Crosstalk between chloroplast thioredoxin systems in regulation of photosynthesis

Thioredoxins (TRXs) mediate light‐dependent activation of primary photosynthetic reactions in plant chloroplasts by reducing disulphide bridges in redox‐regulated enzymes. Of the two plastid TRX systems, the ferredoxin‐TRX system consists of ferredoxin‐thioredoxin reductase (FTR) and multiple TRXs, while the NADPH‐dependent thioredoxin reductase (NTRC) contains a complete TRX system in a single polypeptide. Using Arabidopsis plants overexpressing or lacking a functional NTRC, we have investigated the redundancy and interaction between the NTRC and Fd‐TRX systems in regulation of photosynthesis in vivo. Overexpression of NTRC raised the CO₂ fixation rate and lowered non‐photochemical quenching and acceptor side limitation of PSI in low light conditions by enhancing the activation of chloroplast ATP synthase and TRX‐regulated enzymes in Calvin–Benson cycle (CBC). Overexpression of NTRC with an inactivated NTR or TRX domain partly recovered the phenotype of knockout plants, suggesting crosstalk between the plastid TRX systems. NTRC interacted in planta with fructose‐1,6‐bisphosphatase, phosphoribulokinase and CF₁γ subunit of the ATP synthase and with several chloroplast TRXs. These findings indicate that NTRC‐mediated regulation of the CBC and ATP synthesis occurs both directly and through interaction with the ferredoxin‐TRX system and is crucial when availability of light is limiting photosynthesis.     

Reduction of ferredoxin:thioredoxin reductase by artificial electron donors

The ferredoxin:thioredoxin reductase is an essential enzyme of the light dependent regulatory system in oxygenic photosynthesis. It is composed of two dissimilar subunits and contains a 4Fe-4S cluster and a redox-active disulfide bridge. Artificial electron donors of redox potentials below –300 mV are capable of reducing the disulfide bridge. Based on our results we speculate that a group of more negative potential than the disulfide bridge is the first acceptor of the electrons in FTR. The chemical reduction of FTR has been used successfully for the detection of the enzyme during its purification.Abbreviation FBPase fructose 1,6-bisphosphatase - FTR ferredoxin:thioredoxin reductase - MV methyl viologen Dedicated to Prof. D.I. Arnon.     

The ferredoxin-thioredoxin system of a green alga,Chlamydomonas reinhardtii

The components of the ferredoxin-thioredoxin (FT) system of Chlamydomonas reinhardtii have been purified and characterized. The system resembled that of higher plants in consisting of a ferredoxin-thioredoxin reductase (FTR) and two types of thioredoxin, a single f and two m species, m1 and m2. The Chlamydomonas m and f thioredoxins were antigenically similar to their higher-plant counterparts, but not to one another. The m thioredoxins were recognized by antibodies to both higher-plant m and bacterial thioredoxins, whereas the thioredoxin f was not. Chlamydomonas thioredoxin f reacted, although weakly, with the antibody to spinach thioredoxin f. The algal thioredoxin f differed from thioredoxins studied previously in behaving as a basic protein on ion-exchange columns. Purification revealed that the algal thioredoxins had molecular masses (M_rs) typical of thioredoxins from other sources, m1 and m2 being 10700 and f 11 500. Chlamydomonas FTR had two dissimilar subunits, a feature common to all FTRs studied thus far. One, the 13-kDa (similar) subunit, resembled its counterpart from other sources in both size and antigenicity. The other, 10-kDa (variable) sub-unit was not recognized by antibodies to any FTR tested. When combined with spinach, (Spinacia oleracea L.) thylakoid membranes, the components of the FT system functioned in the light activation of the standard target enzymes from chloroplasts, corn (Zea mays L.) NADP-malate dehydrogenase (EC 1.1.1.82) and spinach fructose 1,6-bisphosphatase (EC 3.1.3.11) as well as the chloroplast-type fructose 1,6-bisphosphatase from Chlamydomonas. Activity was greatest if ferredoxin and other components of the FT system were from Chlamydomonas. The capacity of the Chlamydomonas FT system to activate autologous FBPase indicates that light regulates the photosynthetic carbon metabolism of green algae as in other oxygenic photosynthetic organisms.Abbreviations DEAE diethylaminoethyl - ELISA enzyme-linked immunosorption assay - FBPase fructose 1,6-bisphosphatase - Fd ferredoxin - FPLC fast protein liquid chromatography - FTR ferredoxin-thioredoxin reductase - FT system ferredoxin-thioredoxin system - kDa kilodaltons - M_r relative molecular mass - NADP-MDH NADP-malate dehydrogenase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work was supported in part by a grant from the National Aeronautics and Space Administration. We would like to thank Don Carlson and Jacqueline Girard for their assistance with cell cultures.     

E292 is important for the aminoacylation activity of Escherichia coli leucyl-tRNA synthetase

Escherichia coli leucyl-tRNA synthetase (LeuRS) has a large connecting polypeptide (CP1) inserted into its active site. It was demonstrated that the peptide bond between E292–A293 was crucial for the aminoacylation activity of E. coli LeuRS. To investigate the effect of E292 on the function of Escherichia coli LeuRS, E292 was mutated to K, F, S, D, Q and A. These mutations at 292 did not change the specific activity of the amino acid activation reaction. Though the conformational change of these mutants was not detected in CD, their aminoacylation activities were impaired to varying extents. The mutation of E to K decreased the aminoacylation activity to the largest extent. Analysis of the K_m values of these mutants for the three substrates showed that the E292 was not involved in the binding of leucine and that all mutants had stronger binding with ATP.     

Characterization of ferredoxin:thioredoxin reductase modified by site-directed mutagenesis

Ferredoxin:thioredoxin reductase (FTR) is a key regulatory enzyme of oxygenic photosynthetic cells involved in the reductive regulation of important target enzymes. It catalyzes the two-electron reduction of the disulfide of thioredoxins with electrons from ferredoxin involving a 4Fe-4S cluster and an adjacent active-site disulfide. We replaced Cys-57, Cys-87, and His-86 in the active site of Synechocystis FTR by site-directed mutagenesis and studied the properties of the mutated proteins. Mutation of either of the active-site cysteines yields inactive enzymes, which have different spectral properties, indicating a reduced Fe-S cluster when the inaccessible Cys-87 is replaced and an oxidized cluster when the accessible Cys-57 is replaced. The oxidized cluster in the latter mutant can be reversibly reduced with dithionite showing that it is functional. The C57S mutant is a very stable protein, whereas the C87A mutant is more labile because of the missing interaction with the cluster. The replacement of His-86 greatly reduces its catalytic activity supporting the proposal that His-86 increases the nucleophilicity of the neighboring cysteine. Ferredoxin forms non-covalent complexes with wild type (WT) and mutant FTRs, which are stable except with the C87A mutant. WT and mutant FTRs form stable covalent heteroduplexes with active-site modified thioredoxins. In particular, heteroduplexes formed with WT FTR represent interesting one-electron-reduced reaction intermediates, which can be split by reduction of the Fe-S cluster. Heteroduplexes form non-covalent complexes with ferredoxin demonstrating the ability of FTR to simultaneously dock thioredoxin and ferredoxin, which is in accord with the proposed reaction mechanism and the structural analyses.     

Ferredoxin/thioredoxin system plays an important role in the chloroplastic NADP status of Arabidopsis

NADP is a key electron carrier for a broad spectrum of redox reactions, including photosynthesis. Hence, chloroplastic NADP status, as represented by redox status (ratio of NADPH to NADP⁺) and pool size (sum of NADPH and NADP⁺), is critical for homeostasis in photosynthetic cells. However, the mechanisms and molecules that regulate NADP status in chloroplasts remain largely unknown. We have now characterized an Arabidopsis mutant with imbalanced NADP status (inap1), which exhibits a high NADPH/NADP⁺ ratio and large NADP pool size. inap1 is a point mutation in At2g04700, which encodes the catalytic subunit of ferredoxin/thioredoxin reductase. Upon illumination, inap1 demonstrated earlier increases in NADP pool size than the wild type did. The mutated enzyme was also found in vitro to inefficiently reduce m‐type thioredoxin, which activates Calvin cycle enzymes, and NADP‐dependent malate dehydrogenase to export reducing power to the cytosol. Accordingly, Calvin cycle metabolites and amino acids diminished in inap1 plants. In addition, inap1 plants barely activate NADP‐malate dehydrogenase, and have an altered redox balance between the chloroplast and cytosol, resulting in inefficient nitrate reduction. Finally, mutants deficient in m‐type thioredoxin exhibited similar light‐dependent NADP dynamics as inap1. Collectively, the data suggest that defects in ferredoxin/thioredoxin reductase and m‐type thioredoxin decrease the consumption of NADPH, leading to a high NADPH/NADP⁺ ratio and large NADP pool size. The data also suggest that the fate of NADPH is an important influence on NADP pool size.     

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.     

Heterogeneous photochemical and chloroplasts mediated activation of spinach fructose-1,6-bisphosphatase

A heterogeneous photochemical electron relay system was constructed, mimicking the chloroplast electron transport reaction, in order to activate fructose-1,6-bisphosphatase in light. The photocatalyst acridine orange or proflavin sensitizes EDTA dependent reduction of ferredoxin. In a complete system, consisting of a dye-donor couple, ferredoxin, thioredoxin and ferredoxin-thioredoxin reductase, light activation of purified spinach fructose-1,6-bisphosphatase was observed in vitro. The ferredoxin was not essential for activation of fructose-1,6-bisphosphatase using heterogeneous photochemical system while chloroplasts mediated redox activation essentially required ferredoxin. The heterogeneous photochemical system activated fructose-1,6-bisphosphatase by about 6 fold similar to chloroplasts mediated ferredoxin dependent redox activation. These observations suggest that a thiol mediator is essential for the reductive activation of carboxylating enzymes of photosynthesis. The mechanism of activation is discussed.     

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.     

Multi‐level regulation of the chloroplast ATP synthase: the chloroplast NADPH thioredoxin reductase C (NTRC) is required for redox modulation specifically under low irradiance

The chloroplast ATP synthase is known to be regulated by redox modulation of a disulfide bridge on the γ‐subunit through the ferredoxin–thioredoxin regulatory system. We show that a second enzyme, the recently identified chloroplast NADPH thioredoxin reductase C (NTRC), plays a role specifically at low irradiance. Arabidopsis mutants lacking NTRC (ntrc) displayed a striking photosynthetic phenotype in which feedback regulation of the light reactions was strongly activated at low light, but returned to wild‐type levels as irradiance was increased. This effect was caused by an altered redox state of the γ‐subunit under low, but not high, light. The low light‐specific decrease in ATP synthase activity in ntrc resulted in a buildup of the thylakoid proton motive force with subsequent activation of non‐photochemical quenching and downregulation of linear electron flow. We conclude that NTRC provides redox modulation at low light using the relatively oxidizing substrate NADPH, whereas the canonical ferredoxin–thioredoxin system can take over at higher light, when reduced ferredoxin can accumulate. Based on these results, we reassess previous models for ATP synthase regulation and propose that NTRC is most likely regulated by light. We also find that ntrc is highly sensitive to rapidly changing light intensities that probably do not involve the chloroplast ATP synthase, implicating this system in multiple photosynthetic processes, particularly under fluctuating environmental conditions.     

Structural Basis of Redox Signaling in Photosynthesis: Structure and Function of Ferredoxin:thioredoxin Reductase and Target Enzymes

The role of the ferredoxin:thioredoxin system in the reversible light activation of chloroplast enzymes by thiol-disulfide interchange with thioredoxins is now well established. Recent fruitful collaboration between biochemists and structural biologists, reflected by the shared authorship of the paper, allowed to solve the structures of all of the components of the system, including several target enzymes, thus providing a structural basis for the elucidation of the activation mechanism at a molecular level. In the present Review, these structural data are analyzed in conjunction with the information that was obtained previously through biochemical and site-directed mutagenesis approaches. The unique 4Fe-4S cluster enzyme ferredoxin:thioredoxin reductase (FTR) uses photosynthetically reduced ferredoxin as an electron donor to reduce the disulfide bridge of different thioredoxin isoforms. Thioredoxins in turn reduce regulatory disulfides of various target enzymes. This process triggers conformational changes on these enzymes, allowing them to reach optimal activity. No common activation mechanism can be put forward for these enzymes, as every thioredoxin-regulated protein undergoes specific structural modifications. It is thus important to solve the structures of the individual target enzymes in order to fully understand the molecular mechanism of the redox regulation of each of them.     

Photoactivation of NADP-Malate Dehydrogenase by a Heterogeneous Photochemical System in Zea mays

A heterogeneous photochemical electron relay system was constructed, mimicking the chloroplast electron transport reaction in order to activate the NADP-malate dehydrogenase in light. The photocatalyst acridine orange or proflavin sensitized EDTA-dependent reduction of ferredoxin. In a complete system, consisting of a dye donor couple, ferredoxin, thioredoxin and ferredoxin-thioredoxin reductase, light activation of purified NADP-MDH was observed in vitro. The chloroplast mediated redox activation of enzyme essentially required ferredoxin, while heterogeneous photochemical mediated activation of enzyme need not require ferredoxin. The heterogeneus photochemical system activated NADP-MDH by eight fold similar to chloroplasts mediated ferredoxin dependent redox activation but was not affected by the presence of disalicylinden propanediamine-1, 2-disulphonic acid while there was complete inhibition of chloroplasts mediated activation of NADP-MDH in presence of this inhibitor. These observations suggest that a thiol mediator is essential for reductive activation of NADP-MDH and ferredoxin is not required for photochemical activation.     

Human thioredoxin reactivity-structure/function relationship

The reactivity of human thioredoxin (HTR) was tested in several reactions. HTR was as efficient as E. coli or plant and algal thioredoxins when assayed with E. coli ribonucleotide reductase or for the reduction of insulin. On the other hand, HTR was poorly reduced by NADPH and the E. coli flavoenzyme NADPH thioredoxin reductase as monitored in the DTNB reduction test. When reduced with dithiothreitol (DTT), HTR was much less efficient than thioredoxin m and thioredoxin f, the respective specific thioredoxins for the chloroplast enzymes NADP-malate dehydrogenase (NADP-MDH) and fructose 1,6 bisphosphatase (FBPase). Finally, HTR could be used in the photoactivation of NADP-MDH although less efficiently than thioredoxin m, proving nevertheless that it can be reduced by the iron sulfur enzyme ferredoxin thioredoxin reductase in the presence of photoreduced ferredoxin. Based on sequence comparisons, it was expected that HTR would display a reactivity similar to chloroplast thioredoxin f rather than to thioredoxin m. However the observed behavior of FTR did not exactly fit this prediction. The results are discussed in relation to the structural data available for the proteins.     

Oxidation-reduction properties of thioredoxins and thioredoxin-regulated enzymes

Oxidation-reduction midpoint potentials have been measured for the two chloroplast thioredoxins, thioredoxin f and m , for ferredoxin:thioredoxin reductase (FTR) and for the thioredoxin-regulated enzymes fructose-1,6-bisphosphatase (FBPase), phosphoribulokinase and NADP-malate dehydrogenase. The effects of pH on the midpoint potentials of these chloroplast proteins have been measured so that the effect of the light-induced increase in chloroplast stromal pH on the redox properties of the proteins can be calculated. Spectroscopic measurements on FTR and on an N-ethylmaleimide-modified derivative of the enzyme have been used to elucidate the role of the [4Fe-4S] cluster of FTR during the reduction of the enzyme's active-site disulfide by ferredoxin.     

Site-specific mutations in the D1 polypeptide affect the susceptibility of Synechocystis 6803 cells to photoinhibition

Photoinhibition of photosystem II in the cyanobacterium Synechocystis 6803 was followed after site-specific mutagenesis of the D1 polypeptide. Mutations were created in the stromal/cytosolic loop connecting helices D and E. Two mutations E243K and CA1, a deletion of the three glutamates 242–244 and a substitution Q241H, were made in the putative cleavage area of the D1 polypeptide. A third mutation E229D was made in the PEST-like sequence. Mutants and control cells were illuminated and F_V/F_M was recorded. Compared to the control, the mutants were less photoinhibited. Fluorescence relaxation after a single flash was delayed in CA1. Restoration of F_V/F_M after photoinhibition in the mutants was totally dependent on protein synthesis while control cells were able to recover partially also when protein synthesis was inhibited. In addition, the protein synthesis-dependent recovery of CA1 was slowed down. Our results indicate a correlation between the mutated amino acids and photoinhibition of photosystem II.     

Unexpected diversity of ferredoxin-dependent thioredoxin reductases in cyanobacteria

Thioredoxin reductases control the redox state of thioredoxins (Trxs)—ubiquitous proteins that regulate a spectrum of enzymes by dithiol–disulfide exchange reactions. In most organisms, Trx is reduced by NADPH via a thioredoxin reductase flavoenzyme (NTR), but in oxygenic photosynthetic organisms, this function can also be performed by an iron-sulfur ferredoxin (Fdx)-dependent thioredoxin reductase (FTR) that links light to metabolic regulation. We have recently found that some cyanobacteria, such as the thylakoid-less Gloeobacter and the ocean-dwelling green oxyphotobacterium Prochlorococcus, lack NTR and FTR but contain a thioredoxin reductase flavoenzyme (formerly tentatively called deeply-rooted thioredoxin reductase or DTR), whose electron donor remained undefined. Here, we demonstrate that Fdx functions in this capacity and report the crystallographic structure of the transient complex between the plant-type Fdx1 and the thioredoxin reductase flavoenzyme from Gloeobacter violaceus. Thereby, our data demonstrate that this cyanobacterial enzyme belongs to the Fdx flavin-thioredoxin reductase (FFTR) family, originally described in the anaerobic bacterium Clostridium pasteurianum. Accordingly, the enzyme hitherto termed DTR is renamed FFTR. Our experiments further show that the redox-sensitive peptide CP12 is modulated in vitro by the FFTR/Trx system, demonstrating that FFTR functionally substitutes for FTR in light-linked enzyme regulation in Gloeobacter. Altogether, we demonstrate the FFTR is spread within the cyanobacteria phylum and propose that, by substituting for FTR, it connects the reduction of target proteins to photosynthesis. Besides, the results indicate that FFTR acquisition constitutes a mechanism of evolutionary adaptation in marine phytoplankton such as Prochlorococcus that live in low-iron environments.     

Contrasting modes of photosynthetic enzyme regulation in oxygenic and anoxygenic prokaryotes

Enzymes that are regulated by the ferredoxin/thioredoxin system in chloroplasts — fructose-1,6-bisphosphatase (FBPase), sedoheptulose-1,7-bisphosphatase purified from two different types of photosynthetic prokaryotes (cyanobacteria, purple sulfur bacteria) and tested for a response to thioredoxins. Each of the enzymes from the cyanobacterium Nostoc muscorum, an oxygenic organism known to contain the ferredoxin/thioredoxin system, was activated by thioredoxins that had been reduced either chemically by dithiothreitol or photochemically by reduced ferredoxin and ferredoxin-thioredoxin reductase. Like their chloroplast counterparts, N. muscorum FBPase and SBPase were activated preferentially by reduced thioredoxin f. SBPase was also partially activated by thioredoxin m. PRK, which was present in two regulatory forms in N. muscorum, was activated similarly by thioredoxins f and m. Despite sharing the capacity for regulation by thioredoxins, the cyanobacterial FBPase and SBPase target enzymes differed antigenically from their chloroplast counterparts. The corresponding enzymes from Chromatium vinosum, an anoxygenic photosynthetic purple bacterium found recently to contain the NADP/thioredoxin sytem, differed from both those of cyanobacteria and chloroplasts in showing no response to reduced thioredoxin. Instead, C. vinosum FBPase, SBPase, and PRK activities were regulated by a metabolite effector, 5-AMP. The evidence is in accord with the conclusion that thioredoxins function in regulating the reductive pentose phosphate cycle in oxygenic prokaryotes (cyanobacteria) that contain the ferredoxin/thioredoxin system, but not in anoxygenic prokaryotes (photosynthetic purple bacteria) that contain the NADP/thioredoxin system. In organisms of the latter type, enzyme effectors seem to play a dominant role in regulating photosynthetic carbon dioxide assimilation.     

Chloroplast phenylalanine ammonia-lyase from spinach leaves

Phenylalanine ammonia-lyase (PAL) from spinach (Spinacia oleracea L.) leaves was resolved into three forms by diethyl-aminoethyl(DEAE)-cellulose chromatography. Two forms were found in isolated chloroplasts, and the third form (the major component) was located outside of the chloroplasts. One of the chloroplast forms of the enzyme (designated the regulatory form) was activated by reduced thioredoxin. Neither the other chloroplast form nor the extra-chloroplast form showed a response to thioredoxin. After further purification by hydroxyapatite column chromatography and gel filtration, the regulatory form of chloroplast PAL was stimulated approximately 3-fold by thioredoxin reduced either photochemically by chloroplast membranes, via ferredoxin and ferredoxin-thioredoxin reductase, or chemically by dithiothreitol. Once activated, the enzyme required an added oxidant for deactivation. Physiological oxidants-oxidized glutathione (GSSG) and dehydroascorbate-as well as nonphysiological oxidants-sodium tetrathionate and diamide-were effective in deactivation. The results indicate that chloroplast PAL is regulated by light via the ferredoxin/thioredoxin system in a manner similar to that described for regulatory enzymes of CO₂ assimilation. The extra-chloroplast form of the enzyme, by contrast, appears to be regulated by light via the earlier-described phytochrome-linked system.     

Protein modulase appears to be a complex of ferredoxin, ferredoxin/thioredoxin reductase, and thioredoxin

Protein modulase and ferredoxin/thioredoxin reductase are soluble proteins that have been suggested to catalyze the light-dependent modulation of enzyme activity in the stromal compartment of the chloroplast. Protein modulase is active in vitro without additional ferredoxin and thioredoxin, whereas ferredoxin/thioredoxin reductase requires additional ferredoxin and thioredoxin. We hypothesize that protein modulase is a complex protein composed of ferredoxin/thioredoxin reductase, ferredoxin, and thioredoxin. In reconstituted chloroplast systems, antiserum directed against ferredoxin, at concentrations sufficient to inhibit the photoreduction of NADP, had no effect on light modulation. Antiserum directed against thioredoxin gave variable results: one batch of polyclonal antibodies inhibited light modulation, another was stimulatory, and another was without effect. These results suggest that the ferredoxin and thioredoxin active in light modulation are not free in solution. Furthermore, molecular sieve chromatography of stromal proteins results in the elution of four species that catalyze light modulation. Based on whether or not ferredoxin and/or thioredoxin must be added for activity, these four species have been tentatively identified as protein modulase, a complex of ferredoxin/thioredoxin reductase and ferredoxin, a complex of ferredoxin/thioredoxin reductase and thioredoxin, and ferredoxin/thioredoxin reductase. That is, the four correspond to all the possible combinations of ferredoxin, ferredoxin/thioredoxin reductase, and thioredoxin. We suggest that buffer ionic strength affects the interactions among these proteins and in part determines the fate of the protein modulase complex in vitro.