Ferredoxin-Sulfite Reductase from a Cyanobacterium, Spirulina Platensis
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Agric. Biol. Chem., 52 (2), 373-380, 1988 373 Ferredoxin-sulfite Reductase from a Cyanobacterium, Spirulina platensis Osamu Koguchi and Goro Tamura* Department of Agricultural Chemistry, Faculty of Horticulture, Chiba University, Matsudo, Chiba 271, Japan Received August ll, 1987 Ferredoxin-sulfite reductase (Fd-SiR) [hydrogen-sulfide: ferredoxin oxidoreductase, EC 1.8.7. 1] from a cyanobacterium, Spirulina platensis, has been purified to homogeneity by a novel procedure. Subunit analysis by sodium dodecyl sulfate gel electrophoresis yielded a single protein band with a molecular weight of 63,000. In the presence of 0.1 mpotassium sulfate, gel filtration produced a value of 120,000, indicating the presence of a dimer in this ionic environment. Aplot of ferredoxin concentration versus enzymatic (Fd-SiR) activity yielded a sigmoidal curve, giving a Hill coefficient (n) of2.2. Purified Fd-SiR, in the oxidized form, has absorption maxima at 279, 388, and 590 nm. Thus the enzymehas the properties of a siroheme-containing protein. Assimilatory sulfite reductase catalyzes the enzymecould be found in procaryotic photo- reduction of sulfite to sulfide with NADPH,1} autotrophs (cyanobacteria, which have no reduced ferredoxin,2) or methyl viologen free separate chloroplasts). radical3) as electron donor. This communication reports the methods The enzyme is analogous to plant nitrite developed for obtaining electrophoretically reductase17) in that a 6-electron reduction is homogeneous Fd-SiR preparations from the apparently catalyzed by a single protein. cyanobacterium Spirulina platensis, and sug- Studies of the functional group catalyzing this gests that this cyanobacterial Fd-SiR has a reaction have been made with highly purified subunit structure containing siroheme. preparations of NADPH-linkedsulfite reduc- tase from yeast1} and Escherichia coli.A) The MATERIALS AND METHODS enzyme from both sources contains FMN, Materials. Spirulina platensis OU-1strain, kindly sup- FAD,and siroheme (octacarboxylate iron plied by Professor Wadaof Kanazawa University, was tetrahydroporphyrin). Similarly, ferredoxin- cultured by the method of Ogawaand Terui.5) Spinach linked sulfite reductase (Fd-SiR) from spinach and Spirulina ferredoxins were purified by the method of leaves2) has absorption maximaat 279, 385, Tagawa and Arnon6) (A422/A211=0A5). Ferredoxin- Sepharose was prepared by the method of Shin and 588, and 714nm, indicating the presence of Oshino.7) 5'-Deazaflavin was prepared by the method of siroheme. However, this plant Fd-SiR has no O'Brien,8) and the purity of the compound was checked by flavins. These observations suggest the occur- measuring the Rf value on thin layer chromatography. rence of a commonfunctional group, siro- The following chemicals were purchased from com- heme, in the assimilatory sulfite reductases. mercial sources: DEAE-cellulose (DE-52) (Whatman Ltd.); DEAEBIO-GEL (Bio-Rad Laboratories); Sephadex However, no such information from cyano- G-100 (Pharmacia Fine Chemicals); Coomassie brillant bacteria have been obtained so far. In higher blue G 250 (Fluka AG); Calibration proteins kit plants Fd-SiR may be located within the chlo- (ovalbumin, bovine serum albumin, aldolase, catalase, roplast. We wanted to know if the same kind of and ferritin) (Boehringer Mannheim); SDS-Calibration Abbreviation: SiR, sulfite reductase. * To whomreprint requests should be addressed. 374 O. Koguchi and G. Tamura proteins kit (Bio-Rad Laboratories). Toyopearl HW-55 and BUTYL-Toyopearl 650s were kindly supplied by Toyo Soda MFGCo. Other chemicals were of analytical grade. Assay ofsulfite reductase activity. The assay method for Fd-SiR activity was that of Krueger and Siegel9) with slight modifications. The activity was measured with a Hitachi 556 spectrophotometer. The reaction was done anaerobically in a Thunberg-type cuvette at 25°C. The reaction mixture contained in a total volume of 2ml, 2/rniol of Tris-H2SO4 buffer, pH 7.8, 2/miol of EDTA, 0.03 /rniol of 5'-deazaflavin, 1 mg of ferredoxin, 2 /rniol of sodium sulfite, and enzyme preparation. The absorption at 422 nmof the solution in a cuvette was recorded, and then the cuvette was illuminated with a PRR-500-W lamp placed 5cm from the cuvette. After incubation for 2 min, Fig. 1. Elution Profile of Fd-SiR from a DEAE- the reaction was started by adding sodium sulfite from the Cellulose Column. side arm and the change in absorbance at 422nmwas O-O, enzyme activity in units per ml; #-#, amounts followed. The extinction coefficient of E^j, used for of protein in milligrams per ml. Fractions of ll ml were oxidized-minus-reduced ferredoxin, was 5x 103m~1 collected at a flow rate of 30ml/hr. cm"1.91 A unit of Fd-SiR activity was defined as the amount of enzyme which caused the oxidation of 1 jimo\ of reduced ferredoxin per min under the assay condi- dialyzed for about 16hr against the same tions described. buffer mixture. (3) First DEAE-cellulose column chromatog- Other analytical methods. Protein was estimated by the raphy. The dialyzed solution (250ml) was put method of Bradford10) using bovine serum albumin as a on a DEAE-.cellulose column (7 x 40cm) pre- standard. Sulfide ion concentration was measured by the method of Siegel.U) Analytical PAGEwas done by the viously equilibrated with TGK-20 buffer. The method of Ornstein and Davis.12) SDS-PAGEwas done enzyme was eluted with 50mM Tris-H2SO4 by the method of Weber and Osborn.13) buffer, pH 7.8, containing 10% glycerol and 150mM K2SO4 (TGK-150 buffer). The elution RESULTS pattern is shown in Fig. 1. The active fractions (40 ~65) from the column chromatography on Enzymepurification DEAE-cellulose were combined and dialyzed All the purification procedures were done against TGK-20 buffer. in a room maintained at 2~5°C. (4) Second DEAE-cellulose column chroma- (1) Cell extract. Cells were collected by tography. The dialyzed enzyme solution ob- filtration and resuspended in lOOmM tained (310ml) was put on a DEAE-cellulose Tris-H2SO4 buffer (pH 7.8) containing 100 mM column (4x40cm) which had been equili- K2SO4. The cell suspension was sonicated and brated with TGK-20 buffer. A linear concen- then crude cell extract was obtained after tration gradient of eluent was established with removal of cell debris by centrifugation at 600ml of TGK-20 buffer in the mixing vessel 18,000xg for 20min. A total of400g of cells and 600 ml ofTGK-150buffer in the reservoir. and 1600ml of the above buffer were used. A typical chromatographic pattern thus ob- (2) Acetone fractionation. The precipitate tained is shown in Fig. 2. The eluted solution formed from the cell extract by the addition of in tubes from No. 50 to No. 80in Fig. 2 were acetone to a level of 70%was collected by combined and dialyzed against TGK-20 centrifugation at 18,000 x g for 10min and was buffer. resuspended in 300ml of 50mMTris-H2SO4 (5) DEAE BIO-GEL column chromatog- buffer, pH 7.8, containing 10% glycerol and raphy. The dialyzed enzyme solution (360 ml) 20mMK2SO4 (TGK-20 buffer) and was then was put on a DEAE BIO-GEL column Cyanobacterial Sulfite Reductase 375 Fig. 4. Elution Pattern of Sulfite Reductase on a BUTYL-Toyopearl Column. Fig. 2. Elution Pattern ofFd-SiR on a Second DEAE- Cellulose Column.O-O, SiR activity; #-#, protein. Fractions of 5ml O-O, SiR activity; #-#. protein. Fractions of 12ml were collected at a flow rate of 20ml/hr. were collected at a flow rate of 30ml/hr. Fig. 5. Sephadex G-100 Gel Filtration. O-O,SiR activity; #-#, protein. Fractions of 4ml were collected at a flow rate of 6ml/hr. Fig. 3. Elution Profile of Sulfite Reductase from a DEAE Bio-Gel Column. O-O, SiR activity; #-#. protein. Fractions of 6ml viously equilibrated with 50 mMTris-H2SO4 were collected at a flow rate of 12ml/hr. buffer, pH 7.8, containing 35% saturated am- moniumsulfate. A linear concentration gra- (3 x 30 cm) previously equilibrated with TGK- dient of eluent was established with 200 ml of 20 buffer. A linear concentration gradient of 35% saturated ammoniumsulfate solution, eluent was established with 350 ml of TGK-20 containing enough TGK-150 buffer to bring buffer in the mixing vessel and 350 ml ofTGK- the solution to pH 7.8 in the mixing vessel and 150 buffer in the reservoir. The elution pattern 200ml of 50mM Tris-H2SO4 buffer, pH 7.8, in is shown in Fig. 3. The main Fd-SiR fractions the reservoir A typical chromatographic pat- were combined. tern thus obtained is shown in Fig. 4. The (6) BUTYL-Toyopearl column chromatog- eluates in tubes 23~30 in Fig. 4 were com- raphy. The enzymesolution from the preced- bined and concentrated in a dialysis sac ing step (95ml) was diluted with an equal against TGK-150 buffer containing 80% volume of cold distilled water and ammonium saturated ammoniumsulfate. The precipitate sulfate was added to bring them to 35% satu- formed in a dialysis sac was collected by cen- ration. The resulting solution was put on a trifugation at 18,000 x g for 20min, dissolved BUTYL-Toyopearl column (2 x 30cm), pre- in a minimal amount of 50mMTris-H2SO4 376 O. Koguchi and G. Tamura Table I. Summary of the Purification . Activity Protein Specific activity Yield (units) (mg) (units/mg of protein) (%) Crude extract a 720 10,800 0.066 100 Acetone (0 - 70%) 440 595 0.74 61 1 st DEAE-cellulose 375 500 0.75 52 2nd DEAE-cellulose 238 95.2 2.5 33 DEAE BIO-GEL 126 ll.0 ll.4 17.5 BUTYL-Toyopearl 83.7 3.7 22.6 1 1.5 Sephadex G-100 25.4 0.64 39.7 3.5 Fd-Sepharoseb 7.8 0. 16 49.0 1.0 a Started with 400g of cells. b Ferredoxin from Spirulina platensis.