Phosphosulfate Reductase Research Articles

Development of genic cleavage markers in association with seed glucosinolate content in canola.

The orthologues of Arabidopsis involved in seed glucosinolates metabolism within QTL confidence intervals were identified, and functional markers were developed to facilitate breeding for ultra-low glucosinolates in canola. Further reducing the content of seed glucosinolates will have a positive impact on the seed quality of canola (Brassica napus). In this study 43 quantitative trait loci (QTL) for seed glucosinolate (GSL) content in a low-GSL genetic background were mapped over seven environments in Germany and China in a doubled haploid population from a cross between two low-GSL oilseed rape parents with transgressive segregation. By anchoring these QTL to the reference genomes of B. rapa and B. oleracea, we identified 23 orthologues of Arabidopsis involved in GSL metabolism within the QTL confidence intervals. Sequence polymorphisms between the corresponding coding regions of the parental lines were used to develop cleaved amplified polymorphic site markers for two QTL-linked genes, ISOPROPYLMALATE DEHYDROGENASE1 and ADENOSINE 5'-PHOSPHOSULFATE REDUCTASE 3. The genic cleavage markers were mapped in the DH population into the corresponding intervals of QTL explaining 3.36-6.88 and 4.55-8.67 % of the phenotypic variation for seed GSL, respectively. The markers will facilitate breeding for ultra-low seed GSL content in canola.

Design, Synthesis and Evaluation of Fe-S Targeted Adenosine 5′-Phosphosulfate Reductase Inhibitors

Adenosine 5′-phosphosulfate reductase (APR) is an iron-sulfur enzyme that is vital for survival of Mycobacterium tuberculosis during dormancy and is an attractive target for the treatment of latent tuberculosis (TB) infection. The 4Fe-4S cluster is coordinated to APR by sulfur atoms of four cysteine residues, is proximal to substrate, adenosine 5′-phopsphosulfate (APS), and is essential for catalytic activity. Herein, we present an approach for the development of a new class of APR inhibitors. As an initial step, we have employed an improved solid-phase chemistry method to prepare a series of N6-substituted adenosine analogues and their 5′-phosphates as well as adenosine 5′-phosphate diesters bearing different Fe and S binding groups, such as thiols or carboxylic and hydroxamic acid moieties. Evaluation of the resulting compounds indicates a clearly defined spacing requirement between the Fe-S targeting group and adenosine scaffold and that smaller Fe-S targeting groups are better tolerated. Molecular docking analysis suggests that the S atom of the most potent inhibitor may establish a favorable interaction with an S atom in the cluster. In summary, this study showcases an improved solid-phase method that expedites the preparation of adenosine and related 5′-phosphate derivatives and presents a unique Fe-S targeting strategy for the development of APR inhibitors.

Differential expression of sulfur assimilation pathway genes in Acidithiobacillus ferrooxidans under Cd²⁺ stress: evidence from transcriptional, enzymatic, and metabolic profiles.

Acidithiobacillus ferrooxidans is a heavy metal-tolerant acidophilic chemolithotroph found in acidic mine effluent and is used commercially in the bioleaching of sulfide ores. In this work, we investigated the interplay between divalent cadmium (Cd(2+)) resistance and expression of genes involved in the sulfur assimilation pathway (SAP). We also investigated the response of the thiol-containing metal-chelating metabolites, cysteine and glutathione(GSH), to increasing Cd(2+) concentrations. During growth in the presence of 30 mM Cd(2+), the concentrations of mRNA for 5 genes in the SAP pathway increased more than fourfold: these encode ATP sulfurylase (ATPS), adenosine 5'-phosphosulfate (APS) reductase, sulfite reductase (SiR), serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL). Increased transcription was also reflected in increased enzyme activities: those of SAT and adenosylphosphosulfate reductase (APR) reached a peak of 26- and 15.8-fold, respectively, compared to the control culture in the presence of 15 mM Cd(2+). In contrast, the activity of OAS-TL, which is responsible for the biosynthesis of cysteine, was diminished. At the metabolite level, the intracellular cysteine and GSH contents nearly doubled. These results suggested that Cd(2+) induced transcription of SAP genes, while directly inhibiting the activities of some enzymes (e.g., OAS-TL). Overall, these results are consistent with a detoxification/resistance mechanism involving enhanced sulfur uptake and sequestration of Cd(2+) by cysteine and glutathione.

Phylogenetic Diversity of aprA Genes in Subseafloor Sediments on the Northwestern Pacific Margin off Japan.

Markedly diverse sequences of the adenosine-5′-phosphosulfate reductase alpha subunit gene (aprA), which encodes a key enzyme in microbial sulfate reduction and sulfur oxidation, were detected in subseafloor sediments on the northwestern Pacific off Japan. The aprA gene sequences were grouped into 135 operational taxonomic units (90% sequence identity), including genes related to putative sulfur-oxidizing bacteria predominantly detected in sulfate-depleted deep sediments. Our results suggest that microbial ecosystems in the subseafloor biosphere have phylogenetically diverse genetic potentials to mediate cryptic sulfur cycles in sediments, even where sulfate is rarely present.

Sulfate resupply accentuates protein synthesis in coordination with nitrogen metabolism in sulfur deprived Brassica napus

To investigate the regulatory interactions between S assimilation and N metabolism in Brassica napus, de novo synthesis of amino acids and proteins was quantified by 15N and 34S tracing, and the responses of transporter genes, assimilatory enzymes and metabolites pool involving in nitrate and sulfate metabolism were assessed under continuous sulfur supply, sulfur deprivation and sulfate resupply after 3 days of sulfur (S) deprivation. S-deprived plants were characterized by a strong induction of sulfate transporter genes, ATP sulfurylase (ATPS) and adenosine 5′-phosphosulfate reductase (APR), and by a repressed activity of nitrate reductase (NR) and glutamine synthetase (GS). Sulfate resupply to the S-deprived plants strongly increased cysteine, amino acids and proteins concentration. The increase in sulfate and cysteine concentration caused by sulfate resupply was not matched with the expression of sulfate transporters and the activity of ATPS and APR which were rapidly decreased by sulfate resupply. A strong induction of O-acetylserine(thiol)lyase (OASTL), NR and GS upon sulfate resupply was accompanied with the increase in cysteine, amino acids and proteins pool. Sulfate resupply resulted in a strong increase in de novo synthesis of amino acids and proteins, as evidenced by the increases in N and S incorporation into amino acids (1.8- and 2.4-fold increase) and proteins (2.2-and 6.3-fold increase) when compared to S-deprived plants. The results thus indicate that sulfate resupply followed by S-deprivation accelerates nitrate assimilation for protein synthesis.

Microbial sulfur transformations in sediments from Subglacial Lake Whillans.

Diverse microbial assemblages inhabit subglacial aquatic environments. While few of these environments have been sampled, data reveal that subglacial organisms gain energy for growth from reduced minerals containing nitrogen, iron, and sulfur. Here we investigate the role of microbially mediated sulfur transformations in sediments from Subglacial Lake Whillans (SLW), Antarctica, by examining key genes involved in dissimilatory sulfur oxidation and reduction. The presence of sulfur transformation genes throughout the top 34 cm of SLW sediments changes with depth. SLW surficial sediments were dominated by genes related to known sulfur-oxidizing chemoautotrophs. Sequences encoding the adenosine-5′-phosphosulfate (APS) reductase gene, involved in both dissimilatory sulfate reduction and sulfur oxidation, were present in all samples and clustered into 16 distinct operational taxonomic units. The majority of APS reductase sequences (74%) clustered with known sulfur oxidizers including those within the “Sideroxydans” and Thiobacillus genera. Reverse-acting dissimilatory sulfite reductase (rDSR) and 16S rRNA gene sequences further support dominance of “Sideroxydans” and Thiobacillus phylotypes in the top 2 cm of SLW sediments. The SLW microbial community has the genetic potential for sulfate reduction which is supported by experimentally measured low rates (1.4 pmol cm-3d-1) of biologically mediated sulfate reduction and the presence of APS reductase and DSR gene sequences related to Desulfobacteraceae and Desulfotomaculum. Our results also infer the presence of sulfur oxidation, which can be a significant energetic pathway for chemosynthetic biosynthesis in SLW sediments. The water in SLW ultimately flows into the Ross Sea where intermediates from subglacial sulfur transformations can influence the flux of solutes to the Southern Ocean.

Molecular mechanisms of regulation of sulfate assimilation: first steps on a long road.

The pathway of sulfate assimilation, which provides plants with the essential nutrient sulfur, is tightly regulated and coordinated with the demand for reduced sulfur. The responses of metabolite concentrations, enzyme activities and mRNA levels to various signals and environmental conditions have been well described for the pathway. However, only little is known about the molecular mechanisms of this regulation. To date, nine transcription factors have been described to control transcription of genes of sulfate uptake and assimilation. In addition, other levels of regulation contribute to the control of sulfur metabolism. Post-transcriptional regulation has been shown for sulfate transporters, adenosine 5′phosphosulfate reductase, and cysteine synthase. Several genes of the pathway are targets of microRNA miR395. In addition, protein–protein interaction is increasingly found in the center of various regulatory circuits. On top of the mechanisms of regulation of single genes, we are starting to learn more about mechanisms of adaptation, due to analyses of natural variation. In this article, the summary of different mechanisms of regulation will be accompanied by identification of the major gaps in knowledge and proposition of possible ways of filling them.

Oligo-carrageenan kappa increases NADPH, ascorbate and glutathione syntheses and TRR/TRX activities enhancing photosynthesis, basal metabolism, and growth in Eucalyptus trees.

In order to analyze the effect of OC kappa in redox status, photosynthesis, basal metabolism and growth in Eucalyptus globulus, trees were treated with water (control), with OC kappa at 1 mg mL−1, or treated with inhibitors of NAD(P)H, ascorbate (ASC), and glutathione (GSH) syntheses and thioredoxin reductase (TRR) activity, CHS-828, lycorine, buthionine sulfoximine (BSO), and auranofin, respectively, and with OC kappa, and cultivated for 4 months. Treatment with OC kappa induced an increase in NADPH, ASC, and GSH syntheses, TRR and thioredoxin (TRX) activities, photosynthesis, growth and activities of basal metabolism enzymes such as rubisco, glutamine synthetase (GlnS), adenosine 5′-phosphosulfate reductase (APR), involved in C, N, and S assimilation, respectively, Krebs cycle and purine/pyrimidine synthesis enzymes. Treatment with inhibitors and OC kappa showed that increases in ASC, GSH, and TRR/TRX enhanced NADPH synthesis, increases in NADPH and TRR/TRX enhanced ASC and GSH syntheses, and only the increase in NADPH enhanced TRR/TRX activities. In addition, the increase in NADPH, ASC, GSH, and TRR/TRX enhanced photosynthesis and growth. Moreover, the increase in NADPH, ASC and TRR/TRX enhanced activities of rubisco, Krebs cycle, and purine/pyrimidine synthesis enzymes, the increase in GSH, NADPH, and TRR/TRX enhanced APR activity, and the increase in NADPH and TRR/TRX enhanced GlnS activity. Thus, OC kappa increases NADPH, ASC, and GSH syntheses leading to a more reducing redox status, the increase in NADPH, ASC, GSH syntheses, and TRR/TRX activities are cross-talking events leading to activation of photosynthesis, basal metabolism, and growth in Eucalyptus trees.

Significance of redox-active cysteines in human FAD synthase isoform 2

FAD synthase (FMN:ATP adenylyl transferase, FMNAT or FADS, EC 2.7.7.2) is the last enzyme in the pathway converting riboflavin into FAD. In humans, FADS is localized in different subcellular compartments and exists in different isoforms. Isoform 2 (490-amino acids) is organized in two domains: the 3′-phosphoadenosine-5′-phosphosulfate (PAPS) reductase domain, that is the FAD-forming catalytic domain, and one resembling a molybdopterin-binding (MPTb) domain, with a hypothetical regulatory role. hFADS2 contains ten Cys residues, seven of which located in the PAPS reductase domain, with a possible involvement either in FAD synthesis or in FAD delivery to cognate apo-flavoproteins. A homology model of the PAPS reductase domain of hFADS2 revealed a co-ordinated network among the Cys residues in this domain. In this model, C312 and C303 are very close to the flavin substrate, consistent with a significantly lowered FAD synthesis rate in C303A and C312A mutants. FAD synthesis is also inhibited by thiol-blocking reagents, suggesting the involvement of free cysteines in the hFADS2 catalytic cycle. Mass spectrometry measurements and titration with thiol reagents on wt hFADS2 and on several individual cysteine/alanine mutants allowed us to detect two stably reduced cysteines (C139 and C241, one for each protein domain), two stable disulfide bridges (C399–C402, C303–C312, both in the PAPS domain), and two unstable disulfides (C39–C50; C440–C464). Whereas the C39–C50 unstable disulfide is located in the MPTb domain and appears to have no catalytic relevance, a cysteine-based redox switch may involve formation and breakdown of a disulfide between C440 and C464 in the PAPS domain.

Souring control in fluid samples of oil industry using a multiple ligand simultaneous docking (MLSD) strategy

We have used docking techniques in order to propose potential inhibitors to the enzymes adenosine phosphosulfate reductase and adenosine triphosphate sulfurylase that are responsible, among other deleterious effects, for causing souring of oil and gas reservoirs. Three candidates selected through molecular docking revealed new and improved polar and hydrophobic interactions with the above-mentioned enzymes. Microbiological laboratory assays performed subsequently corroborated the results of computer modelling that the three compounds can efficiently control the biogenic sulfide production.

Impairment in Sulfite Reductase Leads to Early Leaf Senescence in Tomato Plants.

Sulfite reductase (SiR) is an essential enzyme of the sulfate assimilation reductive pathway, which catalyzes the reduction of sulfite to sulfide. Here, we show that tomato (Solanum lycopersicum) plants with impaired SiR expression due to RNA interference (SIR Ri) developed early leaf senescence. The visual chlorophyll degradation in leaves of SIR Ri mutants was accompanied by a reduction of maximal quantum yield, as well as accumulation of hydrogen peroxide and malondialdehyde, a product of lipid peroxidation. Interestingly, messenger RNA transcripts and proteins involved in chlorophyll breakdown in the chloroplasts were found to be enhanced in the mutants, while transcripts and their plastidic proteins, functioning in photosystem II, were reduced in these mutants compared with wild-type leaves. As a consequence of SiR impairment, the levels of sulfite, sulfate, and thiosulfate were higher and glutathione levels were lower compared with the wild type. Unexpectedly, in a futile attempt to compensate for the low glutathione, the activity of adenosine-5'-phosphosulfate reductase was enhanced, leading to further sulfite accumulation in SIR Ri plants. Increased sulfite oxidation to sulfate and incorporation of sulfite into sulfoquinovosyl diacylglycerols were not sufficient to maintain low basal sulfite levels, resulting in accumulative leaf damage in mutant leaves. Our results indicate that, in addition to its biosynthetic role, SiR plays an important role in prevention of premature senescence. The higher sulfite is likely the main reason for the initiation of chlorophyll degradation, while the lower glutathione as well as the higher hydrogen peroxide and malondialdehyde additionally contribute to premature senescence in mutant leaves.

ACTIVITY AND KINETIC PROPERTIES OF ADENOSINE 5′-PHOSPHOSULFATE REDUCTASE IN THE INTESTINAL SULFATE-REDUCING BACTERIA

Adenosine 5′-phosphosulfate (APS) reductase activity and the kinetic properties of the enzyme from intestinal sulfate-reducing bacteria Desulfovibrio piger Vib-7 and Desulfomicrobium sp. Rod-9 has never been well-characterized and has not been studied yet. The aim and background of this work was to investigate the dissimilatory APS reductase activity in cell-free extract of intestinal sulfate-reducing bacteria D. piger Vib-7 and Desulfomicrobium sp. Rod-9 and to carry out the kinetic analysis of enzymatic reaction. Methods. Microbiological, biochemical, and biophysical methods of the studies, and statistical processing of the results were used; the obtained data were compared with those from literature. Results. Dissimilatory APS reductase activity in the sulfate-reducing bacteria isolated from human intestine was studied. The highest activity of the enzyme (0.34±0.029 U×mg-1 protein) was measured in the cell-free extract prepared from D. piger Vib-7 cells then from Desulfomicrobium sp. Rod-9 (0.22±0.018 U×mg-1 protein). The optimal temperature (+35 oC) and pH (8.0) for APS reductase reaction were determined. The analysis of the kinetic properties of the bacterial APS reductase was carried out. The APS reductase activity, initial (instantaneous) reaction rate (V0) and maximum rate of the APS reductase reaction (Vmax) in both D. piger Vib-7 and Desulfomicrobium sp. Rod-9 bacterial strains were defined. Michaelis constants (Km) of the enzyme reaction (4.33±0.47 and 3.57±0.32 mM for D. piger Vib-7 and Desulfomicrobium sp. Rod-9, respectively) were determined. Conclusion. The described results of these studies can be the prospects to clarify the etiological role of these bacteria in the development of inflammatory bowel diseases humans and animals.

Vertical distribution of major sulfate-reducing bacteria in a shallow eutrophic meromictic lake

The vertical distribution of sulfate-reducing bacteria was investigated in a shallow, eutrophic, meromictic lake, Lake Harutori, located in a residential area of Kushiro, Japan. A steep chemocline, characterized by gradients of oxygen, sulfide and salinity, was found at a depth of 3.5–4.0m. The sulfide concentration at the bottom of the lake was high (up to a concentration of 10.7mM). Clone libraries were constructed using the aprA gene, which encodes adenosine-5′-phosphosulfate reductase subunit A, in order to monitor sulfate-reducing bacteria. In the aprA clone libraries, the most abundant sequences were those from the Desulfosarcina–Desulfococcus (DSS) group. A primer set for a DSS group-specific 16S rRNA gene was used to construct another clone library, analysis of which revealed that the uncultured group of sulfate-reducing bacteria, SEEP SRB-1, accounted for nearly half of the obtained sequences. Quantification of the major bacterial groups by catalyzed reporter deposition-fluorescence in situ hybridization demonstrated that the DSS group accounted for 3.2–4.8% of the total bacterial community below the chemocline. The results suggested that the DSS group was one of the major groups of sulfate-reducing bacteria and that these presumably metabolically versatile bacteria might play an important role in sulfur cycling in Lake Harutori.

Evidence for Proteomic and Metabolic Adaptations Associated with Alterations of Seed Yield and Quality in Sulfur-limited Brassica napus L

In Brassica napus, seed yield and quality are related to sulfate availability, but the seed metabolic changes in response to sulfate limitation remain largely unknown. To address this question, proteomics and biochemical studies were carried out on mature seeds obtained from plants grown under low sulfate applied at the bolting (LS32), early flowering (LS53), or start of pod filling (LS70) stage. The protein quality of all low-sulfate seeds was reduced and associated with a reduction of S-rich seed storage protein accumulation (as Cruciferin Cru4) and an increase of S-poor seed storage protein (as Cruciferin BnC1). This compensation allowed the protein content to be maintained in LS70 and LS53 seeds but was not sufficient to maintain the protein content in LS32 seeds. The lipid content and quality of LS53 and LS32 seeds were also affected, and these effects were primarily associated with a reduction of C18-derivative accumulation. Proteomics changes related to lipid storage, carbohydrate metabolism, and energy (reduction of caleosins, phosphoglycerate kinase, malate synthase, ATP-synthase β-subunit, and thiazole biosynthetic enzyme THI1 and accumulation of β-glucosidase and citrate synthase) provide insights into processes that may contribute to decreased oil content and altered lipid composition (in favor of long-chain fatty acids in LS53 and LS32 seeds). These data indicate that metabolic changes associated with S limitation responses affect seed storage protein composition and lipid quality. Proteins involved in plant stress response, such as dehydroascorbate reductase and Cu/Zn-superoxide dismutase, were also accumulated in LS53 and LS32 seeds, and this might be a consequence of reduced glutathione content under low S availability. LS32 treatment also resulted in (i) reduced germination vigor, as evidenced by lower germination indexes, (ii) reduced seed germination capacity, related to a lower seed viability, and (iii) a strong decrease of glyoxysomal malate synthase, which is essential for the use of fatty acids during seedling establishment.

Do PFT1 and HY5 interact in regulation of sulfate assimilation by light in Arabidopsis?

Sulfate assimilation pathway is highly responsive to changes in environment, but the mechanisms of such regulation are only slowly beginning to unravel. Here we show evidence that PHYTOCHROME AND FLOWERING TIME 1 (PFT1) may be another component of the sulfate assimilation regulatory circuit. Transcriptional regulation by light of the key enzyme of sulfate assimilation, adenosine 5'phosphosulfate (APS) reductase, is disturbed in pft1-2 mutants. PFT1, however, affects also APS reductase enzyme activity, flux through the sulfate assimilation pathway and accumulation of glutathione. In addition, our data suggest a possible interplay of PFT1 with another transcription factor, HY5, in regulation of APS reductase by light.

Microbial communities in semi-consolidated carbonate sediments of the Southwest Indian Ridge

White semi-consolidated carbonate sediments attached to black ferromanganese oxide films were collected approximately 50 km west of a newly discovered hydrothermal field near the Southwest Indian Ridge (SWIR). The biodiversity of the prokaryotic communities within the field was examined using clone library-based culture-independent analysis of the exterior black oxides and the interior white carbonates. Subsequent 16S rRNA gene analysis suggested that Gamma-proteobacteria, Acidobacteria, and Thaumarchaeota members dominated the bacterial and archaeal clone libraries. To further characterize the metabolic processes within the microbial community, analyses of the amoA (coding the alpha subunit of the ammonia monooxygenase for Archaea) and aprA (coding the alpha subunit of the dissimilatory adenosine-5'-phosphosulfate reductase for the sulfate-reducing and sulfur-oxidizing prokaryotes) functional genes were conducted. The functional gene analysis results suggested that Thaumarchaeota and Alphaproteobacteria members were the potential players that participated in N and S cycles in this marine carbonate sedimentary environment. This paper is the first to describe the microbial communities and their potential metabolic pathways within the semi-consolidated carbonate sediments of the SWIR.

Whole-genome sequencing reveals novel insights into sulfur oxidation in the extremophile Acidithiobacillus thiooxidans.

BackgroundAcidithiobacillus thiooxidans (A. thiooxidans), a chemolithoautotrophic extremophile, is widely used in the industrial recovery of copper (bioleaching or biomining). The organism grows and survives by autotrophically utilizing energy derived from the oxidation of elemental sulfur and reduced inorganic sulfur compounds (RISCs). However, the lack of genetic manipulation systems has restricted our exploration of its physiology. With the development of high-throughput sequencing technology, the whole genome sequence analysis of A. thiooxidans has allowed preliminary models to be built for genes/enzymes involved in key energy pathways like sulfur oxidation.ResultsThe genome of A. thiooxidans A01 was sequenced and annotated. It contains key sulfur oxidation enzymes involved in the oxidation of elemental sulfur and RISCs, such as sulfur dioxygenase (SDO), sulfide quinone reductase (SQR), thiosulfate:quinone oxidoreductase (TQO), tetrathionate hydrolase (TetH), sulfur oxidizing protein (Sox) system and their associated electron transport components. Also, the sulfur oxygenase reductase (SOR) gene was detected in the draft genome sequence of A. thiooxidans A01, and multiple sequence alignment was performed to explore the function of groups of related protein sequences. In addition, another putative pathway was found in the cytoplasm of A. thiooxidans, which catalyzes sulfite to sulfate as the final product by phosphoadenosine phosphosulfate (PAPS) reductase and adenylylsulfate (APS) kinase. This differs from its closest relative Acidithiobacillus caldus, which is performed by sulfate adenylyltransferase (SAT). Furthermore, real-time quantitative PCR analysis showed that most of sulfur oxidation genes were more strongly expressed in the S0 medium than that in the Na2S2O3 medium at the mid-log phase.ConclusionSulfur oxidation model of A. thiooxidans A01 has been constructed based on previous studies from other sulfur oxidizing strains and its genome sequence analyses, providing insights into our understanding of its physiology and further analysis of potential functions of key sulfur oxidation genes.

Oxidation and reduction of sulfite contribute to susceptibility and detoxification of SO2 in Populus × canescens leaves

The critical level for SO 2 susceptibility of Populus × canescens is approximately 1.2 μL L −1 SO 2 . Both sulfite oxidation and sulfite reduction and assimilation contribute to SO 2 detoxification. In the present study, uptake, susceptibility and metabolism of SO2 were analyzed in the deciduous tree species poplar (Populus × canescens). A particular focus was on the significance of sulfite oxidase (SO) for sulfite detoxification, as SO has been characterized as a safety valve for SO2 detoxification in herbaceous plants. For this purpose, poplar plants were exposed to different levels of SO2 (0.65, 0.8, 1.0, 1.2 μL L−1) and were characterized by visible injuries and at the physiological level. Gas exchange parameters (stomatal conductance for water vapor, CO2 assimilation, SO2 uptake) of the shoots were compared with metabolite levels (sulfate, thiols) and enzyme activities [SO, adenosine 5′-phosphosulfate reductase (APR)] in expanding leaves (80–90 % expanded). The critical dosage of SO2 that confers injury to the leaves was 1.2 μL L−1 SO2. The observed increase in sulfur containing compounds (sulfate and thiols) in the expanding leaves strongly correlated with total SO2 uptake of the plant shoot, whereas SO2 uptake rate was strongly correlated with stomatal conductance for water vapor. Furthermore, exposure to high concentration of SO2 revealed channeling of sulfite through assimilatory sulfate reduction that contributes in addition to SO-mediated sulfite oxidation to sulfite detoxification in expanding leaves of this woody plant species.

The X-ray crystal structure of APR-B, an atypical adenosine 5′-phosphosulfate reductase from Physcomitrella patens

Sulfonucleotide reductases catalyse the first reductive step of sulfate assimilation. Their substrate specificities generally correlate with the requirement for a [Fe4S4] cluster, where adenosine 5′-phosphosulfate (APS) reductases possess a cluster and 3′-phosphoadenosine 5′-phosphosulfate reductases do not. The exception is the APR-B isoform of APS reductase from the moss Physcomitrella patens, which lacks a cluster. The crystal structure of APR-B, the first for a plant sulfonucleotide reductase, is consistent with a preference for APS. Structural conservation with bacterial APS reductase rules out a structural role for the cluster, but supports the contention that it enhances the activity of conventional APS reductases.

Arabidopsis thaliana Nfu2 Accommodates [2Fe-2S] or [4Fe-4S] Clusters and Is Competent for in Vitro Maturation of Chloroplast [2Fe-2S] and [4Fe-4S] Cluster-Containing Proteins

Nfu-type proteins are essential in the biogenesis of iron-sulfur (Fe-S) clusters in numerous organisms. A number of phenotypes including low levels of Fe-S cluster incorporation are associated with the deletion of the gene encoding a chloroplast-specific Nfu-type protein, Nfu2 from Arabidopsis thaliana (AtNfu2). Here, we report that recombinant AtNfu2 is able to assemble both [2Fe-2S] and [4Fe-4S] clusters. Analytical data and gel filtration studies support cluster/protein stoichiometries of one [2Fe-2S] cluster/homotetramer and one [4Fe-4S] cluster/homodimer. The combination of UV-visible absorption and circular dichroism and resonance Raman and Mössbauer spectroscopies has been employed to investigate the nature, properties, and transfer of the clusters assembled on Nfu2. The results are consistent with subunit-bridging [2Fe-2S](2+) and [4Fe-4S](2+) clusters coordinated by the cysteines in the conserved CXXC motif. The results also provided insight into the specificity of Nfu2 for the maturation of chloroplastic Fe-S proteins via intact, rapid, and quantitative cluster transfer. [2Fe-2S] cluster-bound Nfu2 is shown to be an effective [2Fe-2S](2+) cluster donor for glutaredoxin S16 but not glutaredoxin S14. Moreover, [4Fe-4S] cluster-bound Nfu2 is shown to be a very rapid and efficient [4Fe-4S](2+) cluster donor for adenosine 5'-phosphosulfate reductase (APR1), and yeast two-hybrid studies indicate that APR1 forms a complex with Nfu2 but not with Nfu1 and Nfu3, the two other chloroplastic Nfu proteins. This cluster transfer is likely to be physiologically relevant and is particularly significant for plant metabolism as APR1 catalyzes the second step in reductive sulfur assimilation, which ultimately results in the biosynthesis of cysteine, methionine, glutathione, and Fe-S clusters.