhibitor, antimycin A. Depending on the extent of photoinhibition, changing the balance of linear to cyclic electron flow by adding oxaloacetate and antimycin A either did not relieve, or partially relieved the photoinhibitory effect. The decrease in CO2 fixation appeared to be the consequence of either a limitation by photosystem-II activity (in the case of moderate inhibition) or, at least partially an unfavourable balance between the linear and cyclic electron flows (in the case of strong inhibition). The light activation of NADP-malate dehydrogenase (EC 1.1.1.82) was decreased only in the presence of CO2, i.e. when there was strong competition for reducing power; otherwise, it was unaffected by photoinhibitory treatments, in accordance with its low energy requirement.

Studies on Enzyme Photoactivation by the Ferredoxin/Thioredoxin System

The ferredoxin/thioredoxin system functions in oxygenic photosynthesis by linking light to the regulation of selected target enzymes Fig. 1 (1).

Regulation of NADP-Malate Dehydrogenase Light-Activation by the Reducing Power. I Functional Studies

Chloroplastic NADP dependent malate dehydrogenase (NADP-MDH) is regulated by light via the ferredoxin-thioredoxin system (1). In vitro, light activation of NADP-MDH can be achieved using a reconstituted system, comprising thylakoid membranes, ferredoxin, ferredoxin-thioredoxin reductase and thioredoxin (2). The activation process can also be mimicked by thioredoxin in the presence of a reductant such as dithiothreitol (DTT) (3). It has been shown that the proteins of the ferredoxin-thioredoxin system undergo thiol/disulfide interchange reactions, during the light activation process (4). The critical disulfide bridge of spinach thioredoxin is now well characterized and its primary structure is known (5). However, the corresponding cysteinyl residues on NADP-MDH have not been identified yet. In this work, we studied the structure of the light-dependent regulatory site of NADP-MDH and we investigated the effect of NADP on the light activation of the enzyme.