/ . Biochem., 79, 661-671 (1976)
Properties of Purified Hydrogenase from the Particulate Fraction of Desulfovibrio vulgaris, 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 vulgaris 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 absorption spectrum of the enzyme was characteristic of iron-sulfur proteins. The millimolar 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 Hi in the presence of cytochrome Cg, 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 pchloromercuribenzenesulfonate. 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 participate in the production or consumption of Hj in bacterial metabolism. Three kinds of hydrogenases have been reported, with dif• This study was supported in part by a grant (No. 88024, 1971) from the Ministry of Education, Science and Culture, of Japan. Abbreviation: SDS, sodium dodecyl sulfate. Vol. 79, No. 3, 1976
fering specificities for the electron acceptors, NAD+ (1, 2), cytochrome c% (3, 4), and ferredoxin (5, 6), though still another hydrogenase of unknown specificity ( 7 ) is known. We have reported solubilization and purification procedures for the particulate hydrogenase of Desulfovibrio vulgans, Miyazaki [EC 1.12. 2.1], which is specific to cytochrome c3 (4), and derived some kinetic constants for
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the paraHj-orthoHi conversion and isotope exchange reactions catalyzed by the purified hydrogenase (8). In the present communication, some properties of this enzyme and the effects of some metabolic inhibitors on the enzyme are reported. MATERIALS AND METHODS Chemicals and Reagents — Trypsin [EC 3.4.21.4], twice recrystallized, was a product of Worthington. Cytochrome c, was purified as described by Yagi and Maruyama (9). Its molar concentration was expressed on a protein basis instead of a heme basis. Methylviologen was a product of BDH. Hydrogenase [Ht : ferricytochrome cs oxidoreductase, EC 1.12. 2.1] was purified from the cells of Desulfovibrio vulgaris, Miyazaki, by the following procedure, which is a modification of that reported in our previous paper (4). The particulate fraction of the bacterial sonicate was digested with trypsin (1 g for 400 ml of suspension containing 100 g wet precipitate) at 4° for 20 hr with stirring under Nj. The solubilized hydrogenase was then precipitated with ammonium sulfate at 70% saturation, and the precipitate was dissolved in HjO, concentrated by ultrafiltration using a Diaflo cell (Aminco Corporation) with a PM30 membrane, and chromatographed on a Sephadex G-150 column (2.7x72 cm) with 0.02 M Tris-HCl (pH 7.3) containing 0.08 M NaCl as an eluting buffer. The effluent fractions containing hydrogenase activity were collected, concentrated by means of a Diaflo cell with a PM-30 membrane, and charged onto a column (1.3x12 cm) of DEAE-Sephadex A-50 previously equilibrated with the above buffer. Then the enzyme was eluted from the column by the concentration gradient technique starting with the above buffer and ending with 0.05 M Tris-HCl (pH 7.3) containing 0.2 M NaCl. The active fractions of the effluent were concentrated by partial lyophilization, chromatographed again on a column of Sephadex G-150, and the resulting active brownish fractions were collected (see Fig. 1), dialyzed thoroughly, and lyophilized. The preparation thus ob-
tained was nearly homogeneous as determined by disc electrophoresis (10) at pH 7.3 (see Fig. 2). The specific activity of this preparation was about 330 units/Azsonm, or 610 units/mg. Assay of Protein and Intensity of the Brown Color—The concentration of protein was expressed in terms of the absorbance at 280 nm (A^nm). The intensity of the brown color was expressed in terms of the absorbance at 400 nm (Ajoonm). Desulfoviridin and cytochromes which strongly absorb light at 400 nm were eliminated in the early stages of the purification and did not interfere with the assay of the intensity of the brown color. Assay of Hydrogenase—Activity of hydrogenase was assayed either by the Hj-evolution technique (4) or by the enzymic electric cell method (11, 12) recently developed in our laboratories. Molecular Weight—The molecular weight and partial specific volume of the enzyme protein were kindly determined by Prof. N. Ui of Gunma University by the low-speed sedimentation equilibrium method (13) with a photoelectric scanner (14). The molecular weight of the enzyme subunit was determined by electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS) and 2-mercaptoethanol as described by Weber and Osborn (75). Amino Acid Analysis—The protein sample was dried in a stream of N t and hydrolyzed with 6 M HC1 in evacuated glass tubes at 110° for 24 hr or 48 hr. The hydrolysates were analyzed for amino acid contents with a Hitachi KLA-3B, or JEOL automatic amino acid analyzer by the procedure described by Spackman et al. (16). Tryptophan content was determined spectrophotometrically (17). Metal Analysis—Metal contents were determined by atomic absorption spectrometry. Sulfur Analysis—The sulfur atoms in the enzyme were converted into HjS by the tin(II>strong phosphoric acid reduction method described in this paper, and the H^S thus obtained was led into an absorbent solution containing cadmium acetate by passing N t . It was determined by the colorimetric method using N, N'-dimethyl-/>-phenylenediamine (18). J. Biochem.
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PROPERTIES OF DESULFOVIBRIO HYDROGENASE
The assay method for labile sulfide is described later in the text. Electrofocusing^-This was carried out as described in the instruction manual for LKB 8100 Ampholine, with 0.8% carrier ampholite (pH 5-7) on an LKB 8101 electrofocusing column. RESULTS Molecular Weight of the Hydrogenase— Low-speed sedimentation equilibrium analysis of the hydrogenase preparation revealed linear In c : r* relations (73) in both H,0 and 90% DjO media containing phosphate-NaCl buffer (pH 6.5, /i = 0.2). This indicates that the enzyme is homogeneous, and from the slopes of the plots (i.e., d\n c/d r 2 ), the partial specific volume and the molecular weight of the enzyme protein were calculated to be 0.75i, and 89,000, respectively. Electrophoresis on polyacrylamide gel in the presence of SDS and 2-mercaptoethanol revealed the presence of two subunits in the
m enzyme preparation (Fig. 3). The molecular weight of the larger component was estimated to be 59,000, and that of the smaller component, 28,000 (Fig. 4). These two components were always detected upon electrophoresis in the presence of SDS, even in the absence of 2-mercaptoethanol. Spectral Properties—The absorption spectrum of the purified hydrogenase was shown in Fig. 5, Curve 1. The specific absorbance coefficients were 1.84 at 280 nm, and 0.53 at 400 nm. This means that the millimolar absorbance coefficients of the enzyme were about
0.3
20 22 24 26 Fraction Number (Ifr.8ml) Fig. 1. Elution profile of the hydrogenase from a Sephadex G-150 column. The hydrogenase preparation (5 ml) was chromatographed on a column (2.7 X 72 cm) of Sephadex G-150. The elution buffer was 0.02 M Tris-HCl (pH 7.3) containing 0.08 M NaCl. • , concentration of brown pigment expressed in terms of absorbance at 400 nm; O, protein concentration expressed in terms of absorbance at 280 nm; and A, the activity of hydrogenase assayed by the enzymic electric cell method expressed as unit-ml" 1 of the effluent.
Fig. 2. Disc electrophoretic patterns of hydrogenase. Three tubes, A, B, and C (5x50 mm), of polyacrylamide gel were prepared. In tube A, hydrogenase (52//g) was electrophoresed for 50min at 130 volts, then the gel was stained with Amido Black solution in 7fa acetic acid for 20 min and destained by washing with 7% acetic acid. In tubes B and C, hydrogenase (100 ftg per tube) was electrophoresed under the same conditions. The gel was removed from tube C, immersed in 0.6 mM methylviologen in 0.02 M phosphate buffer (pH 6.9) which had been saturated with H,. When the blue color developed as a result of enzymatic reduction of methylviologen, the gel was quickly removed from the solution and a photograph was taken immediately. The gel in tube B was removed from the tube and a photograph was taken immediately. The mobility of the brownish pigment in tube B was identical to that of hydrogenase activity in tube C, and this was the only area stained by Amido Black in tube A.
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T. YAGI, K. KIMURA, H. DAIDOJI, F. SAKAI, S. TAMURA, and H. INOKUCHI
164 at 280 nm, and 47 at 400 nm. The absorbance ratio, A«o nm/^42so nm, of the most highly purified hydrogenase preparation was 0.29.
The brownish proteinaceous component was not separable from the hydrogenase activity either by Sephadex G-150 column chromatography (Fig. 1), or by disc electrophoresis
0.4 05 Relative Mobility
Fig. 3. SDS-gel electrophoretic pattern of hydrogenase. The SDS-gel electrophoretic pattern of hydrogenase (8 mg) was obtained by the procedure of Weber and Osborn (15) in the presence of 2mercaptoethanol. Omission of 2-mercaptoethanol from the procedure did not affect the results.
300
Fig. 4. Relative mobility of hydrogenase compared with reference proteins upon SDS-gel electrophoresis in the presence of 2-mercaptoethanol. The concentration of the cross-linker was half of that used in the standard conditions described by Weber and Osborn (15). O : marker proteins, i.e., a, alcohol dehydrogenase [EC 1.1.1.1] (subunit weight=37,000); b, bovine serum albumin (mol. wt. =68,000); g, glutamate dehydrogenase [EC 1.4.1.3] (mol. wt.= 53,000), and • : hydrogenase subunits.
400 500 Wavelength (nm)
600
Fig. 5. Absorption spectra of hydrogenase. Curve 1: Hydrogenase (0.59 mg-ml"1 in 20 mM phosphate buffer, pH 7.0). No spectral change was observed upon incubating the solution under an atmosphere of H, (36 Torr) at room temperature for 36 hr. Curve 2 : Hydrogenase (0.59 mg-ml"1 in 20 mM phosphate buffer, pH 7.0) containing 6 nM cytochrome c3, incubated for 12 hr under an atmosphere of H, (36 Torr) at room temperature. The spectrum of the Na2S^D4-reduced hydrogenase was identical with Curve 2 in the visible region, and that of the reoxidized enzyme was similar to Curve 1. J. Downloaded from https://academic.oup.com/jb/article-abstract/79/3/661/2185681 by Mount Royal College user on 12 February 2018
Biochtm.
PROPERTIES OF DESULFOVIBRIO HYDROGENASE
665
on polyacrylamide gel at pH 8.3, as shown in Fig. 2. Upon electrofocusing, both the activity and the brownish protein were concentrated at p / 6.2, as shown in Fig. 6. The absorption spectrum of the enzyme was hardly affected by dialyzing the enzyme against 0.1 mM 1,10-phenanthroline for 24 hr. When the hydrogenase preparation was incubated under Hi in the presence of cytochrome Cs, both the enzyme and the cytochrome were reduced, as determined from the spectral change. Figure 5 shows the absorption spectrum of the enzyme (Curve 1) and that of the reduced form of the enzyme prepared by reduction with H2 in the presence of a trace amount of cytochrome c% (Curve 2). No spectral change was observed even after incubation of the enzyme for 24 hr under H t in the absence of cytochrome c,. Metal Contents—The iron content of purified hydrogenase preparation which had been thoroughly dialyzed against glass - distilled water was 0.62% (9.9 moles Fe/89 kg protein). The iron content of enzyme which had been dialyzed against 1 mM 1,10-phenanthroline was
0.58% (9.2 moles Fe/89 kg protein), while that of enzyme which had been dialyzed against 1 mM tiron (sodium catechol-3,5-disulfonate) was 0.45% (7.2 moles Fe/89 kg protein). There was a tendency for the enzyme to absorb iron from the environment. When the enzyme was concentrated by means of a Diaflo cell with a PM-30 membrane, its iron content rose to 3.0%, though when this preparation was thoroughly dialyzed against glass - distilled water, the iron content fell to 1.3%. When the same preparation was dialyzed against 1 mM 1,10-phenanthroline, the iron content fell to the original level, i.e., 0.60%. Molybdenum was not detected in the preparation. Amino Acid Composition—The amino acid composition of the purified hydrogenase preparation is shown in Table I.
1.0
0.5
0.0
10 15 20 25 Fraction Number(lfr:3ml)
Fig. 6. Elution pattern of the purified hydrogenase from the electrofocusing column (LKB 8101) after isoelectric focusing with 0.8% carrier ampholite (pH 5-8) for 5 days at 600 volts. The absorbance of each fraction was corrected by subtracting that of the corresponding fraction obtained when the electrofocusing was run without the enzyme. • , absorbance at 400 nm; O, absorbance at 280 nm; and A, activity of hydrogenase in units-ml"1.
TABLE I. Amino acid composition of the hydrogenase. Amino acid residue Lysine Histidine Arginine Aspartate Threonine Serine Glutamate Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan
Amino acid composition (a)
(b)
5.51
44
2.95
24
3.25
26
9.36
76
6.13
49
4.62
37
8.61
69
7.01
57
8.79
71
10.48
85
2.36
19
7.67
62
1.40
11
4.60
37
6.63
54
3.29
27
4.04
33
3.29»
27
(a) Percentage of amino acid residues in the protein. (b) Moles per mole of protein to the nearest integer. 1 Determined spectrophotometrically.
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T. YAGI, K. KIMURA, H. DAIDOJI, F. SAKAI, S. TAMURA, and H. INOKUCHI
666
Sulfur Contents—{a) Total sulfur: The enzyme was first decomposed with chromium(TV)-strong phosphoric acid at 150° to oxidize all the sulfur atoms to sulfate ions, which were then reduced to HjS by adding tin(II> strong phosphoric acid at 300° after decomposing the chromium(VT)-strong phosphoric acid to the chromium(IH) compound (19). The sulfur content thus determined was 1.6% (44 moles S/89 kg protein). If the enzyme was TABLE n . Effect of metabolic inhibitors on the hydrogenase activity. The enzyme and cytochrome c, were preincubated with the inhibitor in 3.0 ml of 20 mM phosphate buffer, pH 7.0, for 30 min, and the activity was measured by the H,-evolution technique. Inhibitors KCN
Concen- Residual tration activity (mM) (%)
1 10 10 NaN3 100 1 EDTA 10 1 1,10-Phenanthroline 1 Tiron 1 Iodoacetamide 10 N-Ethylmaleimide 0.1 2 £-Chloromercuribenzoate 0.17 £-Chloromercuribenzenesulfonate 1
107 93 95 98 91 90 99-100* 89-95" 99 27i> 79 40b
reduced directly with tin(II)-strong phosphoric acid, only 1/8 of the total sulfur atoms could be recovered as HjS. (b) Labile sulfide: Different assay methods gave different results. ( i ) Assay of HiS derived from the acidified enzyme. The enzyme was first incubated in 0.2 M NaOH at 50° for 60 min, then acidified by the addition of 0.2 M HC1, and the H.S produced was determined. The content of labile sulfide ions thus determined was 0.14% (3.9 moles S*"/89 kg protein). If the enzyme was directly acidified with 2 M HC1, only a half of the labile sulfide ions could be liberated as HjS. ( » ) Direct assay. The enzyme was incubated with zinc acetate-NaOH for 2 hr, and then dimethyl-£-phenylenediamine and FeCl8 reagents were added as described by Suhara et al. (20). Any turbidity was removed by centrifugation, and the blue color developed due to the labile sulfide of protein was determined colorimetrically. The content of labile sulfide ions thus determined was 0.25-0.28% (6.9-7.9 moles S!"/89 kg protein). (c) Sulfur in the apoprotein: The sulfur content calculated from the number of cysteine and methionine residues in the hydrogenase molecule was 1.08% (30 moles S/89 kg protein). Effects of Metabolic Inhibitors and Chemical Modifications—-The enzyme is not inhibited
65 13c
• The activity was measured by the Hj-evolution technique and the enzymic electric cell method, both of which gave similar results. The figures are cited from Ref. 12. b The blue color of the reduced methylviologen disappeared when these compounds were added to the reaction mixture in the Hi-evolution technique. Probably, these inhibitors oxidized the reduced methylviologen, and thus it is not clear whether these reagents prevented the evolution of H» by blocking the SH-groups of the enzyme or by eliminating the electron donor of the reaction system. e The H,-evolution technique and the enzymic electric cell method gave inconsistent results. We chose the latter for the reasons discussed in Ref. 12.
Fig. 7. Inhibition of hydrogenase by CO. Hydrogenase activities were assayed by the H,-evolution technique under the standard assay conditions except that N, in the gas phase was replaced by CON, mixtures, pco is the partial pressure of CO in the gas phase; p 0 , the activity when pco is 0; and PI , the activity when CO is present. /. Biochem.
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PROPERTIES OF DESULFOVIBRIO HYDROGENASE
667 100
1 0 0 l600nm (see also Fig. 14 in Ref. 12). relative to the original intensity. Tlm« (hr)
by metal-complexing agents such as cyanide, azide, 1,10-phenanthroline, tiron, and EDTA, as shown in Table II. The absorption spectra and the specific activities of enzyme which had been treated with 1,10-phenanthroline or tiron were not very different from those of the untreated enzyme. The enzyme was inhibited to some extent by SH-blocking reagents such as iodoacetamide, iodoacetate, N-methylmaleimide, p - chloromercuribenzenesulfonate, or /xhloromercuribenzoate (Table II). CO strongly inhibited the Hj-evolution reaction. The reciprocal of the residual activity was plotted against the partial pressure of CO, as shown in Fig. 7. From this figure, the inhibitor constant (K{) for CO was calculated to be 8.9 Torr. Upon flushing out CO with a stream of Ni, the reaction mixture recovered the original Hi evolution activity, as in the case of the soluble hydrogenase of the same organism ( 3 ) . Hydrogenase was gradually inactivated upon prolonged incubation with dilute HjOt at
25°, and the concomitant spectral change is shown in Fig. 8. The activity of hydrogenase, like those of many other enzymes, decreased when measured in the presence of urea (Fig. 9). However, when the enzyme was incubated in 9 M urea for 24 hr at room temperature and the activity was measured immediately after 15fold dilution, no inactivation at all was observed. The enzyme was also inactivated by 86— 99% in 0.1% SDS (12). When the enzyme was incubated in 0.1% SDS for 120 min at 30° either in the presence or absence of cytochrome d, and the activity was measured immediately after 300-fold dilution, 60% of the original activity was recovered. DISCUSSION Hydrogenase catalyzes the transfer of electrons reversibly between H, and an electron acceptor as well as the conversion between paraHi and
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TABLE Ilf.
Comparison of the properties of hydrogenase preparations of various origins.
Source of hydrogenase
Hydrogenomonas H16« NAD +
Electron acceptor
1.12.1.2
NCIB 8303»
Hildenborough
cytochrome c,
cytochrome c3
1
Miyazaki
0
cytochrome c5
Clostridium pasteurianum W5» ferredoxin
Chromatiumb not NAD\ nor ferredoxin
1.12.2.1
1.12.2.1
1.12.7.1
45,000
60,000
89,000
60,000
98,000
30,000x2
59,000+28,000
monomer
50,000x2
0.72
3.5
7-9
12
4
0.35
3.2
7-8
12
4
408
near 400
near 400
near 400
410
-4«o=0.31
/l«o=O.53
AtK=0A2
Properties of Purified Hydrogenase from the Particulate Fraction of Desulfovibrio vulgaris, 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 vulgaris 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 absorption spectrum of the enzyme was characteristic of iron-sulfur proteins. The millimolar 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 Hi in the presence of cytochrome Cg, 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 pchloromercuribenzenesulfonate. 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 participate in the production or consumption of Hj in bacterial metabolism. Three kinds of hydrogenases have been reported, with dif• This study was supported in part by a grant (No. 88024, 1971) from the Ministry of Education, Science and Culture, of Japan. Abbreviation: SDS, sodium dodecyl sulfate. Vol. 79, No. 3, 1976
fering specificities for the electron acceptors, NAD+ (1, 2), cytochrome c% (3, 4), and ferredoxin (5, 6), though still another hydrogenase of unknown specificity ( 7 ) is known. We have reported solubilization and purification procedures for the particulate hydrogenase of Desulfovibrio vulgans, Miyazaki [EC 1.12. 2.1], which is specific to cytochrome c3 (4), and derived some kinetic constants for
661
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T. YAGI, K. KIMURA, H. DAIDOJI, F. SAKAI, S. TAMURA, and H. INOKUCHI
the paraHj-orthoHi conversion and isotope exchange reactions catalyzed by the purified hydrogenase (8). In the present communication, some properties of this enzyme and the effects of some metabolic inhibitors on the enzyme are reported. MATERIALS AND METHODS Chemicals and Reagents — Trypsin [EC 3.4.21.4], twice recrystallized, was a product of Worthington. Cytochrome c, was purified as described by Yagi and Maruyama (9). Its molar concentration was expressed on a protein basis instead of a heme basis. Methylviologen was a product of BDH. Hydrogenase [Ht : ferricytochrome cs oxidoreductase, EC 1.12. 2.1] was purified from the cells of Desulfovibrio vulgaris, Miyazaki, by the following procedure, which is a modification of that reported in our previous paper (4). The particulate fraction of the bacterial sonicate was digested with trypsin (1 g for 400 ml of suspension containing 100 g wet precipitate) at 4° for 20 hr with stirring under Nj. The solubilized hydrogenase was then precipitated with ammonium sulfate at 70% saturation, and the precipitate was dissolved in HjO, concentrated by ultrafiltration using a Diaflo cell (Aminco Corporation) with a PM30 membrane, and chromatographed on a Sephadex G-150 column (2.7x72 cm) with 0.02 M Tris-HCl (pH 7.3) containing 0.08 M NaCl as an eluting buffer. The effluent fractions containing hydrogenase activity were collected, concentrated by means of a Diaflo cell with a PM-30 membrane, and charged onto a column (1.3x12 cm) of DEAE-Sephadex A-50 previously equilibrated with the above buffer. Then the enzyme was eluted from the column by the concentration gradient technique starting with the above buffer and ending with 0.05 M Tris-HCl (pH 7.3) containing 0.2 M NaCl. The active fractions of the effluent were concentrated by partial lyophilization, chromatographed again on a column of Sephadex G-150, and the resulting active brownish fractions were collected (see Fig. 1), dialyzed thoroughly, and lyophilized. The preparation thus ob-
tained was nearly homogeneous as determined by disc electrophoresis (10) at pH 7.3 (see Fig. 2). The specific activity of this preparation was about 330 units/Azsonm, or 610 units/mg. Assay of Protein and Intensity of the Brown Color—The concentration of protein was expressed in terms of the absorbance at 280 nm (A^nm). The intensity of the brown color was expressed in terms of the absorbance at 400 nm (Ajoonm). Desulfoviridin and cytochromes which strongly absorb light at 400 nm were eliminated in the early stages of the purification and did not interfere with the assay of the intensity of the brown color. Assay of Hydrogenase—Activity of hydrogenase was assayed either by the Hj-evolution technique (4) or by the enzymic electric cell method (11, 12) recently developed in our laboratories. Molecular Weight—The molecular weight and partial specific volume of the enzyme protein were kindly determined by Prof. N. Ui of Gunma University by the low-speed sedimentation equilibrium method (13) with a photoelectric scanner (14). The molecular weight of the enzyme subunit was determined by electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS) and 2-mercaptoethanol as described by Weber and Osborn (75). Amino Acid Analysis—The protein sample was dried in a stream of N t and hydrolyzed with 6 M HC1 in evacuated glass tubes at 110° for 24 hr or 48 hr. The hydrolysates were analyzed for amino acid contents with a Hitachi KLA-3B, or JEOL automatic amino acid analyzer by the procedure described by Spackman et al. (16). Tryptophan content was determined spectrophotometrically (17). Metal Analysis—Metal contents were determined by atomic absorption spectrometry. Sulfur Analysis—The sulfur atoms in the enzyme were converted into HjS by the tin(II>strong phosphoric acid reduction method described in this paper, and the H^S thus obtained was led into an absorbent solution containing cadmium acetate by passing N t . It was determined by the colorimetric method using N, N'-dimethyl-/>-phenylenediamine (18). J. Biochem.
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PROPERTIES OF DESULFOVIBRIO HYDROGENASE
The assay method for labile sulfide is described later in the text. Electrofocusing^-This was carried out as described in the instruction manual for LKB 8100 Ampholine, with 0.8% carrier ampholite (pH 5-7) on an LKB 8101 electrofocusing column. RESULTS Molecular Weight of the Hydrogenase— Low-speed sedimentation equilibrium analysis of the hydrogenase preparation revealed linear In c : r* relations (73) in both H,0 and 90% DjO media containing phosphate-NaCl buffer (pH 6.5, /i = 0.2). This indicates that the enzyme is homogeneous, and from the slopes of the plots (i.e., d\n c/d r 2 ), the partial specific volume and the molecular weight of the enzyme protein were calculated to be 0.75i, and 89,000, respectively. Electrophoresis on polyacrylamide gel in the presence of SDS and 2-mercaptoethanol revealed the presence of two subunits in the
m enzyme preparation (Fig. 3). The molecular weight of the larger component was estimated to be 59,000, and that of the smaller component, 28,000 (Fig. 4). These two components were always detected upon electrophoresis in the presence of SDS, even in the absence of 2-mercaptoethanol. Spectral Properties—The absorption spectrum of the purified hydrogenase was shown in Fig. 5, Curve 1. The specific absorbance coefficients were 1.84 at 280 nm, and 0.53 at 400 nm. This means that the millimolar absorbance coefficients of the enzyme were about
0.3
20 22 24 26 Fraction Number (Ifr.8ml) Fig. 1. Elution profile of the hydrogenase from a Sephadex G-150 column. The hydrogenase preparation (5 ml) was chromatographed on a column (2.7 X 72 cm) of Sephadex G-150. The elution buffer was 0.02 M Tris-HCl (pH 7.3) containing 0.08 M NaCl. • , concentration of brown pigment expressed in terms of absorbance at 400 nm; O, protein concentration expressed in terms of absorbance at 280 nm; and A, the activity of hydrogenase assayed by the enzymic electric cell method expressed as unit-ml" 1 of the effluent.
Fig. 2. Disc electrophoretic patterns of hydrogenase. Three tubes, A, B, and C (5x50 mm), of polyacrylamide gel were prepared. In tube A, hydrogenase (52//g) was electrophoresed for 50min at 130 volts, then the gel was stained with Amido Black solution in 7fa acetic acid for 20 min and destained by washing with 7% acetic acid. In tubes B and C, hydrogenase (100 ftg per tube) was electrophoresed under the same conditions. The gel was removed from tube C, immersed in 0.6 mM methylviologen in 0.02 M phosphate buffer (pH 6.9) which had been saturated with H,. When the blue color developed as a result of enzymatic reduction of methylviologen, the gel was quickly removed from the solution and a photograph was taken immediately. The gel in tube B was removed from the tube and a photograph was taken immediately. The mobility of the brownish pigment in tube B was identical to that of hydrogenase activity in tube C, and this was the only area stained by Amido Black in tube A.
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664
T. YAGI, K. KIMURA, H. DAIDOJI, F. SAKAI, S. TAMURA, and H. INOKUCHI
164 at 280 nm, and 47 at 400 nm. The absorbance ratio, A«o nm/^42so nm, of the most highly purified hydrogenase preparation was 0.29.
The brownish proteinaceous component was not separable from the hydrogenase activity either by Sephadex G-150 column chromatography (Fig. 1), or by disc electrophoresis
0.4 05 Relative Mobility
Fig. 3. SDS-gel electrophoretic pattern of hydrogenase. The SDS-gel electrophoretic pattern of hydrogenase (8 mg) was obtained by the procedure of Weber and Osborn (15) in the presence of 2mercaptoethanol. Omission of 2-mercaptoethanol from the procedure did not affect the results.
300
Fig. 4. Relative mobility of hydrogenase compared with reference proteins upon SDS-gel electrophoresis in the presence of 2-mercaptoethanol. The concentration of the cross-linker was half of that used in the standard conditions described by Weber and Osborn (15). O : marker proteins, i.e., a, alcohol dehydrogenase [EC 1.1.1.1] (subunit weight=37,000); b, bovine serum albumin (mol. wt. =68,000); g, glutamate dehydrogenase [EC 1.4.1.3] (mol. wt.= 53,000), and • : hydrogenase subunits.
400 500 Wavelength (nm)
600
Fig. 5. Absorption spectra of hydrogenase. Curve 1: Hydrogenase (0.59 mg-ml"1 in 20 mM phosphate buffer, pH 7.0). No spectral change was observed upon incubating the solution under an atmosphere of H, (36 Torr) at room temperature for 36 hr. Curve 2 : Hydrogenase (0.59 mg-ml"1 in 20 mM phosphate buffer, pH 7.0) containing 6 nM cytochrome c3, incubated for 12 hr under an atmosphere of H, (36 Torr) at room temperature. The spectrum of the Na2S^D4-reduced hydrogenase was identical with Curve 2 in the visible region, and that of the reoxidized enzyme was similar to Curve 1. J. Downloaded from https://academic.oup.com/jb/article-abstract/79/3/661/2185681 by Mount Royal College user on 12 February 2018
Biochtm.
PROPERTIES OF DESULFOVIBRIO HYDROGENASE
665
on polyacrylamide gel at pH 8.3, as shown in Fig. 2. Upon electrofocusing, both the activity and the brownish protein were concentrated at p / 6.2, as shown in Fig. 6. The absorption spectrum of the enzyme was hardly affected by dialyzing the enzyme against 0.1 mM 1,10-phenanthroline for 24 hr. When the hydrogenase preparation was incubated under Hi in the presence of cytochrome Cs, both the enzyme and the cytochrome were reduced, as determined from the spectral change. Figure 5 shows the absorption spectrum of the enzyme (Curve 1) and that of the reduced form of the enzyme prepared by reduction with H2 in the presence of a trace amount of cytochrome c% (Curve 2). No spectral change was observed even after incubation of the enzyme for 24 hr under H t in the absence of cytochrome c,. Metal Contents—The iron content of purified hydrogenase preparation which had been thoroughly dialyzed against glass - distilled water was 0.62% (9.9 moles Fe/89 kg protein). The iron content of enzyme which had been dialyzed against 1 mM 1,10-phenanthroline was
0.58% (9.2 moles Fe/89 kg protein), while that of enzyme which had been dialyzed against 1 mM tiron (sodium catechol-3,5-disulfonate) was 0.45% (7.2 moles Fe/89 kg protein). There was a tendency for the enzyme to absorb iron from the environment. When the enzyme was concentrated by means of a Diaflo cell with a PM-30 membrane, its iron content rose to 3.0%, though when this preparation was thoroughly dialyzed against glass - distilled water, the iron content fell to 1.3%. When the same preparation was dialyzed against 1 mM 1,10-phenanthroline, the iron content fell to the original level, i.e., 0.60%. Molybdenum was not detected in the preparation. Amino Acid Composition—The amino acid composition of the purified hydrogenase preparation is shown in Table I.
1.0
0.5
0.0
10 15 20 25 Fraction Number(lfr:3ml)
Fig. 6. Elution pattern of the purified hydrogenase from the electrofocusing column (LKB 8101) after isoelectric focusing with 0.8% carrier ampholite (pH 5-8) for 5 days at 600 volts. The absorbance of each fraction was corrected by subtracting that of the corresponding fraction obtained when the electrofocusing was run without the enzyme. • , absorbance at 400 nm; O, absorbance at 280 nm; and A, activity of hydrogenase in units-ml"1.
TABLE I. Amino acid composition of the hydrogenase. Amino acid residue Lysine Histidine Arginine Aspartate Threonine Serine Glutamate Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan
Amino acid composition (a)
(b)
5.51
44
2.95
24
3.25
26
9.36
76
6.13
49
4.62
37
8.61
69
7.01
57
8.79
71
10.48
85
2.36
19
7.67
62
1.40
11
4.60
37
6.63
54
3.29
27
4.04
33
3.29»
27
(a) Percentage of amino acid residues in the protein. (b) Moles per mole of protein to the nearest integer. 1 Determined spectrophotometrically.
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T. YAGI, K. KIMURA, H. DAIDOJI, F. SAKAI, S. TAMURA, and H. INOKUCHI
666
Sulfur Contents—{a) Total sulfur: The enzyme was first decomposed with chromium(TV)-strong phosphoric acid at 150° to oxidize all the sulfur atoms to sulfate ions, which were then reduced to HjS by adding tin(II> strong phosphoric acid at 300° after decomposing the chromium(VT)-strong phosphoric acid to the chromium(IH) compound (19). The sulfur content thus determined was 1.6% (44 moles S/89 kg protein). If the enzyme was TABLE n . Effect of metabolic inhibitors on the hydrogenase activity. The enzyme and cytochrome c, were preincubated with the inhibitor in 3.0 ml of 20 mM phosphate buffer, pH 7.0, for 30 min, and the activity was measured by the H,-evolution technique. Inhibitors KCN
Concen- Residual tration activity (mM) (%)
1 10 10 NaN3 100 1 EDTA 10 1 1,10-Phenanthroline 1 Tiron 1 Iodoacetamide 10 N-Ethylmaleimide 0.1 2 £-Chloromercuribenzoate 0.17 £-Chloromercuribenzenesulfonate 1
107 93 95 98 91 90 99-100* 89-95" 99 27i> 79 40b
reduced directly with tin(II)-strong phosphoric acid, only 1/8 of the total sulfur atoms could be recovered as HjS. (b) Labile sulfide: Different assay methods gave different results. ( i ) Assay of HiS derived from the acidified enzyme. The enzyme was first incubated in 0.2 M NaOH at 50° for 60 min, then acidified by the addition of 0.2 M HC1, and the H.S produced was determined. The content of labile sulfide ions thus determined was 0.14% (3.9 moles S*"/89 kg protein). If the enzyme was directly acidified with 2 M HC1, only a half of the labile sulfide ions could be liberated as HjS. ( » ) Direct assay. The enzyme was incubated with zinc acetate-NaOH for 2 hr, and then dimethyl-£-phenylenediamine and FeCl8 reagents were added as described by Suhara et al. (20). Any turbidity was removed by centrifugation, and the blue color developed due to the labile sulfide of protein was determined colorimetrically. The content of labile sulfide ions thus determined was 0.25-0.28% (6.9-7.9 moles S!"/89 kg protein). (c) Sulfur in the apoprotein: The sulfur content calculated from the number of cysteine and methionine residues in the hydrogenase molecule was 1.08% (30 moles S/89 kg protein). Effects of Metabolic Inhibitors and Chemical Modifications—-The enzyme is not inhibited
65 13c
• The activity was measured by the Hj-evolution technique and the enzymic electric cell method, both of which gave similar results. The figures are cited from Ref. 12. b The blue color of the reduced methylviologen disappeared when these compounds were added to the reaction mixture in the Hi-evolution technique. Probably, these inhibitors oxidized the reduced methylviologen, and thus it is not clear whether these reagents prevented the evolution of H» by blocking the SH-groups of the enzyme or by eliminating the electron donor of the reaction system. e The H,-evolution technique and the enzymic electric cell method gave inconsistent results. We chose the latter for the reasons discussed in Ref. 12.
Fig. 7. Inhibition of hydrogenase by CO. Hydrogenase activities were assayed by the H,-evolution technique under the standard assay conditions except that N, in the gas phase was replaced by CON, mixtures, pco is the partial pressure of CO in the gas phase; p 0 , the activity when pco is 0; and PI , the activity when CO is present. /. Biochem.
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PROPERTIES OF DESULFOVIBRIO HYDROGENASE
667 100
1 0 0 l600nm (see also Fig. 14 in Ref. 12). relative to the original intensity. Tlm« (hr)
by metal-complexing agents such as cyanide, azide, 1,10-phenanthroline, tiron, and EDTA, as shown in Table II. The absorption spectra and the specific activities of enzyme which had been treated with 1,10-phenanthroline or tiron were not very different from those of the untreated enzyme. The enzyme was inhibited to some extent by SH-blocking reagents such as iodoacetamide, iodoacetate, N-methylmaleimide, p - chloromercuribenzenesulfonate, or /xhloromercuribenzoate (Table II). CO strongly inhibited the Hj-evolution reaction. The reciprocal of the residual activity was plotted against the partial pressure of CO, as shown in Fig. 7. From this figure, the inhibitor constant (K{) for CO was calculated to be 8.9 Torr. Upon flushing out CO with a stream of Ni, the reaction mixture recovered the original Hi evolution activity, as in the case of the soluble hydrogenase of the same organism ( 3 ) . Hydrogenase was gradually inactivated upon prolonged incubation with dilute HjOt at
25°, and the concomitant spectral change is shown in Fig. 8. The activity of hydrogenase, like those of many other enzymes, decreased when measured in the presence of urea (Fig. 9). However, when the enzyme was incubated in 9 M urea for 24 hr at room temperature and the activity was measured immediately after 15fold dilution, no inactivation at all was observed. The enzyme was also inactivated by 86— 99% in 0.1% SDS (12). When the enzyme was incubated in 0.1% SDS for 120 min at 30° either in the presence or absence of cytochrome d, and the activity was measured immediately after 300-fold dilution, 60% of the original activity was recovered. DISCUSSION Hydrogenase catalyzes the transfer of electrons reversibly between H, and an electron acceptor as well as the conversion between paraHi and
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TABLE Ilf.
Comparison of the properties of hydrogenase preparations of various origins.
Source of hydrogenase
Hydrogenomonas H16« NAD +
Electron acceptor
1.12.1.2
NCIB 8303»
Hildenborough
cytochrome c,
cytochrome c3
1
Miyazaki
0
cytochrome c5
Clostridium pasteurianum W5» ferredoxin
Chromatiumb not NAD\ nor ferredoxin
1.12.2.1
1.12.2.1
1.12.7.1
45,000
60,000
89,000
60,000
98,000
30,000x2
59,000+28,000
monomer
50,000x2
0.72
3.5
7-9
12
4
0.35
3.2
7-8
12
4
408
near 400
near 400
near 400
410
-4«o=0.31
/l«o=O.53
AtK=0A2
