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.     

Siroheme sulfite reductase isolated from Chromatium vinosum. Purification and investigation of some of its molecular and catalytic properties

Cells of the phototrophic bacterium Chromatium vinosum strain D were shown to contain a siroheme sulfite reductase after autotrophic growth in a sulfide/bicarbonate medium. The enzyme could not be detected in cells grown heterotrophically in a malate/sulfate medium. Siroheme sulfite reductase was isolated from autotrophic cells and obtained in an about 80% pure preparation which was used to investigate some molecular and catalytic properties of the enzyme. It was shown to consist of two different types of subunits with molecular weights of 37,000 and 42,000, most probably arranged in an ₄₄-structure. The molecular weight of the native enzyme was determined to 280,000, 51 atoms of iron and 47 atoms of acid-labile sulfur were found per enzyme molecule. The absorption spectrum indicated siroheme as prosthetic group; it had maxima at 280 nm, 392 nm, 595 nm, and 724 nm. The molar extinction coefficients were determined as 302×10³ cm²xmmol^-1 at 392 nm, 98×10³ cm² xmmol^-1 at 595 nm and 22×10³ cm²x-mmol^-1 at 724 nm. With reduced viologen dyes as electron donor the enzyme reduced sulfite to sulfide, thiosulfate, and trithionate. The turnover number with 59 (2 e^-/enzyme moleculexmin) was low. The pH-optimum was at 6.0. C. vinosum sulfite reductase closely resembled the corresponding enzyme from Thiobacillus denitrificans and also desulfoviridin, the dismilatory sulfite reductase from Desulfovibrio species. It is proposed that C. vinosum catalyses anaerobic oxidation of sulfide and/or elemental sulfur to sulfite in the course of dissimilatory oxidation of reduced sulfur compounds to sulfate.Non-common abbreviations APS adenylyl sulfate - SDS sodium dodecyl sulfate     

Chemical modification studies of tryptophan,arginine and lysine residues in maize chloroplast ferredoxin:sulfite oxidoreductase

The ferredoxin-dependent sulfite reductase from maize was treated, in separate experiments, with three different covalent modifiers of specific amino acid side chains. Treatment with the tryptophan-modifying reagent, N-bromosuccinimide (NBS), resulted in a loss of enzymatic activity with both the physiological donor for the enzyme, reduced ferredoxin, and with reduced methyl viologen, a non-physiological electron donor. Formation of the 1:1 ferredoxin/sulfite reductase complex prior to treating the enzyme with NBS completely protected the enzyme against the loss of both activities. Neither the secondary structure, nor the oxidation-reduction midpoint potential (E _m) values of the siroheme and [4Fe–4S] cluster prosthetic groups of sulfite reductase, nor the binding affinity of the enzyme for ferredoxin were affected by NBS treatment. Treatment of sulfite reductase with the lysine-modifying reagent, N-acetylsuccinimide, inhibited the ferredoxin-linked activity of the enzyme without inhibiting the methyl viologen-linked activity. Complex formation with ferredoxin protects the enzyme against the inhibition of ferredoxin-linked activity produced by treatment with N-acetylsuccinimide. Treatment of sulfite reductase with N-acetylsuccinimide also decreased the binding affinity of the enzyme for ferredoxin. Treatment of sulfite reductase with the arginine-modifying reagent, phenylglyoxal, inhibited both the ferredoxin-linked and methyl viologen-linked activities of the enzyme but had a significantly greater effect on the ferredoxin-dependent activity than on the reduced methyl viologen-linked activity. The effects of these three inhibitory treatments are consistent with a possible role for a tryptophan residue the catalytic mechanism of sulfite reductase and for lysine and arginine residues at the ferredoxin-binding site of the enzyme.     

Sulfite oxidation by the quinone-reducing molybdenum sulfite dehydrogenase SoeABC from the bacterium Aquifex aeolicus

The microaerophilic bacterium Aquifex aeolicus is a chemolitoautotroph that uses sulfur compounds as electron sources. The model of oxidation of the energetic sulfur compounds in this bacterium predicts that sulfite would probably be a metabolic intermediate released in the cytoplasm. In this work, we purified and characterized a membrane-bound sulfite dehydrogenase, identified as an SoeABC enzyme, that was previously described as a sulfur reductase. It is a member of the DMSO-reductase family of molybdenum enzymes. This type of enzyme was identified a few years ago but never purified, and biochemical data and kinetic properties were completely lacking. An enzyme catalyzing sulfite oxidation using Nitro-blue tetrazolium as artificial electron acceptor was extracted from the membrane fraction of Aquifex aeolicus. The purified enzyme is a dimer of trimer (αβγ)₂ of about 390 kDa. The K_M for sulfite and k_cat values were 34 μM and 567 s⁻¹ respectively, at pH 8.3 and 55 °C. We furthermore showed that SoeABC reduces a UQ₁₀ analogue, the decyl-ubiquinone, as well, with a K_M of 2.6 μM and a k_cat of 52.9 s⁻¹. It seems to specifically oxidize sulfite but can work in the reverse direction, reduction of sulfur or tetrathionate, using reduced methyl viologen as electron donor. The close phylogenetic relationship of Soe with sulfur and tetrathionate reductases that we established, perfectly explains this enzymatic ability, although its bidirectionality in vivo still needs to be clarified. Oxygen-consumption measurements confirmed that electrons generated by sulfite oxidation in the cytoplasm enter the respiratory chain at the level of quinones.     

Purification and characterization of an inducible dissimilatory type sulfite reductase from Clostridium pasteurianum

An inducible sulfite reductase was purified from Clostridium pasteurianum. The pH optimum of the enzyme is 7.5 in phosphate buffer. The molecular weight of the reductase was determined to be 83,600 from sodium dodecyl sulfate gel electrophoresis with a proposed molecular structure: ₂₂. Its absorption spectrum showed a maximum at 275 nm, a broad shoulder at 370 nm and a very small absorption maximum at 585 nm. No siroheme chromophore was isolated from this reductase. The enzyme could reduced the following substrates in preferential order: NH₂OH> SeO ₃ ^2- >NO ₂ ^2- at rates 50% or less of its preferred substrate SO ₃ ^2- . The proposed dissimilatory intermediates, S₃O ₆ ^2- or S₂O ₃ ^2- , were not utilized by this reductase while KCN inhibited its activity. Varying the substrate concentration [SO ₃ ^2- ] from 1 to 2.5 mol affected the stoichiometry of the enzyme reaction by alteration of the ratio of H₂ uptake to S^2- formed from 2.5:1 to 3.1:1. The inducible sulfite reductase was found to be linked to ferredoxin which could be completely replaced by methyl viologen or partially by benzyl viologen. Some of the above-mentioned enzyme properties and physiological considerations indicated that it was a dissimilatory type sulfite reductase.Abbreviations SDS sodium dodecyl sulfate - BSA bovine serum albumin - LDH Lactate dehydrogenase     

Sulfur metabolism in Thiobacillus denitrificans

The relatively high specific sulfite reductase activity of 25 mU/mg protein was found in extracts from Thiobacillus denitrificans. The absorption spectrum of the partially purified enzyme was similar to the siroheme containing sulfite reductases from other sources. It is suggested that the T. denitrificans sulfite reductase may function during the oxidation of reduced sulfur compounds.     

Purification and properties of human erythrocyte membrane NADH-cytochrome b5 reductase

An NADH:(acceptor) oxidoreductase (EC 1.6.99.3) of human erythrocyte membrane was purified by DEAE-cellulose anion exchange, hydroxyapatite adsorption, and 5′-ADP-hexane-agarose affinity chromatographies after solubilization with Triton X-100. The purified reductase preparation was homogeneous and estimated to have an apparent molecular weight of 36,000 on SDS-polyacrylamide slab gel electrophoresis and of 144,000 on Sephadex G-200 gel filtration in the presence of 0.2% Triton X-100, whereas a soluble NADH-cytochrome b₅ reductase of human erythrocyte had a molecular weight of 32,000 by both methods, indicating the existence of a distinct membrane reductase. Digestion of the membrane reductase with cathepsin D yielded a new polypeptide chain which gave the same relative mobility as the soluble reductase on SDS-polyacrylamide slab gel electrophoresis. The membrane enzyme, the cathepsin-digested enzyme, and the soluble enzyme all cross-reacted with the antibody to rat liver microsomal NADH-cytochrome b₅ reductase. The enzyme had one mole FAD per 36,000 as a prosthetic group and could reduce K₃Fe(CN)₆, 2,6-dichlorophenolindophenol, cytochrome c, methemoglobin-ferrocyanide complex, cytochrome b₅ and methemoglobin via cytochrome b₅ when NADH was used as an electron donor. NADPH was less effective as an electron donor than NADH. The specific activity of the purified enzyme was 790 μmol ferricyanide reduced min^?1 mg^?1 and the turnover number was 40,600 mol ferricyanide reduced min^?1 mol^?1 FAD at 25 °C. The apparent K_m values for NADH and cytochrome b₅ were 0.6 and 20 μm, respectively, and the apparent V value was 270 μmol cytochrome b₅ reduced min^?1 mg^?1. These kinetic properties were similar to those of the soluble NADH-cytochrome b₅ reductase. The results indicate that the NADH:(acceptor) oxidoreductase of human erythrocyte membrane could be characterized as a membrane NADH-cytochrome b₅ reductase.     

Isolation and preliminary characterization of a respiratory nitrate reductase from hydrocarbon-degrading bacterium <Emphasis Type="Italic">Gordonia alkanivorans</Emphasis> S7

Gordonia alkanivorans S7 is an efficient degrader of fuel oil hydrocarbons that can simultaneously utilize oxygen and nitrate as electron acceptors. The respiratory nitrate reductase (Nar) from this organism has been isolated using ion exchange chromatography and gel filtration, and then preliminarily characterized. PAGE, SDS-PAGE and gel filtration chromatography revealed that Nar consisted of three subunits of 103, 53 and 25 kDa. The enzyme was optimally active at pH 7.9 and 40°C. K _m values for NO₃ ⁻ (110 μM) and for ClO₃ ⁻ (138 μM) were determined for a reduced viologen as an electron donor. The purified Nar did not use NADH as the electron donor to reduce nitrate or chlorate. Azide was a strong inhibitor of its activity. Our results imply that enzyme isolated from G. alkanivorans S7 is a respiratory membrane-bound nitrate reductase. This is the first report of purification of a nitrate reductase from Gordonia species.     

Purification and properties of an F420-dependent NADP reductase from methanobacterium thermoautotrophicum

The F₄₂₀-dependent NADP reductase of Methanobacterium thermoautotrophicum has been purified employing a combination of DEAE-cellulose ion-exchange chromatography, affinity chromatography with Blue Sepharose, Sephadex G-200 column chromatography and Red Sepharose affinity chromatography. The enzyme, which requires reduced F₄₂₀ as an electron donor, has been purified over 3000-fold with a recovery of 65%. A molecular weight of 112000 was determined by Sephadex G-200 chromatography. A subunit molecular weight of 28 500 was determined by Sephadex G-200 chromatography. A subunit native enzyme is a tetramer. The optimal temperature for enzymatic activity was found to be 60°C with a pH optimum of 8.0. The NADP reductase had an apparent K_m of 128 μMJ for reduced F420 and 40 μM for NADP. The enzyme was stable for at least 4 h at 65°C and pH 7.5. No loss of enzyme activity was detected when purified enzyme was stored aerobically in buffer containing 2-mercaptoethanol for 10 days at 4°C. Neither FMNH₂ nor FADH₂ could serve as electron donors; NAD was not utilized as electron acceptor.     

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     

Purification of a flavoprotein having NADPH-cytochrome c reductase and transhydrogenase activities from Nitrobacter winogradskyi and its molecular and enzymatic properties

A flavoenzyme which showed NADPH-cytochrome c reductase (NADPH-cytochrome c oxidoreductase EC 1.6.2.4) and transhydrogenase (NADPH-NAD⁺ oxidoreductase, EC 1.6.1.1) activities was purified to an electrophoretically homogeneous state from Nitrobacter winogradskyi. The reductase was a flavoprotein which contained one FAD per molecule but no FMN. The oxidized form of the enzyme showed absorption maxima at 272, 375 and 459 nm with a shoulder at 490 nm, its molecular weight was estimated to be 36,000 by SDS polyacrylamide gel electrophoresis, and the enzyme seemed to exist as a dimer in aqueous solution. The enzyme catalyzed reduction of cytochrome c, DCIP and benzylviologen by NADPH, oxidation of NADPH with menadione and duroquinone, and showed transhydrogenase activity. NADH was less effective than NADPH as the electron donor in the reactions catalyzed by the enzyme. The NADPH-reduction catalyzed by the enzyme of N. winogradskyi cytochrome c-550 and horse cytochrome c was stimulated by spinach ferredoxin. The enzyme reduced NADP⁺ with reduced spinach ferredoxin and benzylviologen radical.Abbreviations DCIP dichlorophenolindophenol - Tris trishydroxy-methylaminomethane - Mops 3-(N-morpholino) propanesulfonic acid - SDS sodium dodecylsufate     

Purification and Properties of Sulfite Reductase from Desulfovibrio vulgaris,Miyazaki F

The sulfite reductase of Desulfovibrio vulgaris, strain Miyazaki F (MF), was purified by ammonium sulfate precipitation and chromatography on DEAE-cellulose, Ultrogel AcA34, and hydroxylapatite. The molecular weight was estimated to be 180,000 by gel filtration. It had a subunit structure of α₂β₂; the molecular weight of the α subunit was 50,000 and that of β, 39,000. The absorption spectrum with characteristic peaks at 629 and 409 nm and the amino acid composition resembled those of the sulfite reductase from D. vulgaris, Miyazaki K. The MF enzyme reduced sulfite to trithionate, thiosulfate, and sulfide by hydrogen when coupled with a hydrogenase-methyl viologen system, like other sulfite reductases from Desulfovibrio.     

Two Phenolic Amides in the Seed Balls of Sugar Beet (Beta vulgaris L. var. saccharifera Alefeld)

Ferredoxin-dependent sulfite reductase (Fd-SiR) (EC 1.8.7.1) was purified about 1136-fold, with a yield of 11%, from fresh thalli of Porphyra yezoensis by a procedure involving ammonium sulfate precipitation, DEAE-cellulose chromatography, Buty 1-Toyopearl chromatography, Sephadex G-100 gel filtration and ferredoxin-Sepharose affinity chromatography. The purified enzyme was apparently homogeneous, as judged on polyacrylamide disc gel electrophoresis, with a specific activity of 100 units/mg of protein. The molecular weight of the enzyme was estimated to be 70 kilodaltons by gel filtration. On subunit analysis by SDS-PAGE, a single band corresponding to molecular weight of 65 kilodaltons appeared. The purified enzyme (Fd-SiR) showed 5-times higher ferredoxin-dependent activity than methyl viologen-linked activity. In the oxidized form, the enzyme exhibited absorption maxima at 278, 390 (Soret band), 586 (a band) and 714 (CT band) nm, indicating that siroheme is involved in the catalysis of sulfite reduction. The absorbance ratios, A₃₉₀: A₂₁₈ and A₅₈₆ :A₃₉₀, were 0.32 and 0.31, respectively. A plot of the substrate (sulfite) and electron donor (ferredoxin) concentrations versus enzymatic (Fd-SiR) activity yielded sigmoidal curves, giving Hill coefficients («) of 2.3 (for sulfite) and 2.7 (for ferredoxin), respectively. Antibody against the isolated enzyme was raised in rabbits. Analysis of the antiserum by immunodiffusion suggested that it was specific against isolated Fd-SiR. Using the antiserum, dot immunoblotting was performed to determine the immunological similarity of Fd-SiRs from Porphyra yezoensis, Spirulina platensis, Brassica chinensis and Spinacia oleracea. The tests revealed that the four forms of assimilatory Fd-SiR have antigenic determinants in common.     

Haloalkaliphilic spore-forming sulfidogens from soda lake sediments and description of Desulfitispora alkaliphila gen. nov., sp. nov.

An anaerobic enrichment with pyruvate as electron donor and thiosulfate at pH 10 and 0.6 M Na⁺ inoculated with pasteurized soda lake sediments resulted in a sulfidogenic coculture of two morphotypes of obligately anaerobic haloalkaliphilic endospore-forming clostridia, which were further isolated in pure culture. Strain AHT16 was a thin long rod able to ferment sugars and pyruvate and to respire H₂, formate and pyruvate using thiosulfate and fumarate as electron acceptors and growing optimally at pH 9.5. Thiosulfate was reduced incompletely to sulfide and sulfite. The strain was closely related (99% sequence similarity) to a peptolytic alkaliphilic clostridium Natronincola peptidovorans. Strain AHT17 was a short rod with a restricted respiratory metabolism, growing with pyruvate and lactate as electron donor and sulfite, thiosulfate and elemental sulfur as electron acceptors with a pH optimum 9.5. Thiosulfate was reduced completely via sulfite to sulfide. The ability of AHT17 to use sulfite explained the stability of the original coculture of the two clostridia—one member forming sulfite from thiosulfate and another consuming it. Strain AHT17 formed an independent deep phylogenetic lineage within the Clostridiales and is proposed as a new genus and species Desulfitisporum alkaliphilum gen. nov., sp. nov. (=DSM 22410^T = UNIQEM U794^T).     

Characterization of membrane-bound nitrate reductase from denitrifying bacteriaOchrobactrum anthropi SY509

In this study, we have purified and characterized the membrane bound nitrate reductase obtained from the denitrifying bacteria,Ochrobactrum anthropi SY509, which was isolated from soil samples.O. anthropi SY509 can grow in minimal medium using nitrate as a nitrogen source. We achieved an overall purification rate of 15-fold from the protein extracted from the membrane fraction, with a recovery of approximately 12% of activity. The enzyme exhibited its highest level of activity at pH 5.5, and the activity was increased up to 70°C. Periplasmic and cytochromic proteins, including nitrite and nitrous oxide reductase, were excluded during centrifugation and were verified using enzyme essay. Reduced methyl viologen was determined to be the most efficient electron donor among a variety of anionic and cationic dyestuffs, which could be also used as an electron donor with dimethyl dithionite. The effects of purification and storage conditions on the stability of enzyme were also investigated. The activity of the membrane-bound nitrate reductase was stably maintained for over 2 weeks in solution. To maintain the stability of enzyme, the cell was disrupted using sonication at low temperatures, and enzyme was extracted by hot water without any surfactant. The purified enzyme was stored in solution with no salt to prevent any significant losses in activity levels.     

Studies on nitrite reductase in barley

Summary Nitrite reductase from barley seedlings was purified 50–60 fold by ammonium sulphate precipitation and gel filtration. No differences were established in the characteristics of nitrite reductases isolated in this way from either leaf or root tissues. The root enzyme accepted electrons from reduced methyl viologen, ferredoxin, or an unidentified endogenous cofactor. Enzyme activity in both tissues was markedly increased by growth on nitrate. This activity was not associated with sulphite reductase activity. Microbial contamination could not account for the presence of nitrite reductase activity in roots. Nitrite reductase assayed in vitro with reduced methyl viologen as the electron donor was inhibited by 2,4-dinitrophenol but not by arsenate.Abbreviations DNP 2,4-dinitrophenol - DEAE diethyl amino ethyl     

Purification and properties of nitrite reductase from the blue-green alga Anabaena cylindrical

Nitrite reductase was purified about 40-fold from the blue-greenalga Anabaena cylindrica by acetone precipitation and chromatographyon DEAE-cellulose columns. The nitrite reductase had its pHoptima at about 7.6 with Tris-HCl and at about 7.4 with phosphatewhen reduced methyl viologen was used as an electron donor.The Km's for nitrite, methyl viologen and ferredoxin were 510^–55,210^–4 and 510^–6M, respectively. A stoichiometryof one molecule of ammonia formation per one molecule of nitritedisappearance was confirmed. Ferredoxin which had been reducedeither chemically with dithionite or enzymatically with NADPHin the presence of diaphorase was active as an electron donor.Dithionite-reduced FAD and FMN were inactive. NADPH could notgive electrons directly to nitrite reductase. Hydroxylaminereductase was segregated from nitrite reductase by DEAE-cellulosecolumn chromatography. Purified nitrite reductase showed noactivity for sulfite reduction. A molecular weight of 68,000was estimated for nitrite reductase using a calibrated SephadexG-200 column. ¹This work was supported by grants 4090 and 955008 from theMinistry of Education. ²This work was supported by grants 4090 and 955008 from theMinistry of Education. 2 Present address: Department of Botany,Faculty of Science, University of Tokyo, Tokyo.     

Characterization of a new type of dissimilatory sulfite reductase present in Thermodesulfobacterium commune

A new type of dissimilatory bisulfite reductase, desulfofuscidin, was isolated from the nonsporeforming thermophilic sulfate-reducing microorganism Thermodesulfobacterium commune. The molecular weight of the enzyme was estimated at 167,000 by sedimentation equilibrium, and the protein was pure by both disc electrophoresis and ultracentrifugation. The bisulfite reductase was a tetramer and had two types of subunits with an α₂β₂ structure and an individual molecular weight of 47,000. The enzyme exhibited absorption maxima at 576, 389, and 279 nm, with a weak band at 693 nm. Upon the addition of dithionite, the absorption maxima at 576 and 693 nm were weakened, and a new band appeared at 605 nm. The protein reacted with CO in the presence of dithionite to give a complex with absorption peaks at 593, 548, and 395 nm. The extinction coefficients of the purified enzyme at 576, 389, and 279 nm were 89,000, 310,000, and 663,000 M⁻¹ cm⁻¹, respectively. Siroheme was detected as the prosthetic group. The protein contains 20 to 21 nonheme iron atoms and 16 to 17 acid-labile sulfur groups per molecule. The data suggest the presence of four sirohemes and probably four (4Fe-4S) centers per molecule by comparison with desulfoviridin, the dissimilatory sulfite reductase from Desulfovibrio species. The protein contains 36 cysteine residues and is high in acidic and aromatic amino acids. The N-terminal amino acids of the α and β subunits were threonine and serine, respectively. With reduced methyl viologen as electron donor, the major product of sulfite reduction was trithionate, and the pH optimum for activity was 6.0. The enzyme was stable to 70°C and denatured rapidly above this temperature. The dependence of T. commune bisulfite reductase activity on temperature was linear between 35 and 65°C, and the Q₁₀ values observed were above 3. The presence of this new type of dissimilatory bisulfite reductase in T. commune is discussed in terms of taxonomic significance.     

Sulfate reduction in Catharanthus roseus (L.)

Cell homogenates from Catharanthus roseus (L.) G. Don. grown S-autotrophically on sulfate in the dark are capable of reducing adenylysulfate (APS) to cysteine. This reduction required a particulate protein fraction from the cell extract and reduced ferredoxin as the electron donor. The protein fraction (MW 700,000±50,000) was found to contain Fd:NADP⁺ reductase, glutathione reductase and an unspecific dithiol reductase, and APS-sulfotransferase and thiosulfonate reductase activity. Resolution into these individual enzyme activities led to a non-restorable loss of the APS reducing activity. It was observed that a slow gradual decay of the APS reducing activity was accompanied by a likewise slow generation of a ferredoxin-dependent sulfite reductase.Enzymes and abbreviations APS Adenosine 5-phosphosulfate - APS-kinase E.C.2.7.1.25 - ATP-sulfurylase E.C.2.7.7.4 - Fd ferredoxin - Fd-NADP⁺-reductase E.C.1.6.7.1. - Glutathione reductase E.C.1.6.4.2. - G6P Glucose 6-phosphate - G6PDH glucose 6-phosphate dehydrogenase, E.C.1.1.49 - GSSG oxidized glutathione - GSSO₃H S-sulfoglutathione - MVH reduced methylviologen - OASS O-acetylserine sulfhydrylase-E.C. 4.2.99.8 - Sulfite reductase E.C.1.8.1.2     

Studies on Respiratory Enzymes in Rice Kernel

Glutathione reductase as an acidic flavoprotein, has been isolated from the acidic protein fraction of rice embryos and purified by procedures involving ammonium sulfate fractionation, gel filration on Sephadex G–75 and G–100, ion exchange chromatography on CM-and DEAE-Sephadex and finally hydroxylapatite column chromatography. The preparation was homogeneous when examined by ultracentrifugation and almost pure on polyacrylamide gel electrophoresis. The flavoprotein exhibited an absorption spectrum characteristic of glutathione reductase having absorption maxima at 275, 370, 379, and 463 mμ with a clear double peak between 370 and 380 mμ and shoulders at around 430 and 490 mμ. The absorption ratio of A₂₇₅/A₄₆₃ and A₄₆₃/A₃₇₉ were 8.15 and 1.06, respectively. The purified enzyme was highly specific for NADPH and oxidized glutathione. The preparation had the average catalytic activity of 150 μmoles of NADPH oxidized per min per mg of protein.