Characterization of a HMT2-like enzyme for sulfide oxidation from Pseudomonas putida

The open reading frame pp0053, which has a high homology with the sequence of mitochondrial sulfide dehydrogenase (HMT2) conferring cadmium tolerance in fission yeast, was amplified from Pseudomonas putida KT2440 and expressed in Escherichia coli JM109(DE3). The isolated and purified PP0053-His showed absorption spectra typical of a flavin adenine dinucleotide (FAD)--binding protein. The PP0053-His catalyzed a transfer of sulfide-sulfur to the thiophilic acceptor, cyanide, which decreased the Km value of the enzyme for sulfide oxidation and elevated the sulfide-dependent quinone reduction. Reaction of the enzyme with cyanide elicited a dose-dependent formation of a charge transfer band, and the FAD-cyanide adduct was supposed to work for a sulfur transfer. The pp0053 deletion from P. putida KT2440 led to activity declines of the intracellular catalase and ubiquinone-H2 oxidase. The sulfide-quinone oxidoreductase activity in P. putida KT2440 was attributable to the presence of pp0053, and the activity showed a close relevance to enzymatic activities related to sulfur assimilation.     

排硫硫杆菌D6中硫化氢脱氢酶的纯化及性质

从烟气生物脱硫系统的好氧产硫磁性稳态流化床反应器中,经反复纯化分离出脱硫优势菌排硫硫杆菌菌株D6,采用四步工艺纯化出膜结合型硫化氢脱氢酶。SDS-PAGE测定显示其由α1β1亚基组成,光谱分析表明含有1 mol FAD/mol酶,血红素染色揭示小亚基上结合有1 mol血红素c/mol酶,该酶属于氧还蛋白家族。该酶的最适pH为8.6,对马心细胞色素c和硫化物的表观Km分别为2.5μmol/L和6.1μmol/L,反应计量实验表明其氧化产物为元素硫。硫化氢脱氢酶受到硫和亚硫酸盐的抑制,100μmol/L的氰化钾对该酶抑制率达72%。     

Sulfide:quinone oxidoreductase in membranes of the hyperthermophilic bacterium Aquifex aeolicus (VF5)

The sulfide-dependent reduction of exogenous ubiquinone by membranes of the hyperthermophilic chemotrophic bacterium Aquifex aeolicus (VF5), the sulfide-dependent consumption of oxygen and the reduction of cytochromes by sulfide in membranes were studied. Sulfide reduced decyl-ubiquinone with a maximal rate of up to 3.5 micromol (mg protein)(-1) min(-1) at 20 degrees C. Rates of 220 nmol (mg protein)(-1) min(-1)] for the sulfide-dependent consumption of oxygen and 480 nmol (mg protein)(-1) min(-1) for the oxidation of sulfide at 20 C were estimated. The reactions were sensitive towards 2-n-nonyl-4-hydroxyquinoline-N-oxide, but insensitive towards cyanide. Both reduction of decyl-ubiquinone and consumption of oxygen by sulfide rapidly increased with increasing temperature. For the sulfide-dependent respiratory activity, a sulfide-to-oxygen ratio of 2.3+/-0.2 was measured. This indicates that sulfide was oxidized to the level of zero-valent sulfur. Reduction of cytochromes by sulfide was monitored with an LED-array spectrophotometer. Reduction of cytochrome b was stimulated by 2-n-nonyl-4-hydroxyquinoline-N-oxide in the presence of excess sulfide under oxic conditions. This "oxidant-induced reduction" of cytochrome b suggests that electron transport from sulfide to oxygen in A. aeolicus employs the cytochrome bc complex via the quinone pool. Comparison of the amino acid sequence with the sequence of the sulfide:quinone oxidoreductase from Rhodobacter capsulatus and of the flavocytochrome c from Allochromatium vinosum revealed that the sulfide:quinone oxidoreductase from A. aeolicus belongs to the glutathione reductase family of flavoproteins.     

Identification and analysis of the genes coding for the putative pyruvate dehydrogenase enzyme complex in Acholeplasma laidlawii.

A monospecific antibody recognizing two membrane proteins in Acholeplasma laidlawii identified a plasmid clone from a genomic library. The nucleotide sequence of the 4.6-kbp insert contained four sequential genes coding for proteins of 39 kDa (E1 alpha, N terminus not cloned), 36 kDa (E1 beta), 57 kDa (E2), and 36 kDa (E3; C terminus not cloned). The N termini of the cloned E2, E1 beta, and native A. laidlawii E2 proteins were verified by amino acid sequencing. Computer-aided searches showed that the translated DNA sequences were homologous to the four subenzymes of the pyruvate dehydrogenase complexes from gram-positive bacteria and humans. The plasmid-encoded 57-kDa (E2) protein was recognized by antibodies against the E2 subenzymes of the pyruvate and oxoglutarate dehydrogenase complexes from Bacillus subtilis. A substantial fraction of the E2 protein as well as part of the pyruvate dehydrogenase enzymatic activity was associated with the cytoplasmic membrane in A. laidlawii. In vivo complementation with three different Escherichia coli pyruvate dehydrogenase-defective mutants showed that the four plasmid-encoded proteins were able to restore pyruvate dehydrogenase enzyme activity in E. coli. Since A. laidlawii lacks oxoglutarate dehydrogenase and most likely branched-chain dehydrogenase enzyme complex activities, these results strongly suggest that the sequenced genes code for the pyruvate dehydrogenase complex.     

Mechanism of sulfide-quinone reductase investigated using site-directed mutagenesis and sulfur analysis

Biological sulfide oxidation is a reaction occurring in all three domains of life. One enzyme responsible for this reaction in many bacteria has been identified as sulfide:quinone oxidoreductase (SQR). The enzyme from Rhodobacter capsulatus is a peripherally membrane-bound flavoprotein with a molecular mass of approximately 48 kDa, presumably acting as a homodimer. In this work, SQR from Rb. capsulatus has been modified with an N-terminal His tag and heterologously expressed in and purified from Escherichia coli. Three cysteine residues have been shown to be essential for the reductive half-reaction by site-directed mutagenesis. The catalytic activity has been nearly completely abolished after mutation of each of the cysteines to serine. A decrease in fluorescence on reduction by sulfide as observed for the wild-type enzyme has not been observed for any of the mutated enzymes. Mutation of a conserved valine residue to aspartate within the third flavin-binding domain led to a drastically reduced substrate affinity, for both sulfide and quinone. Two conserved histidine residues have been mutated individually to alanine. Both of the resulting enzymes exhibited a shift in the pH dependence of the SQR reaction. Polysulfide has been identified as a primary reaction product using spectroscopic and chromatographic methods. On the basis of these data, reaction mechanisms for sulfide-dependent reduction and quinone-dependent oxidation of the enzyme and for the formation of polysulfide are proposed.     

New quinoproteins in oxidative fermentation

Several quinoproteins have been newly indicated in acetic acid bacteria, all of which can be applied to fermentative or enzymatic production of useful materials by means of oxidative fermentation. (1) D-Arabitol dehydrogenase from Gluconobacter suboxydans IFO 3257 was purified from the bacterial membrane and found to be a versatile enzyme for oxidation of various substrates to the corresponding oxidation products. It is worthy of notice that the enzyme catalyzes D-gluconate oxidation to 5-keto-D-gluconate, whereas 2-keto-D-gluconate is produced by a flavoprotein D-gluconate dehydrogenase. (2) Membrane-bound cyclic alcohol dehydrogenase was solubilized and purified for the first time from Gluconobacter frateurii CHM 9. When compared with the cytosolic NAD-dependent cyclic alcohol dehydrogenase crystallized from the same strain, the reaction rate in cyclic alcohol oxidation by the membrane enzyme was 100 times stronger than the cytosolic NAD-dependent enzyme. The NAD-dependent enzyme makes no contribution to cyclic alcohol oxidation but contributes to the reduction of cyclic ketones to cyclic alcohols. (3) Meso-erythritol dehydrogenase has been purified from the membrane fraction of G. frateurii CHM 43. The typical properties of quinoproteins were indicated in many respects with the enzyme. It was found that the enzyme, growing cells and also the resting cells of the organism are very effective in producing L-erythrulose. Dihydroxyacetone can be replaced by L-erythrulose for cosmetics for those who are sensitive to dihydroxyacetone. (4) Two different membrane-bound D-sorbitol dehydrogenases were indicated in acetic acid bacteria. One enzyme contributing to L-sorbose production has been identified to be a quinoprotein, while another FAD-containing D-sorbitol dehydrogenase catalyzes D-sorbitol oxidation to D-fructose. D-Fructose production by the oxidative fermentation would be possible by the latter enzyme and it is superior to the well-established D-glucose isomerase, because the oxidative fermentation catalyzes irreversible one-way oxidation of D-sorbitol to D-fructose without any reaction equilibrium, unlike D-glucose isomerase. (5) Quinate dehydrogenase was found in several Gluconobacter strains and other aerobic bacteria like Pseudomonas and Acinetobacter strains. It has become possible to produce dehydroquinate, dehydroshikimate, and shikimate by oxidative fermentation. Quinate dehydrogenase was readily solubilized from the membrane fraction by alkylglucoside in the presence of 0.1 M KCl. A simple purification by hydrophobic chromatography gave a highly purified quinate dehydrogenase that was monodispersed and showed sufficient purity. When quinate dehydrogenase purification was done with Acinetobacter calcoaceticus AC3, which is unable to synthesize PQQ, purified inactive apo-quinate dehydrogenase appeared to be a dimer and it was converted to the monomeric active holo-quinate dehydrogenase by the addition of PQQ.     

Immunological evidence for two types of PQQ-dependent d-glucose dehydrogenase in bacterial membranes and the location of the enzyme in Escherichia coli

Abstract Using an antibody raised against d -glucose dehydrogenase (EC 1.1.99.17) purified from Pseudomonas fluorescens , immuno-cross-reactivity with the enzymes from several bacterial strains and localization of the enzyme in Escherichia coli were examined. The antibody cross-reacted with glucose dehydrogenases from various Gram-negative bacteria examined. As a result, it became apparent that the enzymes from Gluconobacter, Acetobacter, Pseudomonas and Acinetobacter , which existed as holoenzymes in the membranes, had lower molecular weights than those from E. coli and Klebsiella , which were apoenzymes.
Treatment with trypsin of right-side out and inside-out membrane vesicles from E. coli clearly demonstrated that d -glucose dehydrogenase was located on the outer surface of the cytoplasmic membrane of E. coli , as had been suggested for Pseudomonas .     

Reconstitution of pyrroloquinoline quinone-dependent D-glucose oxidase respiratory chain of Escherichia coli with cytochrome o oxidase.

D-Glucose dehydrogenase is a pyrroloquinoline quinone-dependent primary dehydrogenase linked to the respiratory chain of a wide variety of bacteria. The enzyme exists in the membranes of Escherichia coli, mainly as an apoenzyme which can be activated by the addition of pyrroloquinoline quinone and magnesium. Thus, membrane vesicles of E. coli can oxidize D-glucose to gluconate and generate an electrochemical proton gradient in the presence of pyrroloquinoline quinone. The D-glucose oxidase-respiratory chain was reconstituted into proteoliposomes, which consisted of two proteins purified from E. coli membranes, D-glucose dehydrogenase and cytochrome o oxidase, and E. coli phospholipids containing ubiquinone 8. The electron transfer rate during D-glucose oxidation and the membrane potential generation in the reconstituted proteoliposomes were almost the same as those observed in the membrane vesicles when pyrroloquinoline quinone was added. The results demonstrate that the quinoprotein, D-glucose dehydrogenase, can reduce ubiquinone 8 directly within phospholipid bilayer and that the D-glucose oxidase system of E. coli has a relatively simple respiratory chain consisting of primary dehydrogenase, ubiquinone 8, and a terminal oxidase.     

Functional expression, characterization, and purification of the catalytic domain of human 11-beta -hydroxysteroid dehydrogenase type 1

11-beta-hydroxysteroid dehydrogenase type 1 catalyzes the conversion of cortisone to hormonally active cortisol and has been implicated in the pathogenesis of a number of disorders including insulin resistance and obesity. The enzyme is a glycosylated membrane-bound protein that has proved difficult to purify in an active state. Extracted enzyme typically loses the reductase properties seen in intact cells and shows principally dehydrogenase activity. The C-terminal catalytic domain is known to contain a disulfide bond and is located within the lumen of the endoplasmic reticulum, anchored to the membrane by a single N-terminal transmembrane domain. We report here the functional expression of the catalytic domain of the human enzyme, without the transmembrane domain and the extreme N terminus, in Escherichia coli. Moderate levels of soluble active protein were obtained using an N-terminal fusion with thioredoxin and a 6xHis tag. In contrast, the inclusion of a 6xHis tag at the C terminus adversely affected protein solubility and activity. However, the highest levels of active protein were obtained using a construct expressing the untagged catalytic domain. Nonreducing electrophoresis revealed the presence of both monomeric and dimeric disulfide bonded forms; however, mutation of a nonconserved cysteine residue resulted in a recombinant protein with no intermolecular disulfide bonds but full enzymatic activity. Using the optimal combination of plasmid construct and E. coli host strain, the recombinant protein was purified to apparent homogeneity by single step affinity chromatography. The purified protein possessed both dehydrogenase and reductase activities with a K(m) of 1.4 micrometer for cortisol and 9.5 micrometer for cortisone. This study indicates that glycosylation, the N-terminal region including the transmembrane helix, and intermolecular disulfide bonds are not essential for enzyme activity and that expression in bacteria can provide active recombinant protein for future structural and functional studies.     

Expression and purification of His-tagged rat mitochondrial medium-chain acyl-CoA dehydrogenase wild-type and Arg256 mutant proteins

Mitochondrial medium-chain acyl-CoA dehydrogenase is a key enzyme for the beta-oxidation of fatty acids, and the deficiency of this enzyme in patient has been previously reported. We cloned the gene of rat mitochondrial medium-chain acyl-CoA dehydrogenase into a bacterial expression vector pLM1 with six continuous histidine codons attached to the 3' of the gene. The cloned gene was overexpressed in Escherichia coli and the soluble protein was purified with a nickel Hi-Trap chelating metal affinity column in 88% yield to apparent homogeneity. The specific activity of the purified His-tagged rat mitochondrial medium-chain acyl-CoA dehydrogenase was 4.0 U/mg. Arg256 is a highly conserved amino acid, which may play an important role in enzymatic reaction based on the crystal structure of medium-chain acyl-CoA dehydrogenase. We constructed four mutant expression plasmids of the enzyme using site-directed mutagenesis. Mutant proteins were overexpressed in E. coli and purified with a nickel metal affinity column. Kinetic studies of wild-type and mutant proteins were carried out, and the result confirmed that Arg256 is a very important residue of rat mitochondrial medium-chain acyl-CoA dehydrogenase. Our overexpression in E. coli and one-step purification of the highly active rat mitochondrial medium-chain acyl-CoA dehydrogenase greatly facilitated our further investigation of this enzyme, and our result from site-directed mutagenesis increased our understanding of medium-chain acyl-CoA dehydrogenase.     

Mechanism of gallic acid biosynthesis in bacteria (Escherichia coli) and walnut (Juglans regia)

Gallic acid (GA), a key intermediate in the synthesis of plant hydrolysable tannins, is also a primary anti-inflammatory, cardio-protective agent found in wine, tea, and cocoa. In this publication, we reveal the identity of a gene and encoded protein essential for GA synthesis. Although it has long been recognized that plants, bacteria, and fungi synthesize and accumulate GA, the pathway leading to its synthesis was largely unknown. Here we provide evidence that shikimate dehydrogenase (SDH), a shikimate pathway enzyme essential for aromatic amino acid synthesis, is also required for GA production. Escherichia coli (E. coli) aroE mutants lacking a functional SDH can be complemented with the plant enzyme such that they grew on media lacking aromatic amino acids and produced GA in vitro. Transgenic Nicotiana tabacum lines expressing a Juglans regia SDH exhibited a 500% increase in GA accumulation. The J. regia and E. coli SDH was purified via overexpression in E. coli and used to measure substrate and cofactor kinetics, following reduction of NADP(+) to NADPH. Reversed-phase liquid chromatography coupled to electrospray mass spectrometry (RP-LC/ESI-MS) was used to quantify and validate GA production through dehydrogenation of 3-dehydroshikimate (3-DHS) by purified E. coli and J. regia SDH when shikimic acid (SA) or 3-DHS were used as substrates and NADP(+) as cofactor. Finally, we show that purified E. coli and J. regia SDH produced GA in vitro.     

Sulfide dehydrogenase activity of the monomeric flavoprotein SoxF of Paracoccus pantotrophus

Flavocytochrome c-sulfide dehydrogenases (FCSDs) are complexes of a flavoprotein with a c-type cytochrome performing hydrogen sulfide-dependent cytochrome c reduction in vitro. The amino acid sequence analysis revealed that the phylogenetic relationship of different flavoproteins reflected the relationship of sulfur-oxidizing bacteria. The flavoprotein SoxF of Paracoccus pantotrophus is 29-67% identical to the flavoprotein subunit of FCSD of phototrophic sulfur-oxidizing bacteria. Purification of SoxF yielded a homogeneous emerald-green monomeric protein of 42 797 Da. SoxF catalyzed sulfide-dependent horse heart cytochrome c reduction at the optimum pH of 6.0 with a k(cat) of 3.9 s(-1), a K(m) of 2.3 microM for sulfide, and a K(m) of 116 microM for cytochrome c, as determined by nonlinear regression analysis. The yield of 1.9 mol of cytochrome c reduced per mole of sulfide suggests sulfur or polysulfide as the product. Sulfide dehydrogenase activity of SoxF was inhibited by sulfur (K(i) = 1.3 microM) and inactivated by sulfite. Cyanide (1 mM) inhibited SoxF activity at pH 6.0 by 25% and at pH 8.0 by 92%. Redox titrations in the infrared spectral range from 1800 to 1200 cm(-1) and in the visible spectral range from 400 to 700 nm both yielded a midpoint potential for SoxF of -555 +/- 10 mV versus Ag/AgCl at pH 7.5 and -440 +/- 20 mV versus Ag/AgCl at pH 6.0 (-232 mV versus SHE') and a transfer of 1.9 electrons. Electrochemically induced FTIR difference spectra of SoxF as compared to those of free flavin in solution suggested a strong cofactor interaction with the apoprotein. Furthermore, an activation/variation of SoxF during the redox cycles is observed. This is the first report of a monomeric flavoprotein with sulfide dehydrogenase activity.     

Incorporation of glutamic acid into protein by a soluble system

A heat-labile, non-dialyzable factor(s) in soluble fractions from Escherichia coli strains and Bacillus subtilis was found to incorporate the radioactivity of [14C]glutamic acid into 95 degrees C CCl3COOH-insoluble fraction. Incorporation catalyzed by a partially purified factor from E. coli B required ATP, Mg2+, tRNA, casein, carbonate, and 2-mercaptoethanol. A mixture of nineteen amino acids other than glutamic acid had no effect on the incorporation. Heparin, spermine and monovalent cations were inhibitory. Incorporation proceeded via glutamyl-tRNA. The incorporation from [14C]glutamyl-tRNA required Mg2+, casein, carbonate, and 2-mercaptoethanol, and there was no incorporation from [14C]aspartyl-tRNA. The reaction product was identified as protein. The incorporated moiety was the glutamyl moiety of glutamic acid and it retained a free alpha-amino group in the product protein. The incorporating factor of E. coli B was demonstrated to be glutamyl-tRNA synthetase.     

The NADH dehydrogenase of the respiratory chain of Escherichia coli. II. Kinetics of the purified enzyme and the effects of antibodies elicited against it on membrane-bound and free enzyme.

The purified respiratory chain NADH dehydrogenase of Escherichia coli oxidizes NADH with either dichlorophenolindophenol (DCIP). ferricyanide, or menadione as electron acceptors, with values for NADH are similar with the three electron acceptors (approximately 50 muM). The purified enzyme contains no flavin and has an absolute requirement for FAD, with Km values around 4 muM. The pH optimum of the enzyme appears to be between 6.5 and 7; the optimum is difficult to establish because of nonenzymatic reduction of DCIP at the lower pH values. Potassium cyanide stimulates the DCIP reductase activity about 2-fold, but has no effect on ferricyanide reductase. The enzyme exhibits hyperbolic kinetics with respect to NADH concentration in both the ferricyanide and DCIP reductase assays, but cooperatively is seen in the menadione reductase reaction. NAD+ is an effective competitive inhibitor of the reaction (Ki congruent to 20 muM); in the presence of NAD+, the NADH saturation curve becomes cooperative, even in the DCIP reductase assay. Many adenine containing nucleotides are competitive inhibitors of the enzyme. The apparent Ki values for these nucleotides as inhibitors of the purified enzyme, the membrane-bound NADH dehydrogenase, and the NADH oxidase are equivalent. An examination of inhibitory effects of a series of adenine nucleotides suggests that the inhibitors act as analogues of NAD+, which is the true physiological inhibitor. The results suggest that the enzyme in situ is always partially inhibited by the levels of NAD- in the E coli cell, and thus behaves in a cooperative fashion to changes in the NAD+/NADH ratio. An antibody has been elicited against the purified NADH dehydrogenase. Immunodiffusion and crossed immunoelectrophoresis show that the antibody is directed principally against the NADH dehydrogenase, with some activity against minor contaminants in the purified preparation. The antibody inhibits NADH dehydrogenase activity 50% at saturating levels. When this antibody preparation is used to examine solubilized membrane preparations, two major immunoprecipitates are found. A parallel inhibition of the membrane-bound NADH dehydrogenase and NADH oxidase activities is seen, supporting the hypothesis that the purified enzyme is indeed a component of the respiratory chain-dependent NADH oxidase pathway.     

Cloning and nucleotide sequence of a heat-stable amylase gene from an anaerobic thermophile, Dictyoglomus thermophilum

A highly heat-stable amylase gene from an obligately anaerobic and extremely thermophilic bacterium, Dictyoglomus thermophilum, was cloned and expressed in Escherichia coli. The nucleotide sequence of the amylase gene predicts a 686-amino-acid protein of relative molecular mass 81,200, which is consistent with that determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the purified enzyme. The NH2-terminal sequence determined using the enzyme purified from E. coli cells corresponds precisely to that predicted from the nucleotide sequence, except for the absence of the NH2-terminal methionine in the mature protein. When the amylase gene was expressed in E. coli cells, the enzyme was localized in the cytoplasmic fraction; this is probably explained by the absence of the signal sequence for secretion. By using the amylase purified from the E. coli transformant, some enzymatic properties, such as optimum pH, optimum temperature, pH-stability and heat-stability, were examined. The amylase was found to be a highly liquefying-type.     

Characterization of a novel thiosulfate dehydrogenase from a marine acidophilic sulfur-oxidizing bacterium,Acidithiobacillus thiooxidans strain SH

A marine acidophilic sulfur-oxidizing bacterium, Acidithiobacillus thiooxidans strain SH, was isolated to develop a bioleaching process for NaCl-containing sulfide minerals. Because the sulfur moiety of sulfide minerals is metabolized to sulfate via thiosulfate as an intermediate, we purified and characterized the thiosulfate dehydrogenase (TSD) from strain SH. The enzyme had an apparent molecular mass of 44 kDa and was purified 71-fold from the solubilized membrane fraction. Tetrathionate was the product of the TSD-oxidized thiosulfate and ferricyanide or ubiquinone was the electron acceptor. Maximum enzyme activity was observed at pH 4.0, 40 °C, and 200 mM NaCl. To our knowledge, this is the first report of NaCl-stimulated TSD activity. TSD was structurally different from the previously reported thiosulfate-oxidizing enzymes. In addition, TSD activity was strongly inhibited by 2-heptyl-4-hydroxy-quinoline N-oxide, suggesting that the TSD is a novel thiosulfate:quinone reductase.     

Isolation of an enzyme complex with carbon monoxide dehydrogenase activity containing corrinoid and nickel from acetate-grown Methanosarcina thermophila.

Fast protein liquid chromatography of cell extract from methanol- or acetate-grown Methanosarcina thermophila resolved two peaks of CO dehydrogenase activity. The activity of one of the CO dehydrogenases was sixfold greater in acetate-grown compared with methanol-grown cells. This CO dehydrogenase was purified to apparent homogeneity (70 mumol of methyl viologen reduced per min per mg of protein) and made up greater than 10% of the cellular protein of acetate-grown cells. The native enzyme (Mr 250,000) formed aggregates with an Mr of approximately 1,000,000. The enzyme contained five subunits (Mrs 89,000, 71,000, 60,000, 58,000, and 19,000), suggesting a multifunctional enzyme complex. Nickel, iron, cobalt, zinc, inorganic sulfide, and a corrinoid were present in the complex. The UV-visible spectrum suggested the presence of iron-sulfur centers. The electron paramagnetic resonance spectrum contained g values of 2.073, 2.049, and 2.028; these features were broadened in enzyme that was purified from cells grown in the presence of medium enriched with 61Ni, indicating the involvement of this metal in the spectrum. The pattern of potassium cyanide inhibition indicated that cyanide binds at or near the CO binding site. The properties of the enzyme imply an involvement in the dissimilation of acetate to methane, possibly by cleavage of acetate or activated acetate.     

Cyanobacterial sulfide-quinone reductase: cloning and heterologous expression

The gene encoding sulfide-quinone reductase (SQR; E.C.1.8.5.'), the enzyme catalyzing the first step of anoxygenic photosynthesis in the filamentous cyanobacterium Oscillatoria limnetica, was cloned by use of amino acid sequences of tryptic peptides as well as sequences conserved in the Rhodobacter capsulatus SQR and in an open reading frame found in the genome of Aquifex aeolicus. SQR activity was also detected in the unicellular cyanobacterium Aphanothece halophytica following sulfide induction, with a V(max) of 180 micromol of plastoquinone-1 (PQ-1) reduced/mg of chlorophyll/h and apparent K(m) values of 20 and 40 microM for sulfide and quinone, respectively. Based on the conserved sequences, the gene encoding A. halophytica SQR was also cloned. The SQR polypeptides deduced from the two cyanobacterial genes consist of 436 amino acids for O. limnetica SQR and 437 amino acids for A. halophytica SQR and show 58% identity and 74% similarity. The calculated molecular mass is about 48 kDa for both proteins; the theoretical isoelectric points are 7.7 and 5.6 and the net charges at a neutral pH are 0 and -14 for O. limnetica SQR and A. halophytica SQR, respectively. A search of databases showed SQR homologs in the genomes of the cyanobacterium Anabaena PCC7120 as well as the chemolithotrophic bacteria Shewanella putrefaciens and Thiobacillus ferrooxidans. All SQR enzymes contain characteristic flavin adenine dinucleotide binding fingerprints. The cyanobacterial proteins were expressed in Escherichia coli under the control of the T7 promoter. Membranes isolated from E. coli cells expressing A. halophytica SQR performed sulfide-dependent PQ-1 reduction that was sensitive to the quinone analog inhibitor 2n-nonyl-4-hydroxyquinoline-N-oxide. The wide distribution of SQR genes emphasizes the important role of SQR in the sulfur cycle in nature.