Sulfite Reduction Research Articles

33] Sulfite reductase and APS reductase from Archaeoglobus fulgidus

Publisher Summary Archaeoglobus fulgidus carries out sulfate reduction via the pathway originally proposed for bacterial species. All steps of sulfate reduction occur in the cytoplasm, implying that sulfate must be transported across the cytoplasmic membrane. Sulfate transport has not been studied in A. fulgidus, but may resemble that of marine bacterial sulfate reducers, which use sodium ions for the symport of sulfate. The enzyme dissimilatory sulfite reductase catalyzes the six-electron reduction of sulfite to sulfide, which is the central energy-conserving step of sulfate respiration. The natural electron donor of sulfite reductase in sulfate reducers is not known. Thiosulfate reduction in A. fulgidus has not been studied biochemically. Analysis of the A. fulgidus genome sequence revealed the presence of genes encoding several putative molybdopterinbinding oxidoreductases with thiosulfate or polysulfide as potential substrates. This chapter focuses on the purification and characterization of APS reductase and sulfite reductase from A. fulgidus.

Mutational analysis of the substrate specificity of plant sulfite reductase

Plants have ferredoxin-dependent sulfite reductase (SiR: E.C.1.8.7.1) and nitrite reductase (NiR: E.C.1.7.7.1) for assimilation of inorganic sulfur and nitrogen, respectively. These enzymes contain common prosthetic groups, one [4Fe-4S] cluster and one siroheme, and have partially overlapped substrate specificity, both catalyzing a six-electron reduction of sulfite and nitrite to sulfide and ammonia. In fact, SiR shows a strong preference for sulfite (Km: ~5 µM) over nitrite (Km:~0.5 mM). The substrate anions are coordinated to the siroheme iron at its distal side where putative substrate-binding basic residues, Arg-193, Lys-276 and Lys-278 are located. We tried to clarify the structural basis for such substrate discrimination by site-directed mutagenesis of maize SiR. By a heterologous expression of SiR cDNA in E. coli cells, four mutants, R193E, R193A, K276Q and K278N, were obtained as a holo-enzyme with the two prosthetic groups assembled. All mutants lost the sulfite-reduction activity almost completely, and interestingly some mutants showed higher activity for nitrite reduction than wild type. Kinetic analysis demonstrated that affinity to sulfite of R193A and R193E was decreased by less than one-hundredth, while affinity to nitrite of R193A was increased remarkably to an extent similar to that of NiR (Km: ~2 µM). K276Q retained a significant affinity to both sulfite and nitrite, but neither substrate was reduced. These data indicated that the substrate specificity of SiR is drastically changed by substitution of the single basic residue at the distal side of the siroheme.

31] Siroheme-sulfite reductase-type protein from Pyrobaculum islandicum

Publisher Summary Elemental sulfur is used as an electron acceptor for chemolithotrophic growth on H 2 of the hyperthermophilic crenarchaeote Pyrobaculum islandicum . This organism has an optimum growth temperature of 100° and was isolated from an Icelandic geothermal power plant. During organotrophic growth, P. islandicum is also able to grow using sulfite, thiosulfate, cystine, and oxidized glutathione as electron acceptors but it is not able to reduce sulfate. In all organisms capable of reducing sulfite during anaerobic respiration investigated thus far, the six-electron reduction of sulfite to sulfde is catalyzed by the enzyme sulfite reductase. The sulfite reductase-type protein from P. islandicum , which is the subject of this article, shares several common characteristics with dissimilatory sulfite reductases. All of these enzymes consist of two different polypeptides in an α 2 β 2 structure and contain siroheme, nonheme iron, and acid-labile sulfide. In addition, the primary sequence of the P. islandicum sulfite reductase and those available from dissimilatory sulfate reducers and the phototrophic sulfur oxidizer A. vinosum show remarkable similarity.

Alternative pathways for siroheme synthesis in Klebsiella aerogenes.

Siroheme, the cofactor for sulfite and nitrite reductases, is formed by methylation, oxidation, and iron insertion into the tetrapyrrole uroporphyrinogen III (Uro-III). The CysG protein performs all three steps of siroheme biosynthesis in the enteric bacteria Escherichia coli and Salmonella enterica. In either taxon, cysG mutants cannot reduce sulfite to sulfide and require a source of sulfide or cysteine for growth. In addition, CysG-mediated methylation of Uro-III is required for de novo synthesis of cobalamin (coenzyme B(12)) in S. enterica. We have determined that cysG mutants of the related enteric bacterium Klebsiella aerogenes have no defect in the reduction of sulfite to sulfide. These data suggest that an alternative enzyme allows for siroheme biosynthesis in CysG-deficient strains of Klebsiella. However, Klebsiella cysG mutants fail to synthesize coenzyme B(12), suggesting that the alternative siroheme biosynthetic pathway proceeds by a different route. Gene cysF, encoding an alternative siroheme synthase homologous to CysG, has been identified by genetic analysis and lies within the cysFDNC operon; the cysF gene is absent from the E. coli and S. enterica genomes. While CysG is coregulated with the siroheme-dependent nitrite reductase, the cysF gene is regulated by sulfur starvation. Models for alternative regulation of the CysF and CysG siroheme synthases in Klebsiella and for the loss of the cysF gene from the ancestor of E. coli and S. enterica are presented.

Oxidation-reduction properties of two enzymes involved in reductive sulfate assimilation in plants

Assimilation of sulfate by plants involves reaction of sulfate with ATP to form 5?-adenylylsulfate (APS), followed by the two-electron reduction of APS to sulfite by glutathione, catalyzed by APS reductase. The subsequent six-electron reduction of sulfite to sulfide, catalyzed by sulfite reductase, utilizes reduced ferredoxin as the electron donor. Arabidopsis thaliana has three isoforms of APS reductase. The activity of the APR1 form is redox-state controlled via a regulatory disulfide. A second APR1 disulfide is the likely electron acceptor at the active site. APR1 becomes more active, through an increase in Vmax and decrease in the Km for APS, when the regulatory cysteines are oxidized to a disulfide. Oxidative activation plays an important role in the plant?s response to oxidative stress. The Em value for this regulatory disulfide, ?330 mV (at pH 8.5), is equal to that for glutathione at this pH. Sulfite reductase contains one siroheme and one [4Fe-4S] cluster as prosthetic groups. It is likely that a sulfur from the [4Fe-4S] cluster serves as an axial ligand to the iron of the siroheme. We have carried out the first the redox titrations of a ferredoxin-dependent sulfite reductase, using the wild-type enzyme from maize, and measured Em values (at pH 7.5) of -205 mV and ?410 mV for the siroheme and [4Fe-4S] cluster, respectively. The effects of cyanide and phosphate binding to the siroheme and of site-specific mutations (R193A and R193E) that alter substrate specificity on the redox properties of the enzyme have also been investigated.

Wood Hydrolysis and Hydrolyzate Detoxification for Subsequent Xylitol Production

This study deals with two different aspects of the transformation of lignocellulosics into xylitol: the optimization of conditions for wood hydrolysis and the setting-up of an adequate hydrolyzate detoxification procedure necessary to obtain high xylitol yields in the successive fermentation process. A comparison between the processes of wood autohydrolysis (steam explosion) and pre-hydrolysis with dilute sulfuric acid, carried out batch-wise in laboratory scale, shows comparable yields, either in terms of final concentrations of xylose and pentose sugars in the hydrolyzate or of solubilised fraction of wood. On the other hand, notwithstanding the longer time required, the pre-hydrolysis with dilute sulfuric acid produced acid hydrolyzates with lower contents of inhibiting substances (furfural, acetic acid, etc.). In order to obtain satisfactory xylitol yields from the hydrolysate produced by steam explosion, samples of this hydrolyzate were submitted to different detoxification techniques and then fermented batch-wise by a Pachysolen tannophilus strain previously adapted to this substrate. The best detoxification was performed by adding to the traditional overliming with Ca(OH)2 and sulfite reduction, three steps of a) filtration to remove insoluble substances, b) stripping of acetic acid and furfural, and c) lignin-derived compounds removal by adsorption on charcoal. The fermentation of this hydrolyzate was very effective, achieving a final xylitol concentration of 39.5 g/l from 89.0 g/l xylose after 96 h, corresponding to a volumetric productivity of 0.41 g/lh and a product yield of 0.63 g/g.

Plant sulfite reductase: molecular structure, catalytic function and interaction with ferredoxin

Plant sulfite reductase contains the siroheme and the [4Fe–4S] cluster as catalytically active redox centers and catalyzes the six-electron reductions of sulfite and nitrite using electrons donated from ferredoxin. A heterologous expression of a cDNA for maize sulfite reductase in E. coli has enabled us to produce the wild-type and mutant enzymes. Putative substrate-binding basic residues, located at the siroheme distal side, have been substituted for other residues with neutral or negatively charged side chains. Kinetic studies of the resulting mutant enzymes have demonstrated that substrate specificity for the two anions is remarkably changed by amino acid substitutions at a single site. We have also produced two classes of ferredoxin mutants with less ability to donate electrons to sulfite reductase: one with a defect in the recognition of the partner enzyme and the other with an unfavorable redox property. This article summarizes our knowledge about the structure function relationships of plant sulfite reductase.

Thermophilic sulfate and sulfite reduction with methanol in a high rate anaerobic reactor

Thermophilic sulfite and sulfate reduction offers good prospects as part of an alternative technology to conventional off-gas desulfurization technologies. Thermophilic sulfate and sulfite reduction with methanol as the sole carbon and energy source for the sulfate reducing bacteria was studied in lab-scale Expanded Granular Sludge Bed (EGSB) reactors operated at 65 °C and pH 7.5. At a hydraulic retention time (HRT) of 4 hr, sulfite and sulfate elimination rates of up to 0.22 mol-S.l-1.day-1 (100% elimination) and 0.15 mol-S.l-1.day-1 (80% elimination), respectively, were achieved. Sulfite and sulfate reduction accounted for 85–90% of the electrons released during degradation of methanol. In addition, 10–13% and 1–2% of the consumed methanol was converted to acetate and methane, respectively. Acetate was not utilized as electron donor for sulfate reduction. Acetate production seemed to be linearly correlated to the amount of sulfite and sulfate reduced. Sulfite disproportionating activity of the sludge was demonstrated by the simultaneous appearance of sulfide and sulfate in batch tests with sulfite. However, sulfite disproportionation rates were 4 times lower than sulfate reduction rates with methanol. The results clearly demonstrate that methanol can be efficiently used as electron and carbon source to obtain high sulfite and sulfate elimination rates in thermophilic bioreactors.

Characterization of a heme c nitrite reductase from a non-ammonifying microorganism, Desulfovibrio vulgaris Hildenborough

A cytochrome c nitrite reductase (NiR) was purified for the first time from a microorganism not capable of growing on nitrate, the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough. It was isolated from the membranes as a large heterooligomeric complex of 760 kDa, containing two cytochrome c subunits of 56 and 18 kDa. This complex has nitrite and sulfite reductase activities of 685 μmol NH 4 +/min/mg and 1.0 μmol H 2/min/mg. The enzyme was studied by UV–visible and electron paramagnetic resonance (EPR) spectroscopies. The overall redox behavior was determined through a visible redox titration. The data were analyzed with a set of four redox transitions, with an E 0′ of +160 mV (12% of total absorption), −5 mV (38% of total absorption), −110 mV (38% of total absorption) and −210 mV (12% of total absorption) at pH 7.6. The EPR spectra of oxidized and partially reduced NiR show a complex pattern, indicative of multiple heme–heme magnetic interactions. It was found that D. vulgaris Hildenborough is not capable of using nitrite as a terminal electron acceptor. These results indicate that in this organism the NiR is not involved in the dissimilative reduction of nitrite, as is the case with the other similar enzymes isolated so far. The possible role of this enzyme in the detoxification of nitrite and/or in the reduction of sulfite is discussed.

Comparison of four routine methods for the confirmation of Clostridium perfringens in food

In order to compare methods for the confirmation of C. perfringens 52 isolates from strain collections and food samples were examined using four different techniques including the detection of lactose degradation and sulfite reduction in lactose sulfite medium (European Standard EN 13401), examination of nitrate reduction, motility, lactose degradation and gelatine liquefaction in motility–nitrate and lactose–gelatine medium (EN 13401), the reverse CAMP test (German Standard DIN 10103) and the acid phosphatase reaction according to Ueno et al. [Ueno, K., Fujii, H., Marui, T., Takahshi, J., Sugitani, T., Ushijima, T., Suzuki, S. 1970. Acid phosphatase in Clostridium perfringens — A new rapid and simple identification method. Jpn. J. Microbiol. 14, 171–173]. Pure cultures of the test strains were suspended in sterile physiological saline and decimal dilutions inoculated into modified SC agar according to Hauschild and Hilsheimer [Hauschild, A.H.W., Hilsheimer, R., 1974. Evaluation and modifications of media for enumeration of Clostridium perfringens. Appl. Microbiol. 27, 521–526] and Eisgruber and Reuter [Eisgruber, H., Reuter, G., 1995. A selective medium for the detection and enumeration of mesophilic sulphite-reducing clostridia in food monitoring programs. Food Res. Internat. 28, 219–226] by pour-plating. Five individual colonies per test strain (in total 260 colonies) were examined. Using either the reverse CAMP test or the acid phosphatase reaction 94.2% of the colonies were confirmed as C. perfringens. The detection of nitratase and motility in combination with lactose and gelatine degradation enabled the identification of C. perfringens in 88.8% of the colonies. The lowest percentage of C. perfringens colonies was detected via lactose sulfite medium: only 42.7% of the tested colonies showed typical sulfite reduction with gas formation from lactose.

Four crystal structures of the 60 kDa flavoprotein monomer of the sulfite reductase indicate a disordered flavodoxin-like module

Escherichia coli NADPH-sulfite reductase (SiR) is a 780 kDa multimeric hemoflavoprotein composed of eight α-subunits (SiR-FP) and four β-subunits (SiR-HP) that catalyses the six electron reduction of sulfite to sulfide. Each β-subunit contains a Fe4S4 cluster and a siroheme, and each α-subunit binds one FAD and one FMN as prosthetic groups. The FAD gets electrons from NADPH, and the FMN transfers the electrons to the metal centers of the β-subunit for sulfite reduction. We report here the 1.94 Å X-ray structure of SiR-FP60, a recombinant monomeric fragment of SiR-FP that binds both FAD and FMN and retains the catalytic properties of the native protein. The structure can be divided into three domains. The carboxy-terminal part of the enzyme is composed of an antiparallel β-barrel which binds the FAD, and a variant of the classical pyridine dinucleotide binding fold which binds NADPH. These two domains form the canonic FNR-like module, typical of the ferredoxin NADP+ reductase family. By analogy with the structure of the cytochrome P450 reductase, the third domain, composed of seven α-helices, is supposed to connect the FNR-like module to the N-terminal flavodoxine-like module. In four different crystal forms, the FMN-binding module is absent from electron density maps, although mass spectroscopy, amino acid sequencing and activity experiments carried out on dissolved crystals indicate that a functional module is present in the protein. Our results clearly indicate that the interaction between the FNR-like and the FMN-like modules displays lower affinity than in the case of cytochrome P450 reductase. The flexibility of the FMN-binding domain may be related, as observed in the case of cytochrome bc1, to a domain reorganisation in the course of electron transfer. Thus, a movement of the FMN-binding domain relative to the rest of the enzyme may be a requirement for its optimal positioning relative to both the FNR-like module and the β-subunit.

Analysis of reductant supply systems for ferredoxin-dependent sulfite reductase in photosynthetic and nonphotosynthetic organs of maize.

Sulfite reductase (SiR) catalyzes the reduction of sulfite to sulfide in chloroplasts and root plastids using ferredoxin (Fd) as an electron donor. Using purified maize (Zea mays L.) SiR and isoproteins of Fd and Fd-NADP(+) reductase (FNR), we reconstituted illuminated thylakoid membrane- and NADPH-dependent sulfite reduction systems. Fd I and L-FNR were distributed in leaves and Fd III and R-FNR in roots. The stromal concentrations of SiR and Fd I were estimated at 1.2 and 37 microM, respectively. The molar ratio of Fd III to SiR in root plastids was approximately 3:1. Photoreduced Fd I and Fd III showed a comparable ability to donate electrons to SiR. In contrast, when being reduced with NADPH via FNRs, Fd III showed a several-fold higher activity than Fd I. Fd III and R-FNR showed the highest rate of sulfite reduction among all combinations tested. NADP(+) decreased the rate of sulfite reduction in a dose-dependent manner. These results demonstrate that the participation of Fd III and high NADPH/NADP(+) ratio are crucial for non-photosynthetic sulfite reduction. In accordance with this view, a cysteine-auxotrophic Escherichia coli mutant defective for NADPH-dependent SiR was rescued by co-expression of maize SiR with Fd III but not with Fd I.

A new method for the determination of sulphide in gastrointestinal contents and whole blood by microdistillation and ion chromatography

Hydrogen sulphide is produced in the human large intestine by the bacterial reduction of dietary inorganic sulphate and sulphite and by fermentation of sulphur amino acids. Sulphide may damage the colonic epithelium and has been implicated in the pathogenesis of ulcerative colitis. The accurate measurement of sulphide in biological samples, particularly in gut contents is difficult due to the volatile nature of the compound, and the viscosity and turbidity of the samples. Here we describe a method for the determination of sulphide in gut contents and whole blood which overcomes these problems. Initially, samples are treated with zinc acetate to trap sulphide. A microdistillation pretreatment is then used, which releases sulphide from its stable, stored state, coupled to ion chromatography with electrochemical detection. The limit of detection of the method was determined as 2.5 μmol/l, which enabled sulphide levels in gut contents and whole blood samples obtained from humans to be accurately determined. A preliminary investigation in healthy human subjects showed blood sulphide ranged from 10 to 100 μmol/l. Whole blood sulphide did not change significantly when increasing amounts of protein from meat were fed. However, faecal sulphide did show a significant increase from 164 to 754 nmol/g in four subjects fed diets which contained 60 and 420 g meat.

Performance of a thermophilic sulfate and sulfite reducing high rate anaerobic reactor fed with methanol.

Thermophilic sulfate and sulfite reduction was studied in lab-scale Expanded Granular Sludge Bed (EGSB) reactors operated at 65 degrees C and pH 7.5 with methanol as the sole carbon and energy source for the sulfate- and sulfite-reducing bacteria. At a hydraulic retention time (HRT) of 10 h, maximum sulfite and sulfate elimination rates of 5.5 g SO3(2-) L(-1) day(-1) (100% elimination) and 5.7 g SO4(2-) L(-1) day(-1) (55% elimination) were achieved, resulting in an effluent sulfide concentration of approximately 1800 mg S L(-1). Sulfate elimination was limited by the sulfide concentration, as stripping of H2S from the reactor with nitrogen gas was found to increase the sulfate elimination rate to 9.9 g SO4(2-) L(-1) day(-1) (100% elimination). At a HRT of 3 h, maximum achievable sulfite and sulfate elimination rates were even 18 g SO3(2-) L(-1) day(-1) (100% elimination) and 11 g SO4(2-) L(-1) day(-1) (50% elimination). At a HRT of 3 h, the elimination rate was limited by the biomass retention of the system. 5.5 +/- 1.8% of the consumed methanol was converted to acetate, which was not further degraded by sulfate reducing bacteria present in the sludge. The acetotrophic activity of the sludge could not be stimulated by cultivating the sludge for 30 days under methanol-limiting conditions. Omitting cobalt as trace element from the influent resulted in a lower acetate production rate, but it also led to a lower sulfate reduction rate. Sulfate degradation in the reactor could be described by zeroth order kinetics down to a threshold concentration of 0.05 g L(-1), while methanol degradation followed Michaelis-Menten kinetics with a Km of 0.037 g COD L(-1).

Overexpression, purification and immunodetection of DsrD from Desulfovibrio vulgaris Hildenborough.

Dissimilatory sulfite reductase (DsrAB) of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough is an alpha2beta2 tetramer of 180 kDa, encoded by the dsr operon. In addition to the dsrA and dsrB genes, this operon contains a gene (dsrD) encoding a protein of only 78 amino acids. Although, the function of DsrD is currently unknown, the presence of a dsrD gene has been demonstrated in a variety of sulfate-reducing bacteria and archaea. DsrD was expressed in Escherichia coli at a very high level and purified to homogeneity. Protein blotting experiments, using antisera raised against purified DsrD, demonstrated that it is expressed constitutively in D. vulgaris and does not copurify with DsrAB. Spectroscopic analysis of DsrD indicated that it does not bind either sulfite or sulfide, the substrate and product, respectively of the reaction catalyzed by DsrAB. Thus, although the conservation of this protein and its demonstrated presence in D. vulgaris, suggest an essential function in dissimilatory sulfite reduction, this function remains to be elucidated.

Sulfur isotope fractionation during bacterial reduction and disproportionation of thiosulfate and sulfite

In bacterial cultures we measured sulfur isotope fractionation during transformations of thiosulfate (S 2O 3 2−) and sulfite (SO 3 2−), pathways which may be of considerable importance in the cycling of sulfur in marine sediments and euxinic waters. We documented isotope fractionations during the reduction and disproportionation of S 2O 3 2− and SO 3 2− by bacterial enrichments and pure bacterial cultures from marine and freshwater environments. We also measured the isotope fractionation associated with the anoxygenic phototrophic oxidation of H 2S to S 2O 3 2− by cyanobacteria. Except for SO 3 2− reduction, isotope fractionations for these processes have not been previously reported. During the dissimilatory reduction of SO 3 2−, H 2S was depleted in 34S by 6‰, and during the reduction of S 2O 3 2− to H 2S, depletions were between 7‰ and 11‰. The largest observed isotope fractionation was associated with the bacterial disproportionation of SO 3 2− which caused a 34S depletion in H 2S of 20–37‰ and a 34S enrichment in sulfate of 7–12‰. During the bacterial disproportionation of S 2O 3 2−, isotope fractionations between the outer sulfane sulfur and H 2S and between the inner sulfonate sulfur and SO 4 2− were <4‰. We observed isotope exchange between the two sulfur atoms of S 2O 3 2− leading to a depletion of 34S in H 2S by up to 12‰ with a comparable enrichment of 34S in SO 4 2−. No isotope fractionation was associated with the anoxygenic phototrophic oxidation of H 2S to S 2O 3 2−. The depletion of 34S into H 2S during the bacterial reduction and disproportionation of S 2O 3 2− and SO 3 2− may, in addition to sulfate reduction and the bacterial disproportionation of elemental sulfur, contribute to the generation of 34S-depleted sedimentary sulfides.

The effect of molecular oxygen on sulfite reduction by Shewanella putrefaciens

Recently, Shewanella putrefaciens isolated from an industrial cooling water system has been shown to play a role in microbially influenced corrosion. In this study S. putrefaciens isolated from industrial cooling waters was shown to produce low levels of hydrogen sulfide from sulfite at dissolved oxygen concentrations up to 1.5 mg l −1 oxygen. Although oxygen is the preferred electron acceptor with the highest energy yield, some of the electrons were diverted to sulfite. When the starting inoculum was from a sulfite-reducing culture, the growth rate and oxygen depletion rate were lower compared to when the inoculum was from an aerobic culture that did not reduce sulfite, indicating that sulfite reduction occurred concurrently with oxygen reduction. The reduction of sulfite by S. putrefaciens under microaerophilic as well as anaerobic conditions points towards a more significant role of this species in microbially influenced corrosion when growing as a biofilm.

The effect of molecular oxygen on sulfite reduction byShewanella putrefaciens

Recently, Shewanella putrefaciens isolated from an industrial cooling water system has been shown to play a role in microbially influenced corrosion. In this study S. putrefaciens isolated from industrial cooling waters was shown to produce low levels of hydrogen sulfide from sulfite at dissolved oxygen concentrations up to 1.5 mg l−1 oxygen. Although oxygen is the preferred electron acceptor with the highest energy yield, some of the electrons were diverted to sulfite. When the starting inoculum was from a sulfite-reducing culture, the growth rate and oxygen depletion rate were lower compared to when the inoculum was from an aerobic culture that did not reduce sulfite, indicating that sulfite reduction occurred concurrently with oxygen reduction. The reduction of sulfite by S. putrefaciens under microaerophilic as well as anaerobic conditions points towards a more significant role of this species in microbially influenced corrosion when growing as a biofilm.

Iron, sulfur, and chlorophyll deficiencies: A need for an integrative approach in plant physiology

Green plants deficient in nitrogen, sulfur, or iron develop a similar yellow coloration. In each case, the yellow coloration is accompanied by a lowered chlorophyll concentration. This review attempts to collate some of the biochemical information concerning these three seemingly diverse nutritive deficiencies and bares a need for a more integrative approach to plant physiology. The biochemical and biological roles of nitrogen, sulfur and iron in living systems are examined, with emphasis on sulfur and iron. Mechanistically, iron and/or sulfur are highly reactive components of many enzymes. Indeed, iron and sulfur sometimes form Fe2S2, Fe3S4, or Fe4S4 clusters which are very active electron transfer agents. Recently, iron‐sulfur clusters have been reported to serve as sensors of oxidative stress, to couple photosynthesis with several metabolic pathways, to participate in the reduction of sulfite and nitrite, and to participate in regulation of gene expression. Thus, there are several mechanisms by which a deficiency of nitrogen, sulfur, or iron could produce the same low‐chlorophyll, yellow phenotype in plants. Unless the interactions and coordination of the various pathways connected to chlorophyll synthesis are elucidated, it is unlikely that we will select the quickest and most direct path to plant improvement.

Redefining reductive sulfate assimilation in higher plants: a role for APS reductase, a new member of the thioredoxin superfamily?

The reaction steps leading from the intermediate adenosine 5′-phosphosulfate (APS) to sulfide within the higher plant reductive sulfate assimilation pathway are the subject of controversy. Two pathways have been proposed: a ‘bound intermediate’ pathway in which the sulfo group of APS is first transferred by APS sulfotransferase to a carrier molecule to form a bound sulfite intermediate and is then further reduced by thiosulfonate reductase to bound sulfide; and a ‘free intermediate’ pathway in which APS is further activated to 3′-phosphoadenosine 5′-phosphosulfate (PAPS) by APS kinase followed by reduction of the sulfo group to free sulfite by PAPS reductase. Sulfite is then reduced to free sulfide by sulfite reductase. Sulfide, either free or bound, is then incorporated into organic form (as cysteine) by the enzyme O-acetylserine (thiol) lyase. In order to better characterize the pathway we attempted to clone PAPS reductase cDNAs by functional complementation of an Escherichia coli cysH mutant to prototrophy. We found no evidence for PAPS reductase cDNAs but did identify cDNAs that encode a small family of novel, chloroplast-localized proteins with APS reductase activity that are new members of the thioredoxin superfamily. We show here that the thioredoxin domain of these proteins is functional. We speculate that rather than proceeding via either of the pathways proposed above, reductive sulfate assimilation proceeds via the reduction of APS to sulfite by APS reductase and the subsequent reduction of sulfite to sulfide by sulfite reductase. In this scheme the product of the APS kinase reaction, PAPS, is not a direct intermediate in the pathway but rather acts as a substrate for sulfotransferase action and perhaps as a store of activated sulfate that can be returned to the pathway as APS via phosphohydrolase action on PAPS. Interactions between enzyme isoforms within the chloroplast stroma may bring about substrate channeling of APS and contribute to the partitioning of APS between sulfotransferase reactions on the one hand and the synthesis of cysteine and related metabolites via the reductive sulfate assimilation pathway on the other.