Identification of the ferredoxin interaction sites on ferredoxin-dependent glutamate synthase from Synechocystis sp. PCC 6803.

Based on in silico docking methods, five amino acids in glutamate synthase (Gln-467, His-1144, Asn-1147, Arg-1162, and Trp-676) likely constitute key binding residues in the interface of a glutamate synthase:ferredoxin complex. Although all interfacial mutants studied showed the ability to form a complex under low ionic strength, these docking mutations showed significantly less ferredoxin-dependent activities, while still retaining enzymatic activity. Furthermore, isothermal titration calorimetry showed a possible 1:2 molar ratio between the wild-type glutamate synthase and ferredoxin. However, each of our interfacial mutants showed only a 1:1 complex with ferredoxin, suggesting that the mutations directly affect the glutamate synthase:ferredoxin heterodimer interface.

Identification of Amino Acids at the Catalytic Site of a Ferredoxin-Dependent Cyanobacterial Nitrate Reductase.

An in silico model of the ferredoxin-dependent nitrate reductase from the cyanobacterium Synechococcus sp. PCC 7942, and information about active sites in related enzymes, had identified Cys148, Met149, Met306, Asp163, and Arg351 as amino acids likely to be involved in either nitrate binding, prosthetic group binding, or catalysis. Site-directed mutagenesis was used to alter each of these residues, and differences in enzyme activity and substrate binding of the purified variants were analyzed. In addition, the effects of these replacements on the assembly and properties of the Mo cofactor and [4Fe-4S] centers were investigated using Mo and Fe determinations, coupled with electron paramagnetic resonance spectroscopy. The C148A, M149A, M306A, D163N, and R351Q variants were all inactive with either the physiological electron donor, reduced ferredoxin, or the nonphysiological electron donor, reduced methyl viologen, as the source of electrons, and all exhibited changes in the properties of the Mo cofactor. Charge-conserving D163E and R351K variants were also inactive, suggesting that specific amino acids are required at these two positions. The implications for the role of these five conserved active-site residues in light of these new results and previous structural, spectroscopic, and mutagenesis studies for related periplasmic nitrate reductases are discussed.

A loop unique to ferredoxin-dependent glutamate synthases is not absolutely essential for ferredoxin-dependent catalytic activity.

It had been proposed that a loop, typically containing 26 or 27 amino acids, which is only present in monomeric, ferredoxin-dependent, "plant-type" glutamate synthases and is absent from the catalytic α-subunits of both NADPH-dependent, heterodimeric glutamate synthases found in non-photosynthetic bacteria and NADH-dependent heterodimeric cyanobacterial glutamate synthases, plays a key role in productive binding of ferredoxin to the plant-type enzymes. Site-directed mutagenesis has been used to delete the entire 27 amino acid-long loop in the ferredoxin-dependent glutamate synthase from the cyanobacterium Synechocystis sp. PCC 6803. The specific activity of the resulting loopless variant of this glutamate synthase, when reduced ferredoxin serves as the electron donor, is actually higher than that of the wild-type enzyme, suggesting that this loop is not absolutely essential for efficient electron transfer from reduced ferredoxin to the enzyme. These results are consistent with the results of an in-silico study that suggests that the loop is unlikely to interact directly with ferredoxin in the energetically most favorable model of a 1:1 complex of ferredoxin with the wild-type enzyme.

Roles of four conserved basic amino acids in a ferredoxin-dependent cyanobacterial nitrate reductase.

The roles of four conserved basic amino acids in the reaction catalyzed by the ferredoxin-dependent nitrate reductase from the cyanobacterium Synechococcus sp. PCC 7942 have been investigated using site-directed mutagenesis in combination with measurements of steady-state kinetics, substrate-binding affinities, and spectroscopic properties of the enzyme's two prosthetic groups. Replacement of either Lys58 or Arg70 by glutamine leads to a complete loss of activity, both with the physiological electron donor, reduced ferredoxin, and with a nonphysiological electron donor, reduced methyl viologen. More conservative, charge-maintaining K58R and R70K variants were also completely inactive. Replacement of Lys130 by glutamine produced a variant that retained 26% of the wild-type activity with methyl viologen as the electron donor and 22% of the wild-type activity with ferredoxin as the electron donor, while replacement by arginine produces a variant that retains a significantly higher percentage of the wild-type activity with both electron donors. In contrast, replacement of Arg146 by glutamine had minimal effect on the activity of the enzyme. These results, along with substrate-binding and spectroscopic measurements, are discussed in terms of an in silico structural model for the enzyme.

In the absence of thioredoxins, what are the reductants for peroxiredoxins in Thermotoga maritima?

Three peroxiredoxins (Prxs) were identified in Thermotoga maritima, which possesses neither glutathione nor typical thioredoxins: one of the Prx6 class; one 2-Cys PrxBCP; and a unique hybrid protein containing an N-terminal 1-Cys PrxBCP domain fused to a flavin mononucleotide-containing nitroreductase (Ntr) domain. No peroxidase activity was detected for Prx6, whereas both bacterioferritin comigratory proteins (BCPs) were regenerated by a NADH/thioredoxin reductase/glutaredoxin (Grx)-like system, constituting a unique peroxide removal system. Only two of the three Grx-like proteins were able to support peroxidase activity. The inability of TmGrx1 to regenerate oxidized 2-Cys PrxBCP probably results from the thermodynamically unfavorable difference in their disulfide/dithiol E(m) values, -150 and -315 mV, respectively. Mutagenesis of the Prx-Ntr fusion, combined with kinetic and structural analyses, indicated that electrons are not transferred between its two domains. However, their separate activities could function in a complementary manner, with peroxide originating from the chromate reductase activity of the Ntr domain reduced by the Prx domain.

Structural and functional characterization of the ga-substituted ferredoxin from Synechocystis sp. PCC6803, a mimic of the native protein.

In photosynthetic organisms, ferredoxin (Fd) interacts with many proteins, acting as a shuttle for electrons from Photosystem I to a group of enzymes involved in NADP(+) reduction, sulfur and nitrogen assimilation, and the regulation of carbon assimilation. The study of the dynamic interactions between ferredoxin and these enzymes by nuclear magnetic resonance is severely hindered by the paramagnetic [2Fe-2S] cluster of a ferredoxin. To establish whether ferredoxin in which the cluster has been replaced by Ga is a suitable diamagnetic mimic, the solution structure of Synechocystis Ga-substituted ferredoxin has been determined and compared with the structure of the native protein. The ensemble of 10 structures with the lowest energies has an average root-mean-square deviation of 0.30 +/- 0.05 A for backbone atoms and 0.65 +/- 0.04 A for all heavy atoms. Comparison of the NMR structure of GaFd with the crystal structure of the native Fd indicates that the general structural fold found for the native, iron-containing ferredoxin is conserved in GaFd. The ferredoxin contains a single gallium and no inorganic sulfide. The distortion of the metal binding loop caused by the single gallium substitution is small. The binding site on Fd for binding ferredoxin:NADP(+) reductase in solution, determined using GaFd, includes the metal binding loop and its surroundings, consistent with the crystal structures of related complexes. The results provide a structural justification for the use of the gallium-substituted analogue in interaction studies.

Enzymatic properties of the ferredoxin-dependent nitrite reductase from Chlamydomonas reinhardtii. Evidence for hydroxylamine as a late intermediate in ammonia production.

The ferredoxin-dependent nitrite reductase from the green alga Chlamydomonas reinhardtii has been cloned, expressed in Escherichia coli as a His-tagged recombinant protein, and purified to homogeneity. The spectra, kinetic properties and substrate-binding parameters of the C. reinhardtii enzyme are quite similar to those of the ferredoxin-dependent spinach chloroplast nitrite reductase. Computer modeling, based on the published structure of spinach nitrite reductase, predicts that the structure of C. reinhardtii nitrite reductase will be similar to that of the spinach enzyme. Chemical modification studies and the ionic-strength dependence of the enzyme's ability to interact with ferredoxin are consistent with the involvement of arginine and lysine residues on C. reinhardtii nitrite reductase in electrostatically-stabilized binding to ferredoxin. The C. reinhardtii enzyme has been used to demonstrate that hydroxylamine can serve as an electron-accepting substrate for the enzyme and that the product of hydroxylamine reduction is ammonia, providing the first experimental evidence for the hypothesis that hydroxylamine, bound to the enzyme, can serve as a late intermediate during the reduction of nitrite to ammonia catalyzed by the enzyme.

Ternary protein complex of ferredoxin, ferredoxin:thioredoxin reductase, and thioredoxin studied by paramagnetic NMR spectroscopy.

In oxygenic photosynthetic cells, carbon metabolism is regulated by a light-dependent redox signaling pathway through which the light signal is transmitted in the form of electrons via a redox chain comprising ferredoxin (Fd), ferredoxin:thioredoxin reductase (FTR), and thioredoxin (Trx). Trx affects the activity of a variety of enzymes via dithiol oxidation and reduction reactions. FTR reduces an intramolecular disulfide bridge of Trx, and Trx reduction involves a transient cross-link with FTR. NMR spectroscopy was used to investigate the interaction of Fd, FTR, and an m-type Trx. NMR titration experiments indicate that FTR uses distinct sites to bind Fd and Trx simultaneously to form a noncovalent ternary complex. The orientation of Trx-m relative to FTR was determined from the intermolecular paramagnetic broadening caused by the [4Fe-4S] cluster of FTR. Two models of the noncovalent binary complex of FTR/Trx-m based on the paramagnetic distance restraints were obtained. The models suggest that either a modest or major rotational movement of Trx must take place when the noncovalent binary complex proceeds to the covalent complex. This study demonstrates the complementarity of paramagnetic NMR and X-ray diffraction of crystals in the elucidation of dynamics in a transient protein complex.

The interaction of spinach nitrite reductase with ferredoxin: a site-directed mutation study.

A series of site-directed mutants of the ferredoxin-dependent spinach nitrite reductase has been characterized and several amino acids have been identified that appear to be involved in the interaction of the enzyme with ferredoxin. In a complementary study, binding constants to nitrite reductase and steady-state kinetic parameters of site-directed mutants of ferredoxin were determined in an attempt to identify ferredoxin amino acids involved in the interaction with nitrite reductase. The results have been interpreted in terms of an in-silico docking model for the 1:1 complex of ferredoxin with nitrite reductase.

Interaction domain on thioredoxin for Pseudomonas aeruginosa 5'-adenylylsulfate reductase.

NMR spectroscopy has been used to map the interaction domain on Escherichia coli thioredoxin for the thioredoxin- dependent 5'-adenylylsulfate reductase from Pseudomonas aeruginosa (PaAPR). Seventeen thioredoxin amino acids, all clustered around Cys-32 (the more surface-exposed of the two active-site cysteines), have been located at the PaAPR binding site. The center of the binding domain is dominated by nonpolar amino acids, with a smaller number of charged and polar amino acids located on the periphery of the site. Twelve of the amino acids detected by NMR have non-polar, hydrophobic side chains, including one aromatic amino acid (Trp-31). Four of the thioredoxin amino acids at the PaAPR binding site have polar side chains (Lys-36, Asp-61, Gln-62 and Arg-73), with three of the four having charged side chains. Site-directed mutagenesis experiments have shown that replacement of Lys-36, Asp-61, and Arg-73 and of the absolutely conserved Trp-31 significantly decreases the V(max) for the PaAPR-catalyzed reduction of 5'-adenylylsulfate, with E. coli thioredoxin serving as the electron donor. The most dramatic effect was observed with the W31A variant, which showed no activity as a donor to PaAPR. Although the thiol of the active-site Cys-256 of PaAPR is the point of the initial nucleophilic attack by reduced thioredoxin, mutagenic replacement of Cys-256 by serine has no effect on thioredoxin binding to PaAPR.

Pattern of expression and substrate specificity of chloroplast ferredoxins from Chlamydomonas reinhardtii.

Ferredoxin (Fd) is the major iron-containing protein in photosynthetic organisms and is central to reductive metabolism in the chloroplast. The Chlamydomonas reinhardtii genome encodes six plant type [Fe2S2] ferredoxins, products of PETF, FDX2-FDX6. We performed the functional analysis of these ferredoxins by localizing Fd, Fdx2, Fdx3, and Fdx6 to the chloroplast by using isoform-specific antibodies and monitoring the pattern of gene expression by iron and copper nutrition, nitrogen source, and hydrogen peroxide stress. In addition, we also measured the midpoint redox potentials of Fd and Fdx2 and determined the kinetic parameters of their reactions with several ferredoxin-interacting proteins, namely nitrite reductase, Fd:NADP+ oxidoreductase, and Fd:thioredoxin reductase. We found that each of the FDX genes is differently regulated in response to changes in nutrient supply. Moreover, we show that Fdx2 (Em = -321 mV), whose expression is regulated by nitrate, is a more efficient electron donor to nitrite reductase relative to Fd. Overall, the results suggest that each ferredoxin isoform has substrate specificity and that the presence of multiple ferredoxin isoforms allows for the allocation of reducing power to specific metabolic pathways in the chloroplast under various growth conditions.

New insights into the catalytic cycle of plant nitrite reductase. Electron transfer kinetics and charge storage.

Nitrite reductase, which reduces nitrite to ammonium in a six-electron reaction, was characterized through kinetic analysis of an electron transfer cascade involving photoexcited Photosystem I and ferredoxin. This cascade was studied at physiological pH by flash-absorption spectroscopy. Two different forms of the enzyme were studied: one isolated from spinach leaf and one histidine-tagged recombinant form. When the enzyme is oxidized in the absence of nitrite, single-enzyme reduction leads mostly to siroheme reduction with the leaf enzyme, whereas the siroheme and the [4Fe-4S] cluster are both reduced in equivalent amounts in the recombinant enzyme. When combined with the results of deazaflavin/EDTA photoreduction experiments, these data support a 50 mV negative shift of the siroheme midpoint potential in the recombinant enzyme. Despite this difference, the two forms of the enzyme exhibit similar values for the rate constant of single reduction by reduced ferredoxin (1200 s(-1)) and for k(cat) (420-450 electrons per second and per nitrite reductase). When nitrite reductase is initially pre-reduced to the state ferrous siroheme-NO(*), the fast kinetics of reduction by ferredoxin and the thermodynamics of ferredoxin binding are equivalent to those observed with oxidized nitrite reductase without nitrite. Spectral and kinetic analyses of single reduction of the recombinant enzyme in the ferrous siroheme-NO(*) state by photoreduced ferredoxin reveal that this process leads to reduction of the [4Fe-4S] cluster with little, if any, NO(*) reduction. These data show that the enzyme must wait for the next reduction step before NO(*) undergoes substantial reduction.

A novel type of thioredoxin dedicated to symbiosis in legumes.

Thioredoxins (Trxs) constitute a family of small proteins in plants. This family has been extensively characterized in Arabidopsis (Arabidopsis thaliana), which contains six different Trx types: f, m, x, and y in chloroplasts, o in mitochondria, and h mainly in cytosol. A detailed study of this family in the model legume Medicago truncatula, realized here, has established the existence of two isoforms that do not belong to any of the types previously described. As no possible orthologs were further found in either rice (Oryza sativa) or poplar (Populus spp.), these novel isoforms may be specific for legumes. Nevertheless, on the basis of protein sequence and gene structure, they are both related to Trxs m and probably have evolved from Trxs m after the divergence of the higher plant families. They have redox potential values similar to those of the classical Trxs, and one of them can act as a substrate for the M. truncatula NADP-Trx reductase A. However, they differ from classical Trxs in that they possess an atypical putative catalytic site and lack disulfide reductase activity with insulin. Another important feature is the presence in both proteins of an N-terminal extension containing a putative signal peptide that targets them to the endoplasmic reticulum, as demonstrated by their transient expression in fusion with the green fluorescent protein in M. truncatula or Nicotiana benthamiana leaves. According to their pattern of expression, these novel isoforms function specifically in symbiotic interactions in legumes. They were therefore given the name of Trxs s, s for symbiosis.

An atypical catalytic mechanism involving three cysteines of thioredoxin.

Unlike other thioredoxins h characterized so far, a poplar thioredoxin of the h type, PtTrxh4, is reduced by glutathione and glutaredoxin (Grx) but not NADPH:thioredoxin reductase (NTR). PtTrxh4 contains three cysteines: one localized in an N-terminal extension (Cys(4)) and two (Cys(58) and Cys(61)) in the classical thioredoxin active site ((57)WCGPC(61)). The property of a mutant in which Cys(58) was replaced by serine demonstrates that it is responsible for the initial nucleophilic attack during the catalytic cycle. The observation that the C4S mutant is inactive in the presence of Grx but fully active when dithiothreitol is used as a reductant indicates that Cys(4) is required for the regeneration of PtTrxh4 by Grx. Biochemical and x-ray crystallographic studies indicate that two intramolecular disulfide bonds involving Cys(58) can be formed, linking it to either Cys(61) or Cys(4). We propose thus a four-step disulfide cascade mechanism involving the transient glutathionylation of Cys(4) to convert this atypical thioredoxin h back to its active reduced form.

A role for cytochrome c and cytochrome c peroxidase in electron shuttling from Erv1.

Erv1 is a flavin-dependent sulfhydryl oxidase in the mitochondrial intermembrane space (IMS) that functions in the import of cysteine-rich proteins. Redox titrations of recombinant Erv1 showed that it contains three distinct couples with midpoint potentials of -320, -215, and -150 mV. Like all redox-active enzymes, Erv1 requires one or more electron acceptors. We have generated strains with erv1 conditional alleles and employed biochemical and genetic strategies to facilitate identifying redox pathways involving Erv1. Here, we report that Erv1 forms a 1:1 complex with cytochrome c and a reduced Erv1 can transfer electrons directly to the ferric form of the cytochrome. Erv1 also utilized molecular oxygen as an electron acceptor to generate hydrogen peroxide, which is subsequently reduced to water by cytochrome c peroxidase (Ccp1). Oxidized Ccp1 was in turn reduced by the Erv1-reduced cytochrome c. By coupling these pathways, cytochrome c and Ccp1 function efficiently as Erv1-dependent electron acceptors. Thus, we propose that Erv1 utilizes diverse pathways for electron shuttling in the IMS.

The role of tryptophan in the ferredoxin-dependent nitrite reductase of spinach.

A system has been developed for expressing a His-tagged form of the ferredoxin-dependent nitrite reductase of spinach in Escherichia coli. The catalytic and spectral properties of the His-tagged, recombinant enzyme are similar, but not identical, to those previously observed for nitrite reductase isolated directly from spinach leaf. A detailed comparison of the spectral, catalytic and fluorescence properties of nitrite reductase variants, in which each of the enzyme's eight tryptophan residues has been replaced using site-directed mutagenesis by either aromatic or non-aromatic amino acids, has been used to examine possible roles for tryptophan residues in the reduction of nitrite to ammonia catalyzed by the enzyme.

Functional, structural, and spectroscopic characterization of a glutathione-ligated [2Fe-2S] cluster in poplar glutaredoxin C1.

When expressed in Escherichia coli, cytosolic poplar glutaredoxin C1 (CGYC active site) exists as a dimeric iron-sulfur-containing holoprotein or as a monomeric apoprotein in solution. Analytical and spectroscopic studies of wild-type protein and site-directed variants and structural characterization of the holoprotein by using x-ray crystallography indicate that the holoprotein contains a subunit-bridging [2Fe-2S] cluster that is ligated by the catalytic cysteines of two glutaredoxins and the cysteines of two glutathiones. Mutagenesis data on a variety of poplar glutaredoxins suggest that the incorporation of an iron-sulfur cluster could be a general feature of plant glutaredoxins possessing a glycine adjacent to the catalytic cysteine. In light of these results, the possible involvement of plant glutaredoxins in oxidative stress sensing or iron-sulfur biosynthesis is discussed with respect to their intracellular localization.

Ferredoxin/ferredoxin-thioredoxin reductase complex: Complete NMR mapping of the interaction site on ferredoxin by gallium substitution.

The reduction of ferredoxin-thioredoxin reductase (FTR) by plant-type ferredoxin plays an important role in redox regulation in plants and cyanobacteria. Nuclear magnetic resonance (NMR) was used to map the binding sites on Synechocystis ferredoxin for FTR. A gallium-substituted structural analog of this [2Fe-2S] ferredoxin was obtained by reconstituting the apoprotein in a refolding buffer containing gallium. For the first time, the complete interaction interface of a [2Fe-2S] ferredoxin with a target enzyme has been mapped by NMR chemical shift perturbation with this diamagnetic structural analog.

Crystallization and structural analysis of GADPH from Spinacia oleracea in a new form.

Two crystalline forms of GADPH (D-glyceraldehyde-3-phosphate dehydrogenase) from Spinacia oleracea were obtained using sitting-drop vapor diffusion. Despite the very low concentration of GADPH in the solutions, two crystalline forms were obtained, one of which was the previously reported C222 space group with unit-cell parameters a = 155.3, b = 181.7, c = 107.6 A and the other of which belonged to a new space group I4(1)22, with unit-cell parameters a = b = 120.9, c = 154.5 A. Diffraction data were measured from both native and derivatives, yielding structures at a resolution limit of 3.0 A. Differences at the NAD(+)/NADP(+)-binding site seen in these structures compared with the previously reported structure with bound coenzyme suggest that conformational changes associated with pyridine-nucleotide binding may play a role in the regulation of this enzyme.

Plant glutathione peroxidases are functional peroxiredoxins distributed in several subcellular compartments and regulated during biotic and abiotic stresses.

We provide here an exhaustive overview of the glutathione (GSH) peroxidase (Gpx) family of poplar (Populus trichocarpa). Although these proteins were initially defined as GSH dependent, in fact they use only reduced thioredoxin (Trx) for their regeneration and do not react with GSH or glutaredoxin, constituting a fifth class of peroxiredoxins. The two chloroplastic Gpxs display a marked selectivity toward their electron donors, being exclusively specific for Trxs of the y type for their reduction. In contrast, poplar Gpxs are much less specific with regard to their electron-accepting substrates, reducing hydrogen peroxide and more complex hydroperoxides equally well. Site-directed mutagenesis indicates that the catalytic mechanism and the Trx-mediated recycling process involve only two (cysteine [Cys]-107 and Cys-155) of the three conserved Cys, which form a disulfide bridge with an oxidation-redox midpoint potential of -295 mV. The reduction/formation of this disulfide is detected both by a shift on sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by measuring the intrinsic tryptophan fluorescence of the protein. The six genes identified coding for Gpxs are expressed in various poplar organs, and two of them are localized in the chloroplast, with one colocalizing in mitochondria, suggesting a broad distribution of Gpxs in plant cells. The abundance of some Gpxs is modified in plants subjected to environmental constraints, generally increasing during fungal infection, water deficit, and metal stress, and decreasing during photooxidative stress, showing that Gpx proteins are involved in the response to both biotic and abiotic stress conditions.