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.
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.
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.
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.
The plant mitochondrial thioredoxin (Trx) system has been described as containing an NADPH-dependent Trx reductase and Trx o. In addition to the mitochondrial isoform, Trx o, plants are known to ...contain several chloroplastic Trx isoforms and the cytosolic Trx h isoforms. We report here the presence in plant mitochondria of a Trx isoform (PtTrxh2) belonging to the Trx h group. Western blot analyses with mitochondrial proteins isolated from both poplar and GFP fusion constructs indicate that PtTrxh2 is targeted to plant mitochondria. The recombinant protein, PtTrxh2, has been shown to be reduced efficiently by the mitochondrial Trx reductase AtNTRA. PtTrxh2 is also able to reduce alternative oxidase homodimers and to allow its activation by pyruvate. In contrast, neither PtTrxh2 nor AtTrxo1 exhibits activity with several poplar glutathione peroxidases and especially a putative mitochondrial isoform. Incubation of PtTrxh2 with glutathione disulfide led to the formation of glutathionylated Trx, identified by mass spectrometry. The formation of a glutathione adduct increases the redox potential of PtTrxh2 from -290 to -225 mV. In addition to Trx o, this study shows that Trx h could also be present in mitochondria. This previously unrecognized complexity is not unexpected, considering the multiple redox-regulated processes found in plant mitochondria.
The plant plastidial thioredoxins (Trx) are involved in the light-dependent regulation of many enzymatic activities, owing to their thiol-disulfide interchange activity. Three different types of ...plastidial Trx have been identified and characterized so far: the m-, f-, and x-types. Recently, a new putative plastidial type, the y-type, was found. In this work the two isoforms of Trx y encoded by the nuclear genome of Arabidopsis (Arabidopsis thaliana) were characterized. The plastidial targeting of Trx y has been established by the expression of a Trx::GFP fusion protein. Then both isoforms were produced as recombinant proteins in their putative mature forms and purified to characterize them by a biochemical approach. Their ability to activate two plastidial light-regulated enzymes, NADP-malate dehydrogenase (NADP-MDH) and fructose-1,6-bisphosphatase, was tested. Both Trx y were poor activators of fructose-1,6-bisphosphatase and NADP-MDH; however, a detailed study of the activation of NADP-MDH using site-directed mutants of its regulatory cysteines suggested that Trx y was able to reduce the less negative regulatory disulfide but not the more negative regulatory disulfide. This property probably results from the fact that Trx y has a less negative redox midpoint potential (-337 mV at pH 7.9) than thioredoxins f and m. The y-type Trxs were also the best substrate for the plastidial peroxiredoxin Q. Gene expression analysis showed that Trx y2 was mainly expressed in leaves and induced by light, whereas Trx y1 was mainly expressed in nonphotosynthetic organs, especially in seeds at a stage of major accumulation of storage lipids.
Peroxiredoxins are ubiquitous thioredoxin- or glutaredoxin-dependent peroxidases, the function of which is to destroy peroxides. Peroxiredoxin Q, one of the four plant subtypes, is a homolog of the ...bacterial bacterioferritin comigratory proteins. We show here that the poplar (Populus tremula × Populus tremuloides) protein acts as a monomer with an intramolecular disulfide bridge between two conserved cysteines. A wide range of electron donors and substrates was tested. Unlike type II peroxiredoxin, peroxiredoxin Q cannot use the glutaredoxin or cyclophilin isoforms tested, but various cytosolic, chloroplastic, and mitochondrial thioredoxins are efficient electron donors with no marked specificities. The redox midpoint potential of the peroxiredoxin Q catalytic disulfide is -325 mV at pH 7.0, explaining why the wild-type protein is reduced by thioredoxin but not by glutaredoxin. Additional evidence that thioredoxin serves as a donor comes from the formation of heterodimers between peroxiredoxin Q and monocysteinic mutants of spinach (Spinacia oleracea) thioredoxin m. Peroxiredoxin Q can reduce various alkyl hydroperoxides, but with a better efficiency for cumene hydroperoxide than hydrogen peroxide and thertiary butyl hydroperoxide. The use of immunolocalization and of a green fluorescence protein fusion construct indicates that the transit sequence efficiently targets peroxiredoxin Q to the chloroplasts and especially to those of the guard cells. The expression of this protein and of type II peroxiredoxin is modified in response to an infection by two races of Melampsora larici-populina, the causative agent of the poplar rust. In the case of an hypersensitive response, the peroxiredoxin expression increased, whereas it decreased during a compatible interaction.
The structure of nitrite reductase, a key enzyme in the process of nitrogen assimilation, has been determined using X-ray diffraction to a resolution limit of 2.8 Å. The protein has a globular fold ...consisting of 3 α/β domains with the siroheme−iron sulfur cofactor at the interface of the three domains. The Fe4S4 cluster is coordinated by cysteines 441, 447, 482, and 486. The siroheme is located at a distance of 4.2 Å from the cluster, and the central iron atom is coordinated to Cys 486. The siroheme is surrounded by several ionizable amino acid residues that facilitate the binding and subsequent reduction of nitrite. A model for the ferredoxin:nitrite reductase complex is proposed in which the binding of ferredoxin to a positively charged region of nitrite reductase results in elimination of exposure of the cofactors to the solvent. The structure of nitrite reductase shows a broad similarity to the hemoprotein subunit of sulfite reductase but has many significant differences in the backbone positions that could reflect sequence differences or could arise from alterations of the sulfite reductase structure that arise from the isolation of this subunit from the native complex. The implications of the nitrite reductase structure for understanding multi-electron processes are discussed in terms of differences in the protein environments of the cofactors.
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.
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 (Cys4) and two (Cys58 and Cys61) in the classical thioredoxin active site (57WCGPC61). The property of a mutant in which Cys58 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 Cys4 is required for the regeneration of PtTrxh4 by Grx. Biochemical and x-ray crystallographic studies indicate that two intramolecular disulfide bonds involving Cys58 can be formed, linking it to either Cys61 or Cys4. We propose thus a four-step disulfide cascade mechanism involving the transient glutathionylation of Cys4 to convert this atypical thioredoxin h back to its active reduced form.
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