Identification and cloning of genes involved in anaerobic sulfite reduction by Salmonella typhimurium.

Transposon Tn5 insertions causing anaerobic cysteine auxotrophy were isolated from a Salmonella typhimurium cysI parent (auxotrophic under aerobic but not anaerobic conditions). Insertions in one mutant group appeared to be in cysG. A second group of insertions, designated asr (anaerobic sulfite reduction), were located near map unit 53 on the S. typhimurium chromosome. They did not cause aerobic or anaerobic auxotrophy in a cys1+ background but did prevent dissimilatory sulfite reduction. Plasmids containing asr DNA cloned from wild-type S. typhimurium conferred anaerobic prototrophy and the ability to produce hydrogen sulfide from sulfite on an Escherichia coli cys1 mutant.     

Cysteine auxotrophs of Salmonella typhimurium which grow without cysteine in a hydrogen/carbon dioxide atmosphere.

Cysteine auxotrophs of Salmonella typhimurium mutated in cysB, cysI or cysJ grew with sulphate as a sulphur source when incubated under a hydrogen/carbon dioxide atmosphere. Yields obtained under these conditions were equivalent to those characteristic of wild-type S. typhimurium. The same mutants failed to grow with sulphate as a sulphur source when incubated aerobically. Auxotrophs mutated in cysA, cysC, cysD, cysE, cysG and cysH required cysteine for growth under both incubation conditions. The results suggest that mutations in cysB (regulation of the several cys operons) and also cysI and cysJ (sulphite reductase activity) can be circumvented during anaerobic growth under hydrogen.     

Cloning of the 3'-phosphoadenylyl sulfate reductase and sulfite reductase genes from Escherichia coli K-12

The structural genes for 3'-phosphoadenylyl sulfate (PAPS) reductase (cysH) and sulfite reductase (alpha and beta subunits; EC 1.8.1.2)(cysI and cysJ) of Escherichia coli K-12 have been cloned by complementation. pCYSI contains two PstI fragments (18.3 and 2.9 kb) which complement cysH-, cysI-, and cysJ- mutants. Subcloning showed that the cysH gene is located on a 1.6-kb ClaI subfragment (pCYSI-3) whereas cysI and most of cysJ are carried on a 3.7-kb ClaI subfragment (pCYSI-5). The PAPS reductase gene is closely linked to the sulfite reductase genes, but its expression is regulated by a unique promoter. The cysI and cysJ genes, on the other hand, are transcribed as an operon and the promoter precedes the cysI gene. Maxicell analysis demonstrated that pCYSI encodes three polypeptides of Mr 27,000, 57,000, and 60,000, in addition to the tetracycline-resistance determinant. The 60- and 57-kDa proteins are most likely the alpha and beta subunits, respectively, of E. coli sulfite reductase while the 27-kDa protein is putatively identified as PAPS reductase. Preliminary data suggest that the alpha and beta subunits of sulfite reductase are encoded by cysI and cysJ, respectively.     

Characterization of the cysJIH regions of Salmonella typhimurium and Escherichia coli B. DNA sequences of cysI and cysH and a model for the siroheme-Fe4S4 active center of sulfite reductase hemoprotein based on amino acid homology with spinach nitrite reductase

The hemoprotein component of Salmonella typhimurium sulfite reductase (NADPH) (EC 1.8.1.2) was purified to homogeneity from cysJ266, a mutant strain lacking sulfite reductase flavoprotein. The siroheme- and Fe4S4-containing enzyme was isolated as a monomeric 63-kDa polypeptide and consisted of a mixture of unligated enzyme and a complex with sulfite. Following reduction with 5'-deazaflavin-EDTA and reoxidation, the complex was converted to the uncomplexed, high spin ferri-siroheme state seen previously with Escherichia coli sulfite reductase hemoprotein preparations. The S. typhimurium hemoprotein exhibited catalytic and physical properties identical to the hemoprotein prepared by urea dissociation of E. coli sulfite reductase holoenzyme and was fully competent in reconstituting NADPH-sulfite reductase activity when combined with excess purified sulfite reductase flavoprotein. The DNA sequences of cysI and cysH from S. typhimurium and E. coli B were determined and, together with previously reported data, confirmed the organization of this region as promoter-cysJ-cysI-cysH with all three genes oriented in the same direction from the promoter. Molecular weights deduced for the cysI-encoded sulfite reductase hemoprotein and for the cysH-encoded 3'-phosphoadenosine 5'-phosphosulfate sulfotransferase were approximately 64,000 and 28,000, respectively. Comparison of the deduced amino acid sequence of sulfite reductase hemoprotein with that of spinach nitrite reductase (Back, E., Burkhart, W., Moyer, M., Privalle, L., and Rothstein, S. (1988) Mol. Gen. Genet. 212, 20-26), which also contains siroheme and an Fe4S4 cluster, showed two groups of cysteine-containing sequences with the structures Cys-(X)3-Cys and Cys-(X)5-Cys, which are homologous in the two enzymes and are postulated to provide the ligands of the Fe4S4 cluster in both proteins. From these sequences and from crystallographic (McRee, D. E., Richardson, D. C., Richardson, J. S., and Siegel, L. M. (1986) J. Biol. Chem. 261, 10277-10281) and spectroscopic data in the literature, a model is proposed for the structure of the active center of these two enzymes.     

Structure-based alteration of substrate specificity and catalytic activity of sulfite oxidase from sulfite oxidation to nitrate reduction

Eukaryotic sulfite oxidase is a dimeric protein that contains the molybdenum cofactor and catalyzes the metabolically essential conversion of sulfite to sulfate as the terminal step in the metabolism of cysteine and methionine. Nitrate reductase is an evolutionarily related molybdoprotein in lower organisms that is essential for growth on nitrate. In this study, we describe human and chicken sulfite oxidase variants in which the active site has been modified to alter substrate specificity and activity from sulfite oxidation to nitrate reduction. On the basis of sequence alignments and the known crystal structure of chicken sulfite oxidase, two residues are conserved in nitrate reductases that align with residues in the active site of sulfite oxidase. On the basis of the crystal structure of yeast nitrate reductase, both positions were mutated in human sulfite oxidase and chicken sulfite oxidase. The resulting double-mutant variants demonstrated a marked decrease in sulfite oxidase activity but gained nitrate reductase activity. An additional methionine residue in the active site was proposed to be important in nitrate catalysis, and therefore, the triple variant was also produced. The nitrate reducing ability of the human sulfite oxidase triple mutant was nearly 3-fold greater than that of the double mutant. To obtain detailed structural data for the active site of these variants, we introduced the analogous mutations into chicken sulfite oxidase to perform crystallographic analysis. The crystal structures of the Mo domains of the double and triple mutants were determined to 2.4 and 2.1 ? resolution, respectively.     

Evidence for sulfite as an essential metabolite for human peripheral lymphocytes

Sulfite has been identified as an essential metabolite by means of growth studies using a chemically-defined, protein-free medium for culture of human peripheral lymphocytes. Sulfite reduced the amount of cysteine required for optimum growth by at least four-fold. In some subjects, sulfite stimulated growth even in the presence of optimal amounts of cysteine indicating that lymphocytes of some individuals are unable to convert cysteine to sulfite in adequate amounts.     

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.     

Sulfite reduction in mycobacteria

Mycobacterium tuberculosis places an enormous burden on the welfare of humanity. Its ability to grow and its pathogenicity are linked to sulfur metabolism, which is considered a fertile area for the development of antibiotics, particularly because many of the sulfur acquisition steps in the bacterium are not found in the host. Sulfite reduction is one such mycobacterium-specific step and is the central focus of this paper. Sulfite reduction in Mycobacterium smegmatis was investigated using a combination of deletion mutagenesis, metabolite screening, complementation, and enzymology. The initial rate parameters for the purified sulfite reductase from M. tuberculosis were determined under strict anaerobic conditions [k(cat) = 1.0 (+/-0.1) electron consumed per second, and K(m(SO(3)(-2))) = 27 (+/-1) microM], and the enzyme exhibits no detectible turnover of nitrite, which need not be the case in the sulfite/nitrite reductase family. Deletion of sulfite reductase (sirA, originally misannotated nirA) reveals that it is essential for growth on sulfate or sulfite as the sole sulfur source and, further, that the nitrite-reducing activities of the cell are incapable of reducing sulfite at a rate sufficient to allow growth. Like their nitrite reductase counterparts, sulfite reductases require a siroheme cofactor for catalysis. Rv2393 (renamed che1) resides in the sulfur reduction operon and is shown for the first time to encode a ferrochelatase, a catalyst that inserts Fe(2+) into siroheme. Deletion of che1 causes cells to grow slowly on metabolites that require sulfite reductase activity. This slow-growth phenotype was ameliorated by optimizing growth conditions for nitrite assimilation, suggesting that nitrogen and sulfur assimilation overlap at the point of ferrochelatase synthesis and delivery.     

Studies on the Sulfite Reducing System of Algae

Sulfite reductase activity by algal extracts was investigated using reduced methylviologen as a hydrogen donor. Sulfite reductase appears to be widely distributed in various algae, but the enzymatic activity was not detected in the brown algae examined. The addition of phosphate buffer to the reaction mixture caused a marked decrease in activity. Sulfite reductase was partially purified from the autolysate of Porphyra tenera and some properties were studied. The optimal pH was 7.5 to 8.5 in Tris-HGl buffer system. The Km for sulfite was 6.65 × 10^?4m. The enzymatic activity was completely inhibited by potassium cyanide at 5 × 10^?4m. The enzyme catalyzed the reduction of sulfite to sulfide. Neither NADPH nor NADH acts as a hydrogen donor. However, it was revealed that ferredoxin can act as an electron carrier in sulfite reduction to sulfide in Porphyra extract.     

Radiation inactivation of hepatic sulfite oxidase

Sulfite oxidase (EC 1.8.3.1), purified from chicken liver, is comprised of two identical subunits of 55 kDa, each of which contains a molybdenum and heme prosthetic group. The functional size of sulfite oxidase was determined by radiation inactivation analysis using both full, sulfite:cytochrome c reductase, and partial, sulfite:ferricyanide reductase, catalytic activities. Inactivation of full enzyme activity indicated a target size of 42 kDa while the partial activity indicated a target size of 25 kDa. These results confirm the earlier findings of two equivalent subunits and suggest the presence of a functional domain within the subunit structure that contains the molybdenum center and exhibits a smaller molecular mass than that of the enzyme subunit.     

Mechanism of dissimilatory sulfite reduction by Desulfovibrio desulfuricans: purification of a membrane-bound sulfite reductase and coupling iwth cytochrome c 3 and hydrogenase

The localization of the dissimilatory sulfite reductase in Desulfovibrio desulfuricans strain Essex 6 was investigated. After treatment of the cells with lysozyme, 90% of the sulfite reductase activity was found in the membrane fraction, compared to 30% after cell rupture with the French press. Sulfite reductase was purified from the membrane (mSiR) and the soluble (sSiR) fractiion. On SDS-PAGE, both mSiR and sSiR exhibited three bands at 50, 45 and 11 kDa, respectively. From their UV/VIS properties (distinct absorption maxima at 391, 410, 583, 630 nm, enzymes as isolated) and the characteristic red fluorescence in alkaline solution, mSiR and sSiR were identified as desulfoviridin. Sulfite reductase (HSO₃ ^-H₂S) activity was reconstituted by coupling of mSiR to hydrogenase and cytochrome c ₃ from D. desulfuricans. The specific activity of mSiR was 103 nmol H₂ min^-1 mg^-1, and sulfide was the major product (72% of theoretical yield). No coupling was found with sSiR under these conditions. Furthermore, carbon monoxide was used to diferentiate between the membrane-bound and the soluble sulfite reductase. In a colorimetric assay, with photochemically reduced methyl viologen as redox mediator, CO stimulated the activity of sSiR significantly. CO had no effect in the case of mSiR. These studies documented that, as isolated, both forms of sulfite reductase behaved differently in vitro. Clearly, in D. desulfuricans, the six electron conversion HSO₃ ^-H₂S was achieved by a membranebound desulfoviridin without the assistance of artificial redox mediators, such as methyl viologen.Abbreviations SiR sulfite reductase - mSiR sulfite reductase purified from membranes - sSiR sulfite reductase purified from the soluble fraction Enzymes Sulfite reductase, EC 1.8.99.1 Cytochrome c ₃ hydrogenase, EC 1.12.2.1     

Binding and reduction of sulfite by cytochrome c nitrite reductase

Pentaheme cytochrome c nitrite reductase (ccNiR) catalyzes the six-electron reduction of nitrite to ammonia as the final step in the dissimilatory pathway of nitrate ammonification. It has also been shown to reduce sulfite to sulfide, thus forming the only known link between the biogeochemical cycles of nitrogen and of sulfur. We have found the sulfite reductase activity of ccNiR from Wolinella succinogenes to be significantly smaller than its nitrite reductase activity but still several times higher than the one described for dissimilatory, siroheme-containing sulfite reductases. To compare the sulfite reductase activity of ccNiR with our previous data on nitrite reduction, we determined the binding mode of sulfite to the catalytic heme center of ccNiR from W. succinogenes at a resolution of 1.7 A. Sulfite and nitrite both provide a pair of electrons to form the coordinative bond to the Fe(III) active site of the enzyme, and the oxygen atoms of sulfite are found to interact with the three active site protein residues conserved within the enzyme family. Furthermore, we have characterized the active site variant Y218F of ccNiR that exhibited an almost complete loss of nitrite reductase activity, while sulfite reduction remained unaffected. These data provide a first direct insight into the role of the first sphere of protein ligands at the active site in ccNiR catalysis.     

Cysteine biosynthesis in a fungus, Histoplasma capsulatum.

We have analyzed a step in cysteine biosynthesis in several strains of the pathogenic dimorphic fungus, Histoplasma capsulatum. Mycelial cells of all strains tested are prototrophic. However, the yeast phase cells of most stains do not grow in the absence of -SH-containing compounds due to the apparent lack of an active form of sulfite reductase, a crucial enzyme in the cysteine biosynthetic pathway. In contrast, the yeast phase cells of one strain (Downs) have been found to have an active sulfite reductase and can grow in the absence of cysteine if serine is added. A different metabolic block must thus exist in this strain. Sulfite reductase in the yeast form of Downs strain is completely repressed by growth on cysteine while the mycelial form seems to be constitutive. The yeast and mycelial phase extracts were analyzed on polyacrylamide gels. A distinct protein band appeared in extracts prepared from the yeast cells incubated in minimal or serine-containing media, but not in extracts from mycelia or from cysteine-grown yeast cells.     

Expression, purification and characterization of the sulfite reductase hemo-subunit, SiR-HP, from Acidithiobacillus ferrooxidans

Sulfite reductase (SiR) is a large and soluble enzyme which catalyzes the transfer of six electrons from NADPH to sulfite to produce sulfide. The sulfite reductase flavoprotein (SiR-FP) contains both FAD and FMN, and the sulfite reductase hemoprotein (SiR-HP) contains an iron-sulfur cluster coupled to a siroheme. The enzyme is arranged so that the redox cofactors in the FAD-FMN-Fe(4)S(4)-Heme sequence make an electron pathway between NADPH and sulfite. Here we report the cloning, expression, and characterization of the SiR-HP of the sulfite reductase from Acidithiobacillus ferrooxidans. The purified SiR-HP contained a [Fe(4)S(4)] cluster. Site-directed mutagenesis results revealed that Cys427, Cys433, Cys472 and Cys476 were in ligating with the [Fe(4)S(4)] cluster of the protein.     

Symbiotic characteristics of cysteine and methionine auxotrophs of Sinorhizobium meliloti

Twenty one cysteine and 13 methionine auxotrophs of Sinorhizobium meliloti Rmd201 were obtained by random mutagenesis with transposon Tn5. The cysteine auxotrophs were sulfite reductase mutants and each of these auxotrophs had a mutation in cysI/cysJ gene. The methionine auxotrophs were metA/metZ, metE and metF mutants. One hundred per cent co-transfer of Tn5-induced kanamycin resistance and auxotrophy from each Tn5-induced auxotrophic mutant indicated that each mutant cell most likely had a single Tn5 insertion. However, the presence of more than one Tn5 insertions in the auxotrophs used in our study cannot be ruled out. All cysteine and methionine auxotrophs induced nodules on alfalfa plants. The nodules induced by cysteine auxotrophs were fully effective like those of the parental strain-induced nodules, whereas the nodules induced by methionine auxotrophs were completely ineffective. The supplementation of methionine to the plant nutrient medium completely restored symbiotic effectiveness to the methionine auxotrophs. These results indicated that the alfalfa host provides cysteine but not methionine to rhizobia during symbiosis. Histological studies showed that the defective symbiosis of methionine auxotrophs with alfalfa plants was due to reduced number of infected nodule cells and incomplete transformation of bacteroids.     

Cysteine transport and sulfite reductase activity in a germination-defective mutant of Histoplasma capsulatum.

A mutant of the dimorphic, zoopathogenic fungus Histoplasma capsulatum, defective in the ability of its blastospores to germinate, was used to show that the nutritional requirement for cysteine and the expression of sulfite reductase activity are temperature dependent and not related to the growth form the fungus, whereas the kinetics of cysteine transport depend on both temperature and the form of the fungus.     

Studies on the Sulfite Reducing System of Algae

Sulfite reductase using reduced methyl viologen as an electron donor was purified about 94-fold from a red alga, Porphyra yezoensis. The enzyme was ultracentrifugically homogenous and could reduce sulfite to sulfide quantitatively with an uptake of six electrons. The enzyme had a pH optimum in the vicinity of 7.5. The Km for sulfite was determined to be 6.5×l0^?4m. The purified preparation of the algal reductase showed its absorption maximum at 385 mμ and slight shoulders at 408, 456, 485, 600 and 664 mμ in addition to an intense peak at 278 mμ. Metal analysis of the purified enzyme suggested the presence of iron and copper in the molecule. NADPH, NADH or the reduced form of spinach ferredoxin could not be a direct electron donor for the purified algal sulfite reductase.