Redox Signaling in Chloroplasts: Cleavage of Disulfides by an Iron-Sulfur Cluster

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Science  28 Jan 2000:
Vol. 287, Issue 5453, pp. 655-658
DOI: 10.1126/science.287.5453.655


Light generates reducing equivalents in chloroplasts that are used not only for carbon reduction, but also for the regulation of the activity of chloroplast enzymes by reduction of regulatory disulfides via the ferredoxin:thioredoxin reductase (FTR) system. FTR, the key electron/thiol transducer enzyme in this pathway, is unique in that it can reduce disulfides by an iron-sulfur cluster, a property that is explained by the tight contact of its active-site disulfide and the iron-sulfur center. The thin, flat FTR molecule makes the two-electron reduction possible by forming on one side a mixed disulfide with thioredoxin and by providing on the opposite side access to ferredoxin for delivering electrons.

Redox signaling and regulation has become an area of increasing interest given that transcription, translation, apoptosis, and enzymatic activity can be regulated in this way (1). This type of regulation was first described for the activation of chloroplast enzymes by light through the reduction of disulfides, thereby changing the metabolism to become anabolic (2). Thioredoxins are the key transmitters of reducing equivalents to target enzymes.

The photosynthetic machinery in plants and other photosynthetic organisms produces reducing equivalents; e.g., nicotinamide adenine dinucleotide phosphate, reduced (NADPH), in the light reactions that together with the generated adenosine 5′-triphosphate are necessary to reduce CO2 to carbohydrates (2). Plants satisfy their energy needs through the light reactions of photosynthesis during light periods. The situation is quite different in the dark, when the plant must use normal catabolic processes as do nonphotosynthetic organisms. The stroma in the chloroplasts contains both assimilatory enzymes of the Calvin cycle and dissimilatory enzymes, which implies that there must be a light-sensitive control to balance these reactions.

One such regulatory mechanism senses the light-dependent redox potential changes of the stroma and translates them into signals that, in the light, stimulate the Calvin cycle and deactivate degradative pathways (2, 3). The enzyme ferredoxin:thioredoxin reductase (FTR) is a key component of this system. During light reactions photosystem I reduces ferredoxin, which is used by NADP+:ferredoxin reductase to produce NADPH for the carbon reduction. The reduced ferredoxin is also used by FTR to produce reduced thioredoxins, which activate fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase, and phosphoribulokinase of the Calvin cycle as well as other chloroplast enzymes by disulfide reduction (3). Recently the structures of several members of this regulatory chain have been determined (Fig. 1). Here we report the structure of the last missing member of the chain, FTR. This enzyme is the central actor that transforms the electron signal received from ferredoxin to a thiol signal that is transmitted to thioredoxin. The results provide the structural framework for this mechanism.

Figure 1

FTR has a key position in light-induced enzyme regulation in chloroplasts. Upon illumination, the photosynthetic electron transfer chain reduces ferredoxin (Fd) (25) by photosystem I (8). Ferredoxin can then reduce ferredoxin:thioredoxin reductase (FTR), which reduces the chloroplast thioredoxins m and f (Trx m and Trx f) (26). Finally, the thioredoxins activate (in some cases deactivate) target enzymes, thereby switching the metabolism to anabolic pathways. Arrows indicate the flow of electrons. The molecular basis for light activation of the target enzyme malate dehydrogenase from C4-plants has recently been revealed from crystal structures (27).

FTR is a unique enzyme, completely different from the bacterial and mammalian thioredoxin reductases, which are flavoproteins that use NADPH as reductant. FTR is an iron-sulfur enzyme, an αβ heterodimer composed of a catalytic β subunit of 13 kD with conserved sequence between species and a variable α subunit of similar size. The β subunit contains a redox-active disulfide and a [4Fe-4S] center. Most biochemical investigations have been done on the spinach enzyme, for which a careful study of the iron-sulfur center has been performed (4). We have determined the structure of FTR fromSynechocystis sp. PCC6803. The Synechocystis FTR shows no functional difference from the spinach enzyme, but it is significantly more stable and can be obtained in larger amounts (5). The structure of the oxidized FTR was determined by multiple isomorphous replacement (MIR) with the help of multiwavelength anomalous dispersion (MAD) data collected on the iron edge for the iron-sulfur center. The structure has been refined at 1.6 Å resolution (6).

The variable chain in FTR has an open β-barrel structure containing five antiparallel strands (Fig. 2A). Two loops between the β strands dominate the interaction area with the catalytic subunit. The structure of the variable chain of FTR is remarkably similar to that of the PsaE protein, which is a stromal subunit of photosystem I (7, 8). Two-thirds of the variable chain (53 Cα atoms of the two proteins) can be superimposed with a root-mean-square (rms) fit of 1.3 Å. The strands of the barrel are very similar in the two structures, while the loops differ. The similarities of the variable subunit of FTR and PsaE are not reflected in sequence similarities nor in functional similarities. The proteins have no common ferredoxin binding site, which might have been anticipated. All conserved residues in the variable chain are either internal or glycines that are conserved for structural reasons, and residues in the interaction area with the catalytic subunit.

Figure 2

(A) Modeling of the electron transport chain from ferredoxin to thioredoxin. FTR is an unusually thin molecule, a concave disk with dimensions 40 Å by 50 Å but only 10 Å across the center of the molecule where the iron-sulfur cluster is located. The disk-shaped structure of the FTR allows docking of a ferredoxin on one side of the molecule (red, to the left), while thioredoxin binds to the other side and forms a heterodisulfide with the enzyme (yellow, to the right). This intermediate can be reduced by an electron from a second ferredoxin molecule. The iron-sulfur centers and disulfide bridges are shown in ball-and-stick representation. The catalytic subunit of FTR (blue) has an overall α-helical structure with loops between the helices containing the iron-sulfur ligands and redox-active cysteines. The variable subunit (green) is heart shaped with the β barrel forming the main body and with two loops forming the upper, outer parts of the heart. (B) Stereo view of the active site of FTR. The irons of the iron-sulfur center are coordinated by cysteines 55, 74, 76, and 85 in a normal cubane-type geometry. The active-site disulfide bridge between residues 57 and 87 is in van der Waals contact with the iron center. The sulfur atom of Cys87 contacts the iron atom bound by Cys55 and the sulfur atom of Cys55, both of which are at 3.1 Å distance. The closest sulfide ion of the cluster is 3.5 Å away from Cys87. Besides the active-site cysteines, Pro75 is also shown in ball-and-stick models. Incoming electrons can pass from ferredoxin onto the main chain of Cys74 to the disulfide bridge by way of the iron center. The oxygen atom in the cis-peptide bond between Cys74 and Pro75 is labeled.

The catalytic subunit has an overall α-helical structure with loops between the helices containing the iron-sulfur ligands and redox-active cysteines (Fig. 2A). The NH2-terminal half of the subunit, together with the COOH-terminal residues, forms an α-helical cap on top of the iron-sulfur center, while the intervening 40 residues contain all the iron ligands and redox-active cysteines. This part contains two additional short helices and intervening loops.

The interaction between the catalytic and variable chains involves the very thin center of the molecule where only a small hydrophobic core is formed. However, most of the interactions between the subunits occur between charged and polar residues that form hydrogen bonds. Practically all residues in the catalytic subunit that participate in subunit interactions are strictly conserved. One important function of the variable subunit seems to be the stabilization of the Fe-S cluster, which might ensue from the observation that most residues involved in subunit interaction are conserved. We have seen that upon treatment of FTR with increasing concentrations of urea or guanidine-HCl, the color of the FTR disappears when the FTR dissociates into subunits. Production of only recombinant catalytic subunit yields neither colored nor active protein.

The irons of the iron-sulfur center (Fig. 2B) are coordinated by cysteines 55, 74, 76, and 85 in a normal cubane-type geometry (9). The sulfur atoms of the liganding cysteines are hydrogen-bonded to main chain nitrogen atoms (10). None of the sulfide ions have a hydrogen bond to a protein atom. The iron center is surrounded exclusively by hydrophobic residues, all coming from the catalytic subunit.

The sequence fingerprints for FTRs differ from those for other iron-sulfur proteins where the liganding cysteines are separated by at least two residues. The common binding motif for [4Fe-4S] clusters, the CXXC motif (11), is absent in FTR. Rather, all ligands are located in short sequence motifs CXC, which is a unique arrangement with the fingerprintCXCx16 CXCx8 CXC (cluster ligands in bold type). Both cysteines in the central CPC motif are ligands to the iron-sulfur center. In the other two motifs, the liganding cysteines are connected to the redox-active cysteines in a CPC and a CHC motif. The latter cysteines in these motifs are forming the disulfide bridge. The active-site disulfide bridge between residues 57 and 87 is in van der Waals contact with the iron center; primarily, the sulfur atom of Cys87 contacts the iron atom bound by Cys55. Cys57 is at the molecular surface and should be the nucleophile in thiol-disulfide exchange reactions during thioredoxin reduction. His86 is very close to the disulfide bridge and might increase the nucleophilicity of the cysteine.

In analogy to known mechanisms, the reduction of thioredoxin has been proposed to proceed by way of a mixed disulfide bond between thioredoxin and FTR (4). Such an intermediate complex would cover one of the sides of the flat FTR molecule, and the second electron for the reduction should be delivered by the next incoming ferredoxin, which has to dock on the opposite side of the flat, disklike heterodimer. The structure of the FTR dimer suggests that the two separate essential interaction surfaces for ferredoxin and thioredoxin involve both FTR subunits. The FTR heterodimer is an unusually thin molecule, a concave disk with only 10 Å across the center of the molecule where the iron-sulfur center is located (Fig. 2A). One side of the iron-sulfur center is covered by the redox-active disulfide that reduces thioredoxin in a thiol-disulfide exchange reaction. FTR (as PsaE) is structurally similar to the SH3 domains, and the peptide binding site of the SH3 domain corresponds in FTR to a part of the binding site for thioredoxin. The opposite side of the disk has shape complementarity to the ferredoxin molecule (12).

The FTR molecule seems ideally suited for electron transfer between ferredoxin and thioredoxin (Fig. 2A). The iron-sulfur ligands on the side of the disk-shaped FTR molecule that is complementary to ferredoxin are connected by Pro75 (the only cis-proline in the structure) in one CPC motif. The main chain of this motif is exposed toward the ferredoxin side and provides excellent candidates for through-bond electron transfer from the bound ferredoxin to the iron-sulfur center (Fig. 2B).

All other biological disulfide reactions occur by means of flavoproteins or thiol-disulfide exchange reactions. The close proximity of iron-sulfur cluster and disulfide is evidently a prerequisite for FTR's unique property of being able to reduce the disulfide by an iron-sulfur center. The disulfide that is adjacent to one iron atom of the cluster can pick up an electron delivered by ferredoxin to the iron-sulfur cluster of FTR but it can also attract one additional electron from the iron-sulfur center to break the disulfide bond. One of the cysteines, Cys57, becomes then the reactive thiol, while the second cysteine thiol is protected by binding to the iron-sulfur cluster. Therefore, the one-electron reduction of FTR by ferredoxin surprisingly results in an oxidation of the [4Fe-4S]2+ cluster to an [4Fe-4S]3+cluster (4) (Fig. 3). Two structures for this intermediate stage have been suggested: with Cys87covalently attached to the cluster either through an Fe atom or a sulfide ion (4). Staples et al. (4) favor an intermediate disulfide of Cys87 with the sulfide ion. The tight interaction by Cys87 with one of the irons of the cluster in the FTR structure instead implicates that Cys87 coordinates the iron in a five-coordinated cluster (Fig. 3). Although there is no example to our knowledge of a cysteine-sulfide bridge in an iron-sulfur cluster, there is some precedence for this type of pentacoordinated iron given that five-coordinated subsites of [4Fe-4S] clusters have been synthesized (13). Higher coordination of iron in iron-sulfur clusters occurs also for aconitase-substrate complexes (14). Furthermore, five-coordinated iron clusters show modified redox potentials (13) analogous to those found for FTR (4). For the FTR intermediate, the redox potential of the [4Fe-4S]3+,2+ couple is lowered from +420 to −210 mV, which is in the same region as the redox potential for the active-site disulfide (4, 5,15). A similar lowering of redox potential by about 300 to 700 mV is observed for five-coordinated iron clusters compared with four-coordinated iron clusters (13).

Figure 3

The proposed mechanism of action of ferredoxin:thioredoxin reductase. Modified from (4).

The one-electron reduced intermediate, with its nucleophilic thiol Cys57, is able to attack the disulfide bridge of thioredoxin to form a hetero-disulfide. The next electron delivered by a new ferredoxin molecule reduces the iron-sulfur center back to its original oxidation state, thereby reducing the disulfide bridge between FTR and thioredoxin and releasing the fully reduced thioredoxin. Such a mechanism, which requires the simultaneous docking of thioredoxin and ferredoxin, is entirely compatible with the disk-shaped structure of FTR, which would allow docking of a second ferredoxin on one side of the molecule, while thioredoxin is bound to the other side by way of the intermolecular disulfide bridge (Fig. 2A).

Note added in proof: Recently, the structure of the redox-regulated target enzyme fructose-1,6-bisphosphate phosphatase was published (28).

  • * Present address: Department of Biological Sciences, 1392 Lilly Hall of Life Sciences, Purdue University, West Lafayette, IN 47907, USA.

  • Present address: Department of Biochemistry, University of Iowa, 51 Newton Road, Iowa City, IA 52242–1109, USA.

  • To whom correspondence should be addressed. E-mail: hasse{at}


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