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APS reductase from Pseudomonas aeruginosa has been shown to form a disulfide-linked adduct with mono-cysteine variants of Escherichia coli thioredoxin and Chlamydomonas reinhardtii thioredoxin h1. These adducts presumably represent trapped versions of the intermediates formed during the catalytic cycle of this thioredoxin-dependent enzyme. The oxidation–reduction midpoint potential of the disulfide bond in the P. aeruginosa APS reductase/C. reinhardtii thioredoxin h1 adduct is −280 mV. Site-directed mutagenesis and mass spectrometry have identified Cys256 as the P. aeruginosa APS reductase residue that forms a disulfide bond with Cys36 of C. reinhardtii TRX h1 and Cys32 of E. coli thioredoxin in these adducts. Spectral perturbation measurements indicate that P. aeruginosa APS reductase can also form a non-covalent complex with E. coli thioredoxin and with C. reinhardtii thioredoxin h1. Perturbation of the resonance Raman and visible-region absorbance spectra of the APS reductase [4Fe–4S] center by either APS or the competitive inhibitor 5′-AMP indicates that both the substrate and product bind in close proximity to the cluster. These results have been interpreted in terms of a scheme in which one of the redox-active cysteine residues serves as the initial reductant for APS bound at or in close proximity to the [4Fe–4S] cluster.

The sulfur oxygenase reductase (SOR) is the initial enzyme in the aerobic sulfur metabolism of the thermoacidophilic and chemolithoautotrophic crenarchaeote Acidianus ambivalens. Single colorless polyhedral crystals were obtained under two crystallization conditions from SOR preparations heterologously overproduced in Escherichia coli. They belonged to space-group I4 and diffraction data were collected up to 1.7 A resolution. Their Patterson symmetry shows additional 4-, 3- and 2-fold non-crystallographic symmetry rotation axes, characteristic of the point group 432. Taking into account the molecular mass of SOR, the crystal unit cell volume, the non-crystallographic symmetry operators and previous electron microscopy studies of the SOR, it was deduced that the quaternary structure of the functionally active enzyme is an icosatetramer with 871 kDa molecular mass.

Molybdenum cofactor deficiency is a fatal neurological disorder, which follows an autosomal-recessive trait and is characterized by combined deficiency of the enzyme, sulfite oxidase, xanthine dehydrogenase and aldehyde oxidase. Early detection of molybdenum cofactor-deficient patients is essential for their proper care and genetic counseling of families at risk. We demonstrate the use of S-sulfonated transthyretin (TTR) as a marker for molybdenum cofactor deficiency. Plasma or sera obtained from 4 patients with molybdenum cofactor deficiency and 57 controls were studied by electrospray ionization mass spectrometry (ESIMS) following selective enrichment of TTR by immunoprecipitation using protein G/A agarose. The data obtained from molybdenum cofactor deficiency samples indicated a strong increase in the peak height of S-sulfonated TTR. A more significant difference was revealed if the peak height ratio of S-sulfonated TTR and the sum of the other oxidized TTR were determined. By accurate determination of the ratio, the samples of molybdenum cofactor deficiency patients could clearly be distinguished from controls without molybdenum cofactor deficiency.

Dibenzothiophene (DBT), a model of organic sulfur compound in petroleum, is microbially desulfurized to 2-hydroxybiphenyl (2-HBP), and the gene operon dszABC was required for DBT desulfurization. The final step in the microbial DBT desulfurization is the conversion of 2'-hydroxybiphenyl-2-sulfinate (HBPSi) to 2-HBP catalyzed by DszB. In this study, DszB of a DBT-desulfurizing bacterium Rhodococcus erythropolis KA2-5-1 was overproduced in Escherichia coli by coexpression with chaperonin genes, groEL/groES, at 25 degrees C. The recombinant DszB was purified to homogeneity and characterized. The optimal temperature and pH for DszB activity were 35 degrees C and about 7.5, respectively. The K(m) and k(cat) values for HBPSi were 8.2 microM and 0.123.s(-1), respectively. DszB has only one cysteine residue, and the mutant enzyme completely lost the activity when the cysteine residue was changed to a serine residue. This result together with experiments using inhibitors showed that the cysteine residue contributes to the enzyme activity. DszB was also inhibited by a reaction product, 2-HBP (K(i)=0.25 mM), and its derivatives, but not by the other reaction product, sulfite. The enzyme showed a narrow substrate specificity: only 2-phenylbenzene sulfinate except HBPSi served as a substrate among the aromatic and aliphatic sulfinates or sulfonates tested. DszB was thought to be a novel enzyme (HBPSi desulfinase) in that it could specifically cleave the carbon-sulfur bond of HBPSi to give 2-HBP and sulfite ion without the aid of any other proteinic components and coenzymes.

A cDNA with an open reading frame of 1929 bp (termed sir) was isolated from a λZapII library of Arabidopsis thaliana leaf tissue. The polypeptide sequence deduced from the cDNA is homologous to the ferredoxin-dependent sulfite reductase (EC 1.8.7.1) from Synechococcus PCC7942 and distantly related to the hemoprotein subunit of Escherichia coli NADPH-dependent sulfite reductase (EC 1.8.1.2). A molecular mass of 71.98 kDa can be predicted for a ferredoxin sulfite reductase from A. thaliana. The polypeptide consists of 642 amino acids including a transit peptide of 66 residues (6.72 kDa) that is assumed to direct the protein into the plastid. For expression and enzymatic characterization of a putative A. thaliana ferredoxin sulfite reductase, the DNA of the transit peptide was deleted by a PCR method. The truncated cDNA clone was expressed as his-tag fusion protein. The modified gene product was enzymatically inactive but specific cross-reaction with polyclonal antibodies against ferredoxin sulfite reductase from Synechococcus is seen as confirmation of its identity as higher plant ferredoxin sulfite reductase.

A 2.4 kilobase cDNA clone of human sulfite oxidase was isolated from a human liver cDNA library in λgt10. Comparison of three sulfite oxidase sequences to several plant and fungal nitrate reductase sequences reveals a single conserved cysteine with highly conserved flanking sequences. The conserved cysteine is postulated to be a ligand of molybdenum in sulfite oxidase and nitrate reductase.

The structural gene of the ferredoxin:sulphite reductase (EC 1.8.7.1) from the cyanobacterium Synechococcus PCC7942 (formerly 'Anacystis nidulans') was cloned and sequenced. The gene termed 'sir' was detected by heterologous Southern hybridisation with the structural gene cysI from Escherichia coli encoding the iron-sulphur haemoprotein of the NADPH:sulphite reductase. The open reading frame is comprised of 1875 bp encoding for a polypeptide of M(r) 70.028. The deduced amino acid sequence is 35.6% identical with the enterobacterial iron-sulphur haemoprotein. This putative fd-dependent sulphite reductase is only distantly related to the fd-dependent nitrite reductase (binary matching coefficient SAB: 0.23) or with the NADPH-sulphite reductase (SAB: 0.32). Highly conserved residues are found within the two Cys clusters forming the reactive Fe4S4-sirohaem centre of the enzyme. Expression of the sir gene using a fusion vector gave a single gene product which is immunologically related with the fd-sulphite reductase from the wild-type bacterium.

We have obtained direct evidence that the majority of the sirohydrochlorin chromophore in the dissimilatory sulfite reductase desulfoviridin from Desulfovibrio gigas, is not associated with any metal. The evidence comes from resonance Raman measurements of native and deuterated samples of desulfoviridin. The breathing mode v4 (or v4*) at 1336 cm-1 in the native enzyme is downshifted to 1326 cm-1 upon deuteration. This mode is not sensitive to deuteration if a metal is present at the center of the chromophore inside protein or in solution. The results also establish the existence of exchangeable core hydrogen(s) at the pyrrolic nitrogen(s).

Mammalian tissues show significant differences in the activity of sulfite oxidase (EC 1.8.3.1) which detoxifies sulfite by oxidation to sulfate. Lung tissue and phagocytic cells such as alveolar macrophages, peritoneal macrophages, Kupffer cells and granulocytes show very low activities of sulfite oxidase. Liver tissue and hepatocytes, however, exhibit high activities of sulfite oxidase. Lung tissue and macrophages show an almost 100% decrease of the intracellular ATP levels when incubated with 1 mM sulfite at pH 6 for 30 min. In addition, the O2 consumption of lung tissue is inhibited by 1 mM sulfite at pH 6 by more than 80%. This sulfite-induced decrease of the ATP level and of the O2 consumption of lung tissue is enhanced between pH 6.0 and pH 7.4 with decreasing pH value of the incubation medium. In contrast, the ATP levels in liver tissue and hepatocytes are not affected by 1 mM sulfite at pH 6. The O2 consumption of liver tissue and hepatocytes is significantly increased by sulfite due to the high activities of sulfite oxidase. Therefore, the activity of the 'sulfite-detoxifying enzyme' sulfite oxidase and the sensitivity of the energy metabolism to sulfite show a reciprocal relationship in the tissues and cells studied.

Desulphoviridin in the oxidized state showed EPR signals around g = 6, consistent with the sirohaem being in the high-spin ferric state. This was unreactive with sulphite, sulphide or cyanide; but readily reduced by methyl viologen. When the enzyme was treated with Na 2 S 2 O 4 the sirohaem was slowly reduced and a spectrum of a reduced iron-sulphur cluster at g = 2.07, 1.93, 1.91 appeared over the course of an hour. An intermediate in this reaction was indicated by a free radical signal which appeared within seconds and then gradually disappeared. On treatment with nitrite and reduced methyl viologen, the enzyme gave a spectrum of a nitroxide derivative similar to that seen with plant nitrite reductase. The midpoint reduction potential of the haem was estimated to be −310 mV or less. The iron-sulphur cluster has a very low potential, being only reduced in the presence of free Na 2 S 2 O 4 around −560 mV. Desulphoviridin can be classed with sirohaem-containing iron-sulphur proteins.

Thiosulfate reductase purified 900-fold from an extract of baker's yeast by a new procedure consisted of three charge-isomeric species, all with catalytic activity. Amino acid analysis of thiosulfate reductase and titrations with 5,5'-dithiobis(2-nitrobenzoate) and iodo[14C]acetate indicated only one cysteine residue per enzyme molecule. Alkylation of this cysteine with either iodoacetate or iodoacetamide did not inactivate the enzyme, indicating that sulfur-sulfur bond cleavage in the thiosulfate substrate does not require an enzymic sulfhydryl group as attacking nucleophile.

Yeast NADPH-sulfite reductase (hydrogen sulfide:NADP+ oxidoreductase, EC 1.8.1.2) is a complex hemoflavoprotein. The Strokes radius was determined to be 80 A by gel filtration and the molecular weight was estimated to be 604,000. The minimal molecular weight calculated from flavin and heme content was 306,000, indicating that this enzyme contains two FAD, two FMN and two hemes per molecule. The enzyme consists of two types of subunit, alpha and beta, having molecular weights of 116,000 and 167,000, respectively. The subunit structure is suggested to be alpha 2 beta 2. The secondary structure, the amino acid composition and the isoelectric point were also investigated. The Km values for sulfite and NADPH were 17 microM and 10 microM, respectively. Sulfite and NADPH affected the reaction velocity to give parallel Lineweaver-Burk plots, indicating the involvement of the 'ping-pong' mechanism in the overall reaction. NADP+ inhibited the reaction competitively with NADPH and noncompetitively with sulfite. Inhibition by sulfide was partially noncompetitive with both substrates but very weak. These results are discussed and compared with sulfite reductase from Escherichia coli.

The effects of ionic strength on the structure of yeast sulfite reductase (EC 1.8.1.2) were investigated. Gel filtration and sedimentation analysis revealed that this enzyme dissociated at ionic strength below 0.1 into two components having molecular weights of 30 0000 and sedimentation coefficients of 7–9 S. The secondary structure and the surroundings of the prosthetic groups were not appreciably affected by ionic strength. This enzyme showed CD spectra in the near-ultraviolet region considered to be due to tyrosine residues. On lowering the ionic strength, they decreased, together with the disappearance of the difference spectra in the same region, indicating that some tyrosine residues became exposed to the environment at low ionic strength. SH groups were also exposed at low ionic strength and reacted with DTNB and PCMB more easily to cause the loss of NADPH-sulfite reductase activity. These essential SH groups as well as some tyrosine residues may exist in the region of contact between the two components constituting the enzyme molecule.

An assay procedure for adenosine 5′-phosphosulfate reductase from Desulfovibrio desulfuricans and Thiobacillus thioparus is described that utilizes the AMP-dependent reduction of ferricyanide occurring in the presence of SO32−, and optimum conditions for the assay are reported. The stoichiometry of this oxidation was determined and indicates that the reaction proceeds as follows: AMP + SO32− + 2Fe(CN)63− → adenosine 5′-phosphosulfate + 2Fe(CN)64−. Other nucleotides, deAMP, IMP, and GMP, can replace AMP in the reaction. Employing this assay, adenosine 5′-phos phosulfate reductase was purified from extracts of D. desulfuricans, and the purified enzyme, essentially homogenous in the ultracentrifuge, contains flavin adenine dinucleotide and iron. Inhibition of the enzyme by p-hydroxymercuribenzoate and reversal of the inhibition by monothiols indicate that one or more sulfhydryl groups are required for activity.