David B. Knaff

The Arabidopsis plastidial thioredoxins: new functions and new insights into specificity

The sequencing of the genome of Arabidopsis thaliana revealed that this plant contained numerous isoforms of thioredoxin (Trx), a protein involved in thiol-disulfide exchanges. On the basis of sequence comparison, seven putative chloroplastic Trxs have been identified, four belonging to the m-type, two belonging to the f-type, and one belonging to a new x-type. In the present work, these isoforms were produced and purified as recombinant proteins without their putative transit peptides. Their activities were tested with two known chloroplast thioredoxin targets: NADP-malate dehydrogenase and fructose-1,6-bisphosphatase and also with a chloroplastic 2-Cys peroxiredoxin. The study confirms the strict specificity of fructose-bisphosphatase for Trx f, reveals that some Trxs are unable to activate NADP-malate dehydrogenase, and shows that the new x-type is the most efficient substrate for peroxiredoxin while being inactive toward the two other targets. This suggests that this isoform might be specifically involved in resistance against oxidative stress. Three-dimensional modeling shows that one of the m-type Trxs, Trx m3, which has no activity with any of the three targets, exhibits a negatively charged surface surrounding the active site. A green fluorescent protein approach confirms the plastidial localization of these Trxs.

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.

Poplar Peroxiredoxin Q. A Thioredoxin-Linked Chloroplast Antioxidant Functional in Pathogen Defense

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 x 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 tertiary 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.

Characterization of Plastidial Thioredoxins from Arabidopsis Belonging to the New y-Type

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.

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.

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.

Glutamate synthase: structural, mechanistic and regulatory properties, and role in the amino acid metabolism.

Ammonium ion assimilation constitutes a central metabolic pathway in many organisms, and glutamate synthase, in concert with glutamine synthetase (GS, EC 6.3.1.2), plays the primary role of ammonium ion incorporation into glutamine and glutamate. Glutamate synthase occurs in three forms that can be distinguished based on whether they use NADPH (NADPH-GOGAT, EC 1.4.1.13), NADH (NADH-GOGAT, EC 1.4.1.14) or reduced ferredoxin (Fd-GOGAT, EC 1.4.7.1) as the electron donor for the (two-electron) conversion of L-glutamine plus 2-oxoglutarate to L-glutamate. The distribution of these three forms of glutamate synthase in different tissues is quite specific to the organism in question. Gene structures have been determined for Fd-, NADH- and NADPH-dependent glutamate synthases from different organisms, as shown by searches in nucleic acid sequence data banks. Fd-glutamate synthase contains two electron-carrying prosthetic groups, the redox properties of which are discussed. A description of the ferredoxin binding by Fd-glutamate synthase is also presented. In plants, including nitrogen-fixing legumes, Fd-glutamate synthase and NADH-glutamate synthase supply glutamate during the nitrogen assimilation and translocation. The biological functions of Fd-glutamate synthase and NADH-glutamate synthase, which show a highly tissue-specific distribution pattern, are tightly related to the regulation by the light and metabolite sensing systems. Analysis of mutants and transgenic studies have provided insights into the primary individual functions of Fd-glutamate synthase and NADH-glutamate synthase. These studies also provided evidence that glutamate dehydrogenase (NADH-GDH, EC 1.4.1.2) does not represent a significant alternate route for glutamate formation in plants. Taken together, biochemical analysis and genetic and molecular data imply that Fd-glutamate synthase incorporates photorespiratory and non-photorespiratory ammonium and provides nitrogen for transport to maintain nitrogen status in plants. Fd-glutamate synthase also plays a role that is redundant, in several important aspects, to that played by NADH-glutamate synthase in ammonium assimilation and nitrogen transport.

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.

Sulfur metabolism in phototrophic organisms

Editorial.-Contents.-Preface-Author Index.-Colour Plates.-I. Sulfate Activation And Reduction, Biosynthesis Of Sulfur Containing Amino Acids.- 1.Introduction To Sulfur Metabolism In Phototrophic Organisms Christiane Dahl, Rudiger Hell,David Knaff, Thomas Leustek.-2.Uptake, Allocation And Subcellular Transport Of Sulfate Malcolm J. Hawkesford .-3.Phylogenetic Analysis Of Sulfate Assimilation And Cysteine Biosynthesis In Phototrophic Organisms Stanislav Kopriva , Nicola Patron, Tom Leustek, Patrick Keeling.-4.Metabolism Of Cysteine In Plants And Phototrophic Bacteria Rudiger Hell, Markus Wirtz .-5.Metabolism Of Methionine In Plants And Phototrophic Bacteria Rainer Hoefgen, Holger Hesse.-6.Sulfotransferases From Plants, Algae And Phototrophic Bacteria Cinta Hernandez-Sebastia, Frederic Marsolais, Luc Varin.-7.Cysteine Desulfurase-Mediated Sulfur Donation Pathways In Plants And Phototrophic Bacteria Lolla Padmavathi, Hong Ye, Elizabeth AH Pilon-Smits, Marinus Pilon .-II Sulfur In Plants And Algae:8.Molecular Biology And Functional Genomics For Identification Of Regulatory Networks Of Plant Sulfate Uptake And Assimilatory Metabolism Hideki Takahashi, Kazuki Saito.-9.Biosynthesis, Compartmentation And Cellular Functions Of Glutathione In Plant Cells Andreas Meyer, Thomas Rausch .-10.Sulfolipid Biosynthesis And Function In Plants Christoph Benning, R. Michael Garavito, Mie Shimojia.- 11.Sulfur-Containing Secondary Metabolites And Their Role In Plant Defense Meike Burow, Jonathan Gershenzon, Ute Wittstock.-12.Sulfite Oxidation In Plants Robert Hansch, Ralf R. Mendel.-13.The State Of Sulfur Metabolism In Algae: From Ecology To Genomics Nakako Shibagaki, Arthur Grossman.- III. Sulfur In Phototrophic Prokaryotes:14.Systematics Of Anoxygenic Phototrophic Bacteria Johannes Imhoff.-15.InorganicSulfur Compounds As Electron Donors In Purple Sulfur Bacteria Christiane Dahl.-16.Sulfide Oxidation From Cyanobacteria To Humans: Sulfide-Quinone Oxidoreductase Yosepha Shahak, Gunther Hauska.-17.Genomic Insights Into The Sulfur Metabolism Of Phototrophic Green Sulfur Bacteria Niels-Ulrik Frigaard, Don Bryant.-18.Genetic And Proteomic Studies Of Sulfur Oxidation In Chlorobium Tepdium Chlorobium Tepidum Leong-Keat Chan, Rachael Morgan-Kiss, Thomas E. Hanson.-IV. Ecology And Biotechnology: 19.Ecology Of Phototrophic Sulfur Bacteria Jorg Overmann.-20.Role Of Sulfur For Algae: Acquisition, Metabolism, Ecology And Evolution Mario Giordano, Alessandra Norici, Simona Ratti, John A. Raven.-21.Role Of Sulfur For Plant Production In Agricultural And Natural Ecosystems Fangjie Zhao, Michael Tausz , Luit De Kok.-22. Using Anoxygenic Photosynthetic Bacteria For The Removal Of Sulfide From Wastewater Timothy J. Hurse, Ulrike Kappler, Jurg Keller.-V. Specific Methods: 23.X-Ray Absorption Spectroscopy As Tool For The Detection And Identification Of Sulfur Compounds In Phototrophic Organisms Alexander Prange, Josef Hormes, Hartwig Modrow.-24.Imaging Thiol-Based Redox Processes In Live Cells Andreas Meyer, Mark D. Fricker.-Index

Signal transduction by the global regulator RegB is mediated by a redox-active cysteine

All living organisms alter their physiology in response to changes in oxygen tension. The photosynthetic bacterium uses the RegB–RegA signal transduction cascade to control a wide variety of oxygen‐responding processes such as respiration, photosynthesis, carbon fixation and nitrogen fixation. We demonstrate that a highly conserved cysteine has a role in controlling the activity of the sensor kinase, RegB. In vitro studies indicate that exposure of RegB to oxidizing conditions results in the formation of an intermolecular disulfide bond and that disulfide bond formation is metal‐dependent, with the metal fulfilling a structural role. Formation of a disulfide bond in vitro is also shown to convert the kinase from an active dimer into an inactive tetramer state. Mutational analysis indicates that a cysteine residue flanked by cationic amino acids is involved in redox sensing in vitro and in vivo. These residues appear to constitute a novel ‘redox‐box’ that is present in sensor kinases from diverse species of bacteria.