Purification and Some Properties of Nitrite Reductase from Clostridium Perfringens1

J. Biochem. 94, 1053-1059 (1983) Purification and Some Properties of Nitrite Reductase from Clostridium perfringens1 Satoshi SEKIGUCHI,- Sachiko SEKI , and Makoto ISHIMOTO Department of Chemical Microbiology , Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo , Hokkaido 060 Received for publication, April 11, 1983 Nitrite reductase from Clostridium perfringens was purified by chromatographies on DEAE-cellulose, DEAE-Sephadex, Sephadex G-150, and hydroxylapatite and by isoelectric focussing to a homogeneous state, showing essentially a single protein band in disc gel electrophoresis and a single immuno-precipitation line in double diffusion against antiserum obtained from immunized rabbits. The reductase was induced in the presence of nitrate. It had a molecular weight of 54,000 and showed no absorption peak in the visible region. The pH optimum was 6.2 and Km for nitrite was 5 mm. Ferredoxin, as well as viologen dyes, was found to be an electron donor. The product of nitrite reduction was hydroxylamine. This reductase was inhibited by o-phenanthroline and azide but not by cyanide or diethyldithiocar bamate. Nitrate reduction in bacteria is carried out by of bacteria (6), fungi (7), and higher plants (8) molybdenum-containing reductase to produce ni reduce nitrate to the level of ammonia and contain trite, but further reduction of nitrite leads to the nitrite reductase which produces ammonia directly formation of nitric oxide, ammonia, or other from nitrite [EC 1.6.6.4, 1.7.7.1]. These enzymes products by different sorts of enzymes (1). In are siroheme proteins with iron-sulfur centers and denitrifying bacteria, two classes of nitrite reduc flavins, and use reduced pyridine nucleotide as an tase have been described, one containing c- and electron donor. d-type hemes [EC 1.9.3.2] (2, 3) and the other A strictly anaerobic bacterium, Clostridium copper [EC 1.7.2.1, 1.7.99.3] (4, 5), both of which perfringens, reduces nitrate to ammonia, promot form nitric oxide. On the other hand, a variety ing the oxidation of organic substrates (9). Ni trate reductase from this organism was purified and ferredoxin was found to play a role as an 1 This study was supported in part by a Grant-in-Aid electron donor (10). for Scientific Research from the Ministry of Education, The present paper describes the purification Science and Culture of Japan and by a grant from and characterization of nitrite reductase from C. Yakult Institute. 2 Present address: Department of Agricultural Chemis perfringens; this enzyme uses ferredoxin as an electron donor and forms hydroxylamine from try, Faculty of Agriculture, The University of Tokyo, nitrite as the main product. Yayoi, Bunkyo-ku, Tokyo 113. Vol. 94, No. 4, 1983 1053 1054 S. SEKIGUCHI, S. SEKI, and M. ISHIMOTO x g for 3 h was subjected to chromatography on EXPERIMENTAL PROCEDURES a DEAE-cellulose column (3.2 x 38 cm) previously equilibrated with TM buffer. The column was Organism and Cultivation-Clostridium per washed with TM buffer containing 0.1 M NaC1 and fringens, strain HM-l, isolated by Prof. H. Iida, then nitrite reductase was eluted with TM buffer was used throughout this work. containing 0.2 M NaCl. Culture medium contained, per liter; Poly Active fractions were pooled, diluted with pepton (Daigo Eiyo), 7.5 g; yeast extract (Daigo 200 ml of TM buffer, and adsorbed on a DEAE- Eiyo), 5.0 g; glucose, 2.5 g; thioglycolic acid, 0.5 Sephadex A-50 column (2.0 x 30 cm) equilibrated ml; and KNO,, 0.02 g. The pH was adjusted to with TM buffer containing 0. 15 M NaCl. Protein 7 by adding NaOH. This medium was inoculated was eluted with 300 ml of a linear gradient system with 0.4% of subculture and incubated without of 0.15-0.3 M NaCl in TM buffer. agitation in deep layer culture at 37°C. After a The active fractions thus eluted were pooled, 10-11 h incubation, cells in the early stationary concentrated to 6 ml by ultrafiltration with a Toyo phase were harvested by continuous centrifugation, membrane filter UK-10, and passed through a washed twice with 20 mm Tris-HCl buffer, pH 7.2, Sephadex G-150 column (2.8 x 88 cm) equilibrated containing 1 mm 2-mercaptoethanol (TM buffer) with TM buffer-0.2 M NaCl. and stored at - 80°C. Usually 500 g of wet cells Active fractions were then adsorbed on a were obtained from a 60 liters culture. hydroxylapatite column (1.2 x 9 cm) equilibrated Preparation of Crude Extract-The bacterial with 50 mm potassium phosphate buffer, pH 7.3, cells were suspended in TM buffer and disrupted containing 1 mm 2-mercaptoethanol. The column by sonic treatment in an Insonator (Kubota, was washed with the same buffer and then nitrite 200M) at 20 kHz at 180 W for 20 min. The reductase was eluted with 50 ml of a linear con supernatant after centrifugation at 17,000 x g was centration gradient system of 50-300 mm phos used as crude extract. phate buffer containing 1 mm 2-mercaptoethanol. Assay of Nitrite Reductase-The assay of The fractions with the highest activity contained nitrite reductase was run in open test tubes with proteins that could not be removed in the next chemically reduced methyl viologen as an electron purification step, so those fractions were not used donor. The reaction mixture contained, in 1 ml; for further purification; the fractions eluted before 0.2 M potassium phosphate buffer, pH 7.2, or Tris and after those fractions were pooled and dialyzed - HCl buffer, pH 6.9, 1 or 2 mm NaNO2, 6 mm against TM buffer. The dialyzed preparation was methyl viologen, 5 mm Na2S2O,, 10 mm KHCO3, subjected to isoelectric focussing on a 110 ml and enzyme. The reaction was started by adding column containing Pharmalyte giving a pH gra Na2S2O4-KHCO3. After 3 or 5 min of incuba dient of 4.5-6.0, and was electrophoresed for 47 h tion at 37°C the reaction was stopped by vigorous at a constant voltage of 1,000 V at 4°C. Enzyme shaking to oxidize reduced methyl viologen and fractions that gave a single protein band in disc the residual nitrite was determined colorimetrically gel electrophoresis were pooled. by the method of Nicholas and Nason (11). Dis- Other Assays-Hydroxylamine was deter- appearance of nitrite in the absence of enzyme or mined by Csaky's method (12). Ammonia was in the presence of boiled enzyme was also mea assayed by Chaney and Marbach's method (13). sured as a control. The activity was obtained Protein was determined by the method of Lowry from the difference between the amount of nitrite et at. (14) or of Bradford (15) with bovine serum decrease in the presence of enzyme and that in the albumin as a standard. absence of enzyme or in the presence of boiled Molecular Weight Estimation-Molecular enzyme. One unit of nitrite reductase was defined weight was estimated by gel filtration as described as the amount of enzyme which catalyzed the by Laurent and Killander (16) using a Sephadex reduction of 1 ƒÊmol of nitrite per min. G-150 column (2.7 x 86 cm) equilibrated with TM Purification of Nitrite Reductase-All opera buffer containing 0.2 M NaC1. As markers, cyto tions were carried out at 4°C. A soluble fraction chrome c (molecular weight 12,500), chymotryp isolated by centrifuging the crude extract at 63,000 sinogen A (25,000), ovalbumin (45,000), and bo- J. Biochem. NITRITE REDUCTASE FROM Clostridium perfringens 1055 vine serum albumin (68,000) were used. obtained from Pharmacia, DEAE-cellulose from Polyacrylamide Gel Electrophoresis-Disc Brown, and hydroxylapatite from Bio Rad (HTP). polyacrylamide get electrophoresis was performed by the method of Williams and Reisfeld (17) using RESULTS 7.5 % polyacrylamide gel (5 x 75 mm) at 2 mA/ tube at 4°C. Protein was stained with Coomassie Induction of Nitrite Reductase in C. perfringens brilliant blue G250 in perchloric acid according -When C. perfringens was grown in a medium to the procedure described by Reisner et al. (18). containing 2 mm or 20 mm nitrate, nitrite accumu Nitrite reductase activity in gels was detected by lated at a concentration of 1 mm at the end of activity staining as described by Vega and Kamin exponential growth and then decreased. While (8). nitrate reductase activity reached its maximum at Preparation of Antibody and Immunological the time of maximal accumulation of nitrite and Tests-Antiserum against nitrite reductase was then decreased sharply, nitrite reductase had its raised in rabbits by subcutaneous injection of 0.22 maximum after the peak of nitrite concentration mg of purified enzyme per rabbit, in emulsion and decreased only partly. Since nitrite reductase containing 50% Freund's complete adjuvant. Im activity was 7-fold higher in the presence of nitrate munization was repeated twice with intervals of added to the medium, cells grown in the medium two and three weeks, A week after the last containing nitrate were used for the preparation treatment antisera were taken. A y-globulin frac of nitrite reductase. tion precipitated at 40% saturation of ammonium Purification of Nitrite Reductase-Nitrite re sulfate was used as the antibody preparation. ductase causing a decrease of nitrite in the presence Double immuno-diffusion was carried out by of dithionite and methyl viologen was extracted the method of Ouchterlony (19). For inhibition from cells by sonic treatment. This enzyme was tests, 0.2 ml of the supernatant at 63,000 x g from soluble, and was purified by DEAE-cellulose, crude extract and 0.8 ml of phosphate buffer- DEAE-Sephadex A-50, Sephadex G-150, and hy saline containing various amounts of antibodies droxylapatite chromatographies and by isoelectric were mixed and incubated at 4°C for 16 h.

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