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Reconstitution of hydrogenase from its apoprotein
Journol of Moleculor Catalysis. 6 (1979) 299 - 301 0 Efsevïer Sequoia S.A., Lausanne - Printed in the Nttherlands
299
Short Communïcation
Reconstitution
of Hydrogenase
ICHIRO OKURA,
KEN-ICHI NAKAMURA
Department of Chemical Tokyo, 152 (daoan) (Received
Engineering,
hom
Tokyo
iLs Apoproteln
and SATOSHI Inslitute
NAKAMURA
of Technotog;r,
nfeguro-Ru.
December 12.1978)
A procedure has been described prevïously for the rcmoval of iron and labde sulfide from clostridial ferredoxin to isolate apoferredoxin, and the reconstitution of ferredoxin Erom this prokin [l - 31. Thïs procedure, which has considerable utility, is being applied to other iron-sulfur protems such as spinach ferredoxin [4], adrenodoxin [SI, and putidaredoxïn [S] , and is also useful in increasing the sensitivity of the Mössbauer method by which proteïn enrïched in “Fe were obtained 17, 81. It bas been shown [9] that the hydrogenase used in this werk contains two Fe,S,£lusters per enzyme molecule from the formation of the Fe,&(thiophenyl), dlanion by the addition of thiophenol. The above-mentioned procedure has been applied to hydrogenasc, and we describe in thE communication methods for the preparation of hydrogenase apoprotein and the reconstitution of hydrogenase from its apoprotein. -411 reagents were obtined from commercial sources and were of the highest, purïty availableHydrogenase iHZ: ferricytochrome C3 osidoreducm, EC 1.12.2.13 from
Desulfouìbrio
udguris,
Miyazaki,
is US&.
Deszdfouibrio
uulgaris
celLs
(whlch were kindly donated by Prof. T. Yagï of Shizuoka Unïv.) were culturd accord’mg to the lïterature [ 101 _The enzyme was purïfied according to Yagï’s procedure [lL] _ Hydrogenase activïty was assayed by the evolution r-ate of hydrogen from reduced methyl viologen at 30 “C. The evolved hydrogen was analyzed by gas chromatography. A reaction mixture contained hydrogenase (5 X l!YIO mol), 7.05 X PO-” mol of methyl viologen and Na2S205 (5 mg) in 3.0 ml of 0.02M phosphate buffer (pH 7.0). The method used for the preparation of apoproteïn was a modification of these of Rabïnowïtz et al. El- 31 and Rao ef al. [7]. Hydrogenax (1.2 X ZO-’ mol) in 6 ml of phosphate buffer was treated w-ith 2 ml of 20% trïchloroacetic acid and flushed wïth nitrogen gas for 3 h to expel HzS. The mixture was centrïfuged ât 20 000 g for 20 min. The precipitate was washecí with water, wïfh 5% trïchloroacetic acid, agaïn with water and resuspended in 4.5 ml of 0.05M Ris-HCl buffer.
The spectrum of the resultïng solution is shown in Fig. 1, curve b_ The solution has no extindion peak at 420 nm attributable to the iron-sulfur cluster, but that at 280 nm remaïns, and üon was not detected in this solution. This ïmplies that the solution contains the protein without an ïronsulfur cluster or apoproteïn.
Fig. 1. Spectra
of hydrogenase
(a), apoprotein
(b) and reconstituted
hydrogenase
(c)_
The determinstion of iron was carrred out by a*omic ahsorption spectrometry and by a spectroscopic method [l, 12]_ The reconstitution of hydrogenase from its apoprotein is carried out as fellows. Four millilitres of the apoproteïn were treated wïth 0.2 ml of 2mercaptoethanol for 2-5 h at room temperature under nitrogen atmosphere. Sodium sulphide, 0.5 ml (4 X lO-‘M), and 0.5 ml of FeCla (4 X LOP2M) or FeSOS(NHa)2SOS were added (the reaction conditions are shown in Table l)_ The reconstituted hydrogenase was separated from excess of reagent by pa&ng bhrough a column (1.5 X 15 cm) of Sephadex G-50. TABLE
1
Run no.
Preparation
system
8
Hydrogenase (holoprotein) Amprotcin Apoprotein + Nag Apoprotcin + Fe Apoprotein + NaaS f Fe*+ Apoprotcin + Na+ + FeC13 + HSCHfiH@H Apoprotem f Na&G L FeSO&NH~)~SO~ + HSCH+H20H Nz# -+ FcSO,(NH~)~SO~ + HSCH&HzOH
Prep.aration
cor,dition
-
Activky (R) 100
0
Room Room Room Room Room
temp. temp temp. temp. temp_
for for for for for
1 1 1 1 1
d d d d d
0
Room
timp.
for 3 h
0
0 0 1.1
1.03
As shown in Table 1, the reconstitution requïred the addition of a s-ource of ionic iron, inorganïc sulfide, and meruptide reducing agent. These
301
were supglied by FeC& or FeSC?+(NH1)2SO~, sodium sulfide and 2-mercaptoethanol. No reconstitution was okerved if any of these components were amitted Though the actitity of the reconstItut& hydrogenase is very low compared tith the orïgma2 hydrogenase, the turnover number per hydrogenase molecuIe was 3.2 min-’ mol-enzyme-l. This resuit shows that only a small portion of the apoprotein is recor&ituted. For thi.s reason, the extinction peak at 420 nm might not he observed, sec Fig. 1, curve c. Further work on thisprojeck k proceeding. The authors are grateful to Prof. T. Yagï OF Shizuoka Univ. for protision of the Desulfouibrio udg~ris cells and for bis help tith the hydrogenase purification.
References 1 W. B. Lovenberg. E. Bucbanan and J. C. Rabinowitz, J. Biel. Chem.. 238 (1963) 3 899. 2 R. Malkin and J. C. Rabinowitz, Biochem. Blophys. Res. Commun., 23 (1966) 822. 3 J. Hong and J. C. Rabïqowi~, Biochem. Biophys_ Res. Commun., 29 (1967) 246. 4 E_ Bayer, D. Josef, P_ Kraus. H Hagenmaier, A_ Roder and A. Trebst, Eiochim_ Eiophys. Acts. 143(1967)435. 5 V. Suzuki, Biochemïstry, 6 (1967) 1335. 6 J C. M. Tsihti. M. J. Namtvedt and I. C. Gu~lus, Biozhem. Biophys. Res. Commun., 30 (1968) 323. 7 K. K. Rao, R. Cammack. D. 0. Hall and C. E. Johnson, Biochem. J., 122 (1971) 257. 8 C. L. Thompmn, C. E. Johnson, D. P. E. Dickson. R. Cammzck, D. 0. Hall, IJ. Weser and K. K. Rao, Biochem. J.. 139 (1974) 97. 9 1. Okura, K. Nakamura and T. Keii, J. Mol. Crlta!., 4 (1978) $53. 10 T. Yagi, M. Honya and N. Tamiya. Biochim. Biophys. Ac@ 153 (1968) 699. 11 T_ Yagï, J_ Biochem., 68 (1970) 649_ 12 A. E. Harvey, J. A. Smart and E. S. Amïs, Anal. Chcm.. 27 (1955) 26.
299
Short Communïcation
Reconstitution
of Hydrogenase
ICHIRO OKURA,
KEN-ICHI NAKAMURA
Department of Chemical Tokyo, 152 (daoan) (Received
Engineering,
hom
Tokyo
iLs Apoproteln
and SATOSHI Inslitute
NAKAMURA
of Technotog;r,
nfeguro-Ru.
December 12.1978)
A procedure has been described prevïously for the rcmoval of iron and labde sulfide from clostridial ferredoxin to isolate apoferredoxin, and the reconstitution of ferredoxin Erom this prokin [l - 31. Thïs procedure, which has considerable utility, is being applied to other iron-sulfur protems such as spinach ferredoxin [4], adrenodoxin [SI, and putidaredoxïn [S] , and is also useful in increasing the sensitivity of the Mössbauer method by which proteïn enrïched in “Fe were obtained 17, 81. It bas been shown [9] that the hydrogenase used in this werk contains two Fe,S,£lusters per enzyme molecule from the formation of the Fe,&(thiophenyl), dlanion by the addition of thiophenol. The above-mentioned procedure has been applied to hydrogenasc, and we describe in thE communication methods for the preparation of hydrogenase apoprotein and the reconstitution of hydrogenase from its apoprotein. -411 reagents were obtined from commercial sources and were of the highest, purïty availableHydrogenase iHZ: ferricytochrome C3 osidoreducm, EC 1.12.2.13 from
Desulfouìbrio
udguris,
Miyazaki,
is US&.
Deszdfouibrio
uulgaris
celLs
(whlch were kindly donated by Prof. T. Yagï of Shizuoka Unïv.) were culturd accord’mg to the lïterature [ 101 _The enzyme was purïfied according to Yagï’s procedure [lL] _ Hydrogenase activïty was assayed by the evolution r-ate of hydrogen from reduced methyl viologen at 30 “C. The evolved hydrogen was analyzed by gas chromatography. A reaction mixture contained hydrogenase (5 X l!YIO mol), 7.05 X PO-” mol of methyl viologen and Na2S205 (5 mg) in 3.0 ml of 0.02M phosphate buffer (pH 7.0). The method used for the preparation of apoproteïn was a modification of these of Rabïnowïtz et al. El- 31 and Rao ef al. [7]. Hydrogenax (1.2 X ZO-’ mol) in 6 ml of phosphate buffer was treated w-ith 2 ml of 20% trïchloroacetic acid and flushed wïth nitrogen gas for 3 h to expel HzS. The mixture was centrïfuged ât 20 000 g for 20 min. The precipitate was washecí with water, wïfh 5% trïchloroacetic acid, agaïn with water and resuspended in 4.5 ml of 0.05M Ris-HCl buffer.
The spectrum of the resultïng solution is shown in Fig. 1, curve b_ The solution has no extindion peak at 420 nm attributable to the iron-sulfur cluster, but that at 280 nm remaïns, and üon was not detected in this solution. This ïmplies that the solution contains the protein without an ïronsulfur cluster or apoproteïn.
Fig. 1. Spectra
of hydrogenase
(a), apoprotein
(b) and reconstituted
hydrogenase
(c)_
The determinstion of iron was carrred out by a*omic ahsorption spectrometry and by a spectroscopic method [l, 12]_ The reconstitution of hydrogenase from its apoprotein is carried out as fellows. Four millilitres of the apoproteïn were treated wïth 0.2 ml of 2mercaptoethanol for 2-5 h at room temperature under nitrogen atmosphere. Sodium sulphide, 0.5 ml (4 X lO-‘M), and 0.5 ml of FeCla (4 X LOP2M) or FeSOS(NHa)2SOS were added (the reaction conditions are shown in Table l)_ The reconstituted hydrogenase was separated from excess of reagent by pa&ng bhrough a column (1.5 X 15 cm) of Sephadex G-50. TABLE
1
Run no.
Preparation
system
8
Hydrogenase (holoprotein) Amprotcin Apoprotein + Nag Apoprotcin + Fe Apoprotein + NaaS f Fe*+ Apoprotcin + Na+ + FeC13 + HSCHfiH@H Apoprotem f Na&G L FeSO&NH~)~SO~ + HSCH+H20H Nz# -+ FcSO,(NH~)~SO~ + HSCH&HzOH
Prep.aration
cor,dition
-
Activky (R) 100
0
Room Room Room Room Room
temp. temp temp. temp. temp_
for for for for for
1 1 1 1 1
d d d d d
0
Room
timp.
for 3 h
0
0 0 1.1
1.03
As shown in Table 1, the reconstitution requïred the addition of a s-ource of ionic iron, inorganïc sulfide, and meruptide reducing agent. These
301
were supglied by FeC& or FeSC?+(NH1)2SO~, sodium sulfide and 2-mercaptoethanol. No reconstitution was okerved if any of these components were amitted Though the actitity of the reconstItut& hydrogenase is very low compared tith the orïgma2 hydrogenase, the turnover number per hydrogenase molecuIe was 3.2 min-’ mol-enzyme-l. This resuit shows that only a small portion of the apoprotein is recor&ituted. For thi.s reason, the extinction peak at 420 nm might not he observed, sec Fig. 1, curve c. Further work on thisprojeck k proceeding. The authors are grateful to Prof. T. Yagï OF Shizuoka Univ. for protision of the Desulfouibrio udg~ris cells and for bis help tith the hydrogenase purification.
References 1 W. B. Lovenberg. E. Bucbanan and J. C. Rabinowitz, J. Biel. Chem.. 238 (1963) 3 899. 2 R. Malkin and J. C. Rabinowitz, Biochem. Blophys. Res. Commun., 23 (1966) 822. 3 J. Hong and J. C. Rabïqowi~, Biochem. Biophys_ Res. Commun., 29 (1967) 246. 4 E_ Bayer, D. Josef, P_ Kraus. H Hagenmaier, A_ Roder and A. Trebst, Eiochim_ Eiophys. Acts. 143(1967)435. 5 V. Suzuki, Biochemïstry, 6 (1967) 1335. 6 J C. M. Tsihti. M. J. Namtvedt and I. C. Gu~lus, Biozhem. Biophys. Res. Commun., 30 (1968) 323. 7 K. K. Rao, R. Cammack. D. 0. Hall and C. E. Johnson, Biochem. J., 122 (1971) 257. 8 C. L. Thompmn, C. E. Johnson, D. P. E. Dickson. R. Cammzck, D. 0. Hall, IJ. Weser and K. K. Rao, Biochem. J.. 139 (1974) 97. 9 1. Okura, K. Nakamura and T. Keii, J. Mol. Crlta!., 4 (1978) $53. 10 T. Yagi, M. Honya and N. Tamiya. Biochim. Biophys. Ac@ 153 (1968) 699. 11 T_ Yagï, J_ Biochem., 68 (1970) 649_ 12 A. E. Harvey, J. A. Smart and E. S. Amïs, Anal. Chcm.. 27 (1955) 26.