J. Biochem., 79, 661-671 (1976) Properties of Purified Hydrogenase from the Particulate Fraction of Desulfovibrio ƒÊulgaris, Miyazaki1 Tatsuhiko YAGI,* Keisaku KIMURA ,** Hidehiro DAIDOJI,*** Fumiko SAKAI,*** Shohei TAMURA ,*** and Hiroo INOKUCHI** *Department of Chemistry , Shizuoka University, Oya-836, Shizuoka 422, **Institute for Molecular Science , Okazaki 444, and ***The Institute for Solid State Physics, the University of Tokyo , Roppongi, Minato-ku, Tokyo 106 Received for publication, September 30 , 1975 The properties of purified hydrogenase [EC 1.12.2.1] solubilized from particulate fraction of sonicated Desulfovibrio ƒÒulgaris cells are described . The enzyme was a brownish iron-sulfur protein of molecular weight 89,000, composed of two different subunits (mol. wt.: 28,000 and 59,000), and it contained 7-9 iron atoms and 7-8 labile sulfide ions. Molybdenum was not detected in the preparation. The absorp tion spectrum of the enzyme was characteristic of iron-sulfur proteins. The milli molar absorbance coefficients of the enzyme were about 164 at 280 nm, and 47 at 400 nm. The absorption spectrum of the enzyme in the visible region changed upon incubating the enzyme under H2 in the presence of cytochrome c3 , but not in its absence. This spectral change was due to the reduction of the enzyme. The absorbance ratio at 400 nm of the reduced and the oxidized forms of the enzyme was 0.66. The activity of the enzyme was hardly affected by metal-complexing agents such as cyanide, azide, 1, 10-phenanthroline, etc., except for CO, which was a strong inhibitor of the enzyme. The activity was inhibited by SH-reagents such as p- chloromercuribenzenesulfonate. The enzyme was significantly resistant to urea, but susceptible to sodium dodecyl sulfate. These properties were very similar to those of clostridial hydrogenase [EC 1.12.7.1], in spite of differences in the acceptor specificity and subunit structure. Hydrogenases are bacterial enzymes which fering specificities for the electron acceptors, participate in the production or consumption NAD+ (1, 2), cytochrome c3 (3, 4), and fer of H2 in bacterial metabolism. Three kinds redoxin (5, 6), though still another hydro of hydrogenases have been reported, with dif- genase of unknown specificity (7) is known. We have reported solubilization and puri 1 This study was supported in part by a grant (No. fication procedures for the particulate hydro 88024, 1971) from the Ministry of Education, Science genase of Desulfovibrio vulgaris, Miyazaki [EC and Culture, of Japan. 1.12.2.1], which is specific to cytochrome c3 Abbreviation : SDS, sodium dodecyl sulfate. (4), and derived some kinetic constants for Vol. 79, No. 3, 1976 661 662 T. YAGI, K. KIMURA, H. DAIDOJI, F. SAKAI, S. TAMURA, and H. INOKUCHI the paraH2-orthoH2 conversion and isotope ex- tained was nearly homogeneous as determined change reactions catalyzed by the purified hy by disc electrophoresis (10) at pH 7.3 (see drogenase (8). Fig. 2). The specific activity of this prepa In the present communication, some prop ration was about 330 units/A28o nm, or 610 erties of this enzyme and the effects of some units/mg. metabolic inhibitors on the enzyme are re- Assay of Protein and Intensity of the ported. Brown Color-The concentration of protein was expressed in terms of the absorbance at MATERIALS AND METHODS 280 nm (A280 nm). The intensity of the brown color was expressed in terms of the absorb Chemicals and Reagents - Trypsin [EC ance at 400 nm (A4oo nm). Desulfoviridin and 3.4.21.41, twice recrystallized, was a product cytochromes which strongly absorb light at of Worthington. Cytochrome c3 was purified 400 nm were eliminated in the early stages as described by Yagi and Maruyama (9 ). Its of the purification and did not interfere with molar concentration was expressed on a pro the assay of the intensity of the brown color. tein basis instead of a heme basis. Methyl Assay of Hydrogenase-Activity of hydro viologen was a product of BDH. genase was assayed either by the H2-evolution Hydrogenase [H2 : ferricytochrome c3 oxi technique (4) or by the enzymic electric cell doreductase, EC 1.12.2.11 was purified from method (11, 12) recently developed in our the cells of Desulfovibrio vulgaris, Miyazaki, laboratories. by the following procedure, which is a modi Molecular Weight-The molecular weight fication of that reported in our previous paper and partial specific volume of the enzyme pro (4 ). The particulate fraction of the bacterial tein were kindly determined by Prof. N. Ui sonicate was digested with trypsin (1 g for of Gunma University by the low-speed sedi 400 ml of suspension containing 100 g wet mentation equilibrium method (13) with a precipitate) at 4? for 20 hr with stirring under photoelectric scanner (14 ). The molecular N2. The solubilized hydrogenase was then weight of the enzyme subunit was determined precipitated with ammonium sulfate at 70% by electrophoresis on polyacrylamide gel in saturation, and the precipitate was dissolved the presence of sodium dodecyl sulfate (SDS) in H2O, concentrated by ultrafiltration using and 2-mercaptoethanol as described by Weber a Diaflo cell (Aminco Corporation) with a PM- and Osborn (15). 30 membrane, and chromatographed on a Se Amino Acid Analysis-The protein sam phadex G-150 column (2.7•~72 cm) with 0.02 ple was dried in a stream of N2 and hydro M Tris-HC1 (pH 7.3) containing 0.08 M NaCl lyzed with 6 M HC1 in evacuated glass tubes as an eluting buffer. The effluent fractions at 110? for 24 hr or 48 hr. The hydrolysates containing hydrogenase activity were collected, were analyzed for amino acid contents with a concentrated by means of a Diaflo cell with a Hitachi KLA-3B, or JEOL automatic amino PM-30 membrane, and charged onto a column acid analyzer by the procedure described by (1.3•~12 cm) of DEAE-Sephadex A-50 previous Spackman et al. (16). Tryptophan content ly equilibrated with the above buffer. Then was determined spectrophotometrically (17 ). the enzyme was eluted from the column by Metal Analysis-Metal contents were de the concentration gradient technique starting termined by atomic absorption spectrometry. with the above buffer and ending with 0.05 M Sulfur Analysis-The sulfur atoms in the Tris-HCl (pH 7.3) containing 0.2 M NaCl. The enzyme were converted into H2S by the tin(II)- active fractions of the effluent were concen strong phosphoric acid reduction method de trated by partial lyophilization, chromato scribed in this paper, and the H2S thus ob graphed again on a column of Sephadex G-150, tained was led into an absorbent solution con and the resulting active brownish fractions taining cadmium acetate by passing N2. It were collected (see Fig. 1), dialyzed thorough- was determined by the colorimetric method ly, and lyophilized. The preparation thus ob- using N, N•Œ-dimethyl-p-phenylenediamine (18). J. Biochem. PROPERTIES OF DESULFOVIBRIO HYDROGENASE 663 The assay method for labile sulfide is de enzyme preparation (Fig. 3). The molecular scribed later in the text. weight of the larger component was estimated Electrofocusing-This was carried out as to be 59,000, and that of the smaller compo described in the instruction manual for LKB nent, 28,000 (Fig. 4). These two components 8100 Ampholine, with 0.8% carrier ampholite were always detected upon electrophoresis (pH 5-7) on an LKB 8101 electrofocusing col in the presence of SDS, even in the absence umn. of 2-mercaptoethanol. Spectral Properties-The absorption spec RESULTS trum of the purified hydrogenase was shown in Fig. 5, Curve 1. The specific absorbance Molecular Weight of the Hydrogenase- coefficients were 1.84 at 280 nm, and 0.53 at Low-speed sedimentation equilibrium analysis 400 nm. This means that the millimolar ab of the hydrogenase preparation revealed linear sorbance coefficients of the enzyme were about In c : ƒÁ2 relations (13) in both H20 and 90% D20 media containing phosphate-NaC1 buffer (pH 6.5, p=0.2). This indicates that the en zyme is homogeneous, and from the slopes of the plots (i.e., d In c/d ƒÁ2), the partial specific volume and the molecular weight of the en zyme protein were calculated to be 0.751, and 89,000, respectively. Electrophoresis on polyacrylamide gel in the presence of SDS and 2-mercaptoethanol revealed the presence of two subunits in the Fig. 2. Disc electrophoretic patterns of hydrogenase. Three tubes, A, B, and C (5 x 50 mM), of polyacryl amide gel were prepared. In tube A, hydrogenase (52 ƒÊg) was electrophoresed for 50 min at 130 volts, then the gel was stained with Amido Black solution in 7% acetic acid for 20 min and destained by washing with 7% acetic acid. In tubes B and C, hydrogenase (100 ƒÊg per tube) was electrophoresed under the same conditions. The gel was removed from tube C, immersed in 0.6 mM methylviologen in Fig. 1. Elution profile of the hydrogenase from a 0.02 M phosphate buffer (pH 6.9) which had been Sephadex G-150 column. The hydrogenase prepara saturated with Ha• When the blue color developed tion (5 ml) was chromatographed on a column (2.7 x as a result of enzymatic reduction of methylviologen, 72 cm) of Sephadex G-150. The elution buffer was the gel was quickly removed from the solution and 0.02 M Tris-HC1 (pH 7.3) containing 0.08 M NaC1. •œ a photograph was taken immediately. The gel in , concentration of brown pigment expressed in tube B was removed from the tube and a photo terms of absorbance at 400 nm ; O , protein concen graph was taken immediately. The mobility of the tration expressed in terms of absorbance at 280 nm ; brownish pigment in tube B was identical to that and 4, the activity of hydrogenase assayed by the of hydrogenase activity in tube C, and this was the enzymic electric cell method expressed as unit•m1-1 only area stained by Amido Black in tube A.