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Inactivation of the hydrogenase from the hydrogen-oxidizing bacterium Alcaligenes eutrophus Z-1 under the action of urea and limited proteolysis
Biochimica etBiophysicaActa, 789 (1984) 210-215
210
Elsevier
BBA 31979
INACTIVATION OF T H E H Y D R O G E N A S E F R O M T H E H Y D R O G E N - O X I D I Z I N G BACTERIUM ALCALIGENES EUTROPHUS Z-1 U N D E R T H E A C T I O N OF UREA A N D LIMITED PROTEOLYSIS VALDIMIR O. POPOV a, ILIYA B. UTKIN b, IRINA G. GAZARYAN b, ALEXANDER N. O V C H I N N I K O V b ALEKSEY M. EGOROV b and ILIYA V. BEREZIN a
" A.N. Bach Institute of Biochemistry, U.S.S.R. Academy of Sciences, Moscow, and ~ Chemical Department of Moscow State University, Moscow (U.S.S.R.) (Received February 1st, 1984) (Revised manuscript received May 16th, 1984)
Key words: Hydrogenase," N A D H dehydrogenase; Urea," Proteolysis," Binding site," (A. eutrophus Z-l)
Inactivation of both the hydrogenase and NADH-dehydrogenase activities of the hydrogenase from the hydrogen-oxidizing bacterium Alcaligenes eutrophus Z-I under the action of urea and limited trypsin proteolysis has been studied. The enzyme activities can be divided into three groups according to their stability towards urea and limited proteolysis. The first group includes the NADH-dehydrogenase activity of the enzyme towards methyl viologen and ferricyanide, which is highly stable and can be activated by NAD. The second group comprises the NADH-dehydrogenase activity towards dichlorophenol indophenol, which is moderately stable but, in the presence of NAD, can be completely stabilized against urea action. The third group includes the hydrogenase activity towards both N A D and methyl viologen as substrates. This type of activity is the most labile and not stabilized by NAD. It is assumed that hydrogenase binds substrates at different spatially independent sites. A hypothetical scheme of the enzyme binding sites arrangement is proposed.
Introduction The NAD-dependent soluble hydrogenase from the hydrogen-oxidizing bacteria Alcaligenes eutrophus has a complex quarternary structure. The hydrogenase from the strain H16 consists of four subunits of three different types and contains two iron-sulphur clusters of Fe4S4 type, two clusters of FezS 2 type, two atoms of nickel and one F M N molecule per enzyme tetramer [1,2]. The hydrogenase from the strain Z-1 has close kinetic and physicochemical properties [3]. A particular feature of the hydrogenase from A. eutrophus is its ability to catalyze both the hydrogenase and NADH-dehydrogenase reactions [4]. N A D is a physiological substrate of the enzyme, but a number of artificial electron acceptors can 0167-4838/84/$03.00 © 1984 Elsevier Science Publishers B.V.
also act as substrates in NADH-dehydrogenase and hydrogenase reactions [4]. In our previous work we have proposed a hypothetical model of N A D H - d e p e n d e n t hydrogenase, that is an oligomeric enzyme consisted of usual hydrogenase and N A D H dehydrogenase [5]. The presence of a special enzyme fragment responsible for the NADH-dehydrogenase activity was demonstrated [5]. It was found that the quarternary structure plays an important role in the stability and activity of the enzyme. The results obtained support the assumption according to which the hydrogenase molecule possesses an intramolecular electron transport chain and several sites of substrate sorption and activation. A study on the enzyme inactivation is an approach to elucidate the mechanism of enzyme ac-
211 tion. The purpose of the present work was to study the effect of urea and limited proteolysis on the hydrogenase and NADH-dehydrogenase activities towards a number of substrates.
Experimental procedure Materials
Methyl viologen (Serva, Switzerland), 2.6-dichlorophenol indophenol (Sigma, USA), NAD, N A D H (Reanal, Hungary), trypsin (Spopha, Czechoslovakia), urea, KH2PO4, K3Fe(CN)6, K O H (analytical grade, U.S.S.R.) were used without further purification. Phenyl-Sepharose was from Pharmacia (Sweden); Toyopearl TSK HW 55F, from Toyo Soda (Japan). The biomass of A. eutrophus Z-1 was kindly supplied by Dr. Ya.V. Fedorova (Institute of Biophysics of the Siberean Branch of the U.S.S.R. Academy of Science, Krasnoyarsk). Methods
Bacterial cells were destroyed by sonication as previously described [6]. Hydrogenase was purified by double hydrophobic chromatography on phenyl-Sepharose and by gel-filtration on Toyopearl HW-55F. The purified hydrogenase preparations were characterized by a specific activity of 15-20 /~mol/min per mg determined with H 2 and NAD as acceptor. Judging by gradient polyacrylamide gel electrophoresis the purity of the enzyme preparations after gel filtration was not less than 80%. They were used for the most of experiments. The similar results were always obtained for homogeneous enzyme preparations. The hydrogenase activity was assayed by spectrophotometric and gas chromatographic methods as described in Ref. 5. The NADH-dehydrogenase activity was measured spectrophotometrically at 340 nm at 37 °C. N A D H (0.2 mM), electron acceptors (methyl viologen (10 mM), dichlorophenol indophenol (DCPIP) (0.15 mM), K3Fe(CN)6 (0.2 mM)) and phosphate buffer were placed in a spectrophotometric cell. The reaction was initiated by adding the enzyme up to the total volume of 2 ml. All measurements were made in 0.05 M potassium phosphate buffer (pH 7.8). The activity was measured with Hitachi 557 spectrophotometer
(Japan) and an L H M - 8 M D gas chromatograph (U.S.S.R.). The molecular weights of the hydrogenase fragments were determined by gel-filtration through Toyopearl TSK HW-55F (1.6 × 90 cm) column or through TSK-G 3000 S.W. (7.5 × 600 mm) column with HPLC instrument (LKB, Sweden). Gradient polyacrylamide gel electrophoresis was performed as described in Ref. 7. Gels were stained for hydrogenase and NADH-dehydrogenase activities according to Ref. 8.
Results and Discussion Hydrogenase inactivation in urea
Figs. 1 and 2 show the inactivation profiles of the enzyme hydrogenase and NADH-dehydrogenase activities in urea solutions. The NADH-dehydrogenase activity measured with methyl viologen and ferricyanide as substrates is stable at low temperatures up to 2 M urea concentrations. Furthermore, the significant activation of the NADH-dehydrogenase activity at the initial stage is observed. Both the magnitude and the duration of the activation period depend on the urea concentration, temperature and presence of NAD. The NADH-dehydrogenase activity towards methyl viologen is readily lost in urea at ambient temperatures (Fig. 2). The descending parts of the inactivation profiles (25 °C) follow the pseudofirst-order reaction kinetics. In this case, the presence of N A D does not significantly stabilize the enzyme activity. In the absence of urea, the enzyme activity is completely stable under experimental conditions. The action of urea on NADH-dehydrogenase activity depends on the origin of the substrate. In particular, the NADH-dehydrogenase activity towards dichlorophenol indophenol rapidly decreases in 2 M urea (Fig. 1), but NAD completely protects the enzyme from inactivation. The hydrogenase activity of the enzyme is the most labile in urea (Fig. 1). The effect of N A D on this activity is insufficient. It should be emphasized that the hydrogenase activity towards both N A D and methyl viologen is simultaneously lost in urea solutions of different concentrations (data not shown). This indicates that the inactivation process occurs on the same stage of both reactions
212
Dissociation does not affect the enzyme NADH-dehydrogenase activity towards one-electron acceptors (methyl viologen, ferricyanide) but results in the inactivation of both the NADH-dehydrogenase activity towards dichlorophenol indophenol and the hydrogenase activity. This is in accord with our earlier observations concerning the role of the enzyme quarternary structure in its catalytic activities [5]. The proposed inactivation mechanism was validated by the results of electrophoresis and HPLC experiments. The electrophoregrams of the hydrogenase inactivated in the presence of 2 M urea reveal the dissociation fragments with molecular weights of 110 000 and 60 000. The staining of the gels for the NADH-dehydrogenase activity towards benzyl viologen reveals not less than three
7
[]
125
,-Ol, 100
75
? 9
50
<
"-,o~ 6 25
5
i 200
1 0
rain
studied. Contrary to the experiments with methyl viologen the inactivation profiles of both the NADH-dehydrogenase activity towards dichlorophenol indophenol and the hydrogenase activity towards N A D cannot be fitted by the simple exponential dependences. The results obtained can be rationalized within the following tentative scheme which assumes the dissociation of the enzyme tetramer to be an important part of the inactivation mechanism:
[-
dis] SOClatlon /oenase/ urea/ f r a g m e n t s / urea/ _ [hydro] /b
/--~ /
kmer
j
/ tetra- /
/
/
.
r
d i. s - 1
. . / /soctaUon/
/ ~
[ rathe /
|fragments/
/
I presence I
"\L-/"
H 2 + NAD +
z+
( k 1, k 2, k 3 -
I00~
~
~
•
,
5
? >
5C
"
•
+
-
_
+ +
+ +
+
NADH + DCPIP NADH+MV
15C
~
~Lof~J
thermo- ~ inactivation products
F i g . 1. Inactivation of the enzyme catalytic a c t i v i t i e s in 2 M urea at 4 ° C in the presence (2, 4, 6) and absence (1, 3, 5) o f 1 m M N A D . NADH-dehydrogenase activity with methyl v i o l o g e n , I , El. NADH-dehydrogenase activity with dichlorophenol indophenol, A, zx. Hydrogenase activity with N A D , O; O . NADH-dehydrogenase activity with methyl viologen in 3 M urea. @ . 0.05 M potassium phosphate buffer ( p H 7.8).
thermoinactivation constants).
I 0
200
•
3
300 min
F i g . 2. Inactivation of the enzyme NADH-dehydrogenase activ-
ity towards methyl viologen in urea solutions at 25 ° C . (1) C3, 1.5 M u r e a ; (2) L 1,5 M u r e a i n the presence of 1 m M N A D ; (3) A, 2 M u r e a ; (4) /,, 3 M u r e a ; (5) O, without urea. 0.05 M potassium phosphate buffer ( p H 7.8).
213
J
15
160
80
40
I
I
I
117
19
M r
2~I
(x
10 3)
VoI. (ml)
Fig. 3. Elution profile patterns of the enzyme on gel filtration on a calibrated HPLC column (0.7 x 60 cm) of TSK-3000 SW in the presence of 2 M urea (0.1 M potassium phosphate buffer (pH 7.2), 25 o C, 0.25 m l / m i n ) . The enzyme was incubated with elution buffer prior to the experiment for 2 h. A 2~0; e, NADH-dehydrogenase activity with methyl viologen; El, NADH-dehydrogenase activity with dichlorophenol indophenol; O, hydrogenase activity with NAD.
active enzyme fragments with molecular weights of 180 000 (native hydrogenase), 110 000 and 60 000. A similar inactivation pattern was obtained in H P L C experiments (Fig. 3). Only the native enzyme tetramer (160 000) preserves the hydrogenase activity towards NAD. The NADH-dehydrogenase activity towards dichlorophenol indophenol is observed in two elution fractions (160000 and 75 000), while the NADH-dehydrogenase activity towards methyl viologen is associated with three elution fractions (160 000, 75 000 and 55 000). The studies on NADH-dehydrogenase inactivation at 4 and 25 o C (Fig. 1 and 2) revealed that the products of enzyme dissociation are more thermolabile than the native tetramer (k2, 3 > kl). The postulated scheme also explains a complex two-exponential form of the enzyme inactivation profiles in the case of the hydrogenase activity as well as NADH-dehydrogenase activity towards dichlorophenol indophenol. The activation effect of urea in the N A D H - d e hydrogenase reaction with one-electron acceptors can emerge from a higher specific activity of the fragments of the native enzyme due to an increase
in the accessibility of certain prosthetic groups during dissociation. The similar effects are known for the N A D H - d e h y d r o g e n a s e of the mitochondrial electron transport chain, which is active towards a number of substrates [9]. During its dissociation, activities towards some of the substrates are decreased, while those towards others are dramatically increased. Meanwhile, the stabilizing effect of the coenzyme on the N A D H - d e h y drogenase activity towards dichlorophenol indophenol may result from the stabilization of intermediates of dissociation, which do not possess the hydrogenase activity but are competent in the NADH-dehydrogenase reaction. The complicated nature of the hydrogenase quarternary structure, its ability to catalyze various enzyme activities, and the presence of different prosthetic groups allows us to assume that the substrate binding sites are spatially independent in the hydrogenase molecule. The effect of urea on different types of hydrogenase catalytic activity provides some indirect evidence supporting this idea. The sorption sites for N A D ( H ) and one-electron carriers (methyl viologen, ferricyanide) appear to be in proximity and may be on the same subunit of the enzyme molecule. This conforms to the assumption that there is a special N A D H - d e hydrogenase fragment in the enzyme molecule [5]. The different type of inactivation of the enzyme hydrogenase and NADH-dehydrogenase activities indicates that the processes of the hydrogen activation and acceptor binding take place at the different sites of the enzyme. The correlation between the hydrogenase inactivation with N A D and methyl viologen as substrates supports an idea of a common inactivation mechanism for both types of the catalytic activity. One of the possible reasons for the enzyme inactivation is a breakdown in the enzyme intramolecule electron transport chain, e.g., as a result of a destruction of the subunits contacts, or the inactivation of the hydrogen binding site of the enzyme. The results obtained indicate that the dichlorophenol indophenol binding site does not coincide with that for N A D or methyl viologen, and is probably located between the binding sites for the coenzyme and hydrogen in the enzyme intramolecule electron transport chain.
214
Inactivation of hydrogenase by #mited proteolysis The hydrogenase and NADH-dehydrogenase activities of the enzyme differ in their stability towards limited proteolysis by trypsin. The NADH-dehydrogenase activity towards methyl viologen is completely stable under experimental conditions, while the hydrogenase activity rapidly falls in the presence of trypsin (Fig. 4). As in the case of the inactivation by urea, the NADH-dehydrogenase activity towards dichlorophenol indophenol is characterized by the intermediate stability (Fig. 4). The results obtained additionally verify the assumption that the binding sites for methyl viologen and dichlorophenol indophenol are different. Fig. 5 presents the results of gel-filtration experiments with the native enzyme and hydrogenase preparations at the late stages of limited proteolysis. The constant elution volume and the shape of the elution profile for the hydrogenase activity in the partially inactivated enzyme preparations indicate the fact that even minor defects in the enzyme structure can lead to the complete loss of the hydrogenase activity. Meanwhile, during the enzyme inactivation the broadening of NADH-dehydrogenase elution profile occurs. The partially hydrolyzed enzyme preparations contain
4
Too
->' 5c
L
TOO
_ _
_ _ _ _
I
200
rain
Fig. 4. Inactivation of the enzyme catalytic activities by trypsin. (1) NADH-dehydrogenase activity with methyl viologen (O); (2) NADH-dehydrogenase activity with dichlorophenol indophenol ([]); (3) Hydrogenase activity with N A D (A); (4). Control - hydrogenase activity without trypsin. 0.05 M potassium phosphate buffer (pH 7.8), 25 o C. Enzyme: trypsin = 40 : 1 (w/w).
140105 I
16
4.5
I
I
18
20
32 I
Mr
( x l O "3)
Vol. ( m l )
Fig. 5. Elution profile patterns of the enzyme on gel-filtration on a calibrated HPLC column ( 0 . 7 × 6 0 cm) TSK-3000 SW after limited proteolysis by trypsin (0.1 M potassium phosphate buffer (pH 7.2) 2 5 ° C , 0.5 m l / m i n ) . - - , A2~o; O, NADH-dehydrogenase activity with methyl viologen: (2), hydrogenase activity with NAD. The enzyme was incubated with trypsin prior to the experiment for 45 rain. Enzyme : trypsin =
80 : 1 (w/w). fragments with the molecular weight of about 100000, which preserve substantial NADH-dehydrogenase activity. The results obtained afford additional evidence for the existence of a special enzyme fragment which is responsible for the NADH-dehydrogenase activity of the enzyme. Thus, the study of the inactivation of the hydrogenase from A. eutrophus Z-1 by urea and limited proteolysis allows us to make several assumptions about the enzyme structure and propose a probable mechanism of its inactivation. It has been shown that the hydrogenase binds substrates at different, probably spatially independent, sites. On the basis of the present study, three sites can be distinguished: the site for hydrogen binding and activation, the site for dichlorophenol indophenol binding and the site for methyl viologen and ferricyanide binding. It is worth mentioning that the existence of several spatially independent subsites for acceptor binding was postulated for the hydrogenase from Clostridium pasteurianum as well [10]. The special attention should be paid to the location of the NADH-binding site. On the basis of
215
the inactivation experiments, one can assume that it is located near the binding sites for one electron acceptors, such as methyl viologen and ferricyanide. Meanwhile, the results of inhibition of hydrogenase from A. eutrophus H16 by dicoumarol give evidence that N A D and methyl viologen bind to the separate sorption sites [11]. We have found that N A D H is a non-competitive inhibitor versus methyl viologen in the reaction of hydrogen evolution from sodium dithionite reduced methyl viologen for the hydrogenase from A. eutrophus Z-1 [5]. This observation also accords with an assumption of the existence of separate sites for acceptor and N A D binding. On the basis of the results obtained, a hypothetical scheme of the arrangement of the binding sites in the molecule of hydrogenase from A. eutrophus Z-1 can be proposed: H2 H+
DCPIPox DCPIPred /,~ NADH [Ni, Fe-S clustersl ~ [fiavln] ~ NAD
\
MVox MVred
The mode suggests a close location of the binding sites for methyl viologen (MV), ferricyanide and NAD, probably on the same N A D H - d e h y d r o genase fragment, while the sorption site for dichlorophenol indophenol is located on another subunit or in the area of subunit contact.
Acknowledgements We are grateful to Dr. Ya.V. Fedorova for supplying with ample amounts of A. eutrophus Z-1 cells. We thank Prof. H.G. Schlegel for providing us with information on the hydrogenase from A. eutrophus H16 prior to publication.
References 1 Schneider, K., Schlegel, H.G., Cammack, R. and Hall, D.O. (1979) Biochim. Biophys. Acta 578, 445-461 2 Friedrich, C.G., Schneider, K. and Friedrich, B. (1982) J. Bacteriol. 152, 42-48 3 Pinchukova, E.V., Varfolomeev, S.D. and Kondratjeva, E.N. (1979) Biochimiya (USSR) 44, 605-614 4 Schneider, K. and Schlegel, H.G. (1976) Biochim. Biophys. Acta 452, 66-80 5 Popov, V.O., Berezin, I.V., Zaks, A.M., Gazaryan, I.G., Utkin, I.B. and Egorov, A.M. (1983) Biochim. Biophys. Acta 744, 298-303 6 Gazaryan, I.G., Zaks, A.M., Egorov, A.M., Popov, V.O. (1983) Prikl. Biokhim. Mikrobiol. (USSR) 19, 751-757 7 Pharmacia Fine Chemicals (1980 Polyacrylamide Gel Electrophoresis, Laboratory Techniques, pp. 7-19, Pharmacia Fine Chemicals, Rahms; Lund 8 Gazaryan, I.G., Avilova, T.V., Semenova, Ya.V., Egorov, A.M. and Popov, V.O. (1981) Prikl. Biochim. Microbiol. USSR 17, 545-554 9 Singer, T.P. and Gutman, M. (1971) Adv. Enzymol. 34, 79-153 10 Khan, S.M., Klibanov, A.M., Kaplan, N.O. and Kamen, M.D. (1981) Biochim. Biophys. Acta 659, 457-465 11 Egerer, P. and Simon, H. (1982) Biochim. Biophys. Acta 703, 158-170
210
Elsevier
BBA 31979
INACTIVATION OF T H E H Y D R O G E N A S E F R O M T H E H Y D R O G E N - O X I D I Z I N G BACTERIUM ALCALIGENES EUTROPHUS Z-1 U N D E R T H E A C T I O N OF UREA A N D LIMITED PROTEOLYSIS VALDIMIR O. POPOV a, ILIYA B. UTKIN b, IRINA G. GAZARYAN b, ALEXANDER N. O V C H I N N I K O V b ALEKSEY M. EGOROV b and ILIYA V. BEREZIN a
" A.N. Bach Institute of Biochemistry, U.S.S.R. Academy of Sciences, Moscow, and ~ Chemical Department of Moscow State University, Moscow (U.S.S.R.) (Received February 1st, 1984) (Revised manuscript received May 16th, 1984)
Key words: Hydrogenase," N A D H dehydrogenase; Urea," Proteolysis," Binding site," (A. eutrophus Z-l)
Inactivation of both the hydrogenase and NADH-dehydrogenase activities of the hydrogenase from the hydrogen-oxidizing bacterium Alcaligenes eutrophus Z-I under the action of urea and limited trypsin proteolysis has been studied. The enzyme activities can be divided into three groups according to their stability towards urea and limited proteolysis. The first group includes the NADH-dehydrogenase activity of the enzyme towards methyl viologen and ferricyanide, which is highly stable and can be activated by NAD. The second group comprises the NADH-dehydrogenase activity towards dichlorophenol indophenol, which is moderately stable but, in the presence of NAD, can be completely stabilized against urea action. The third group includes the hydrogenase activity towards both N A D and methyl viologen as substrates. This type of activity is the most labile and not stabilized by NAD. It is assumed that hydrogenase binds substrates at different spatially independent sites. A hypothetical scheme of the enzyme binding sites arrangement is proposed.
Introduction The NAD-dependent soluble hydrogenase from the hydrogen-oxidizing bacteria Alcaligenes eutrophus has a complex quarternary structure. The hydrogenase from the strain H16 consists of four subunits of three different types and contains two iron-sulphur clusters of Fe4S4 type, two clusters of FezS 2 type, two atoms of nickel and one F M N molecule per enzyme tetramer [1,2]. The hydrogenase from the strain Z-1 has close kinetic and physicochemical properties [3]. A particular feature of the hydrogenase from A. eutrophus is its ability to catalyze both the hydrogenase and NADH-dehydrogenase reactions [4]. N A D is a physiological substrate of the enzyme, but a number of artificial electron acceptors can 0167-4838/84/$03.00 © 1984 Elsevier Science Publishers B.V.
also act as substrates in NADH-dehydrogenase and hydrogenase reactions [4]. In our previous work we have proposed a hypothetical model of N A D H - d e p e n d e n t hydrogenase, that is an oligomeric enzyme consisted of usual hydrogenase and N A D H dehydrogenase [5]. The presence of a special enzyme fragment responsible for the NADH-dehydrogenase activity was demonstrated [5]. It was found that the quarternary structure plays an important role in the stability and activity of the enzyme. The results obtained support the assumption according to which the hydrogenase molecule possesses an intramolecular electron transport chain and several sites of substrate sorption and activation. A study on the enzyme inactivation is an approach to elucidate the mechanism of enzyme ac-
211 tion. The purpose of the present work was to study the effect of urea and limited proteolysis on the hydrogenase and NADH-dehydrogenase activities towards a number of substrates.
Experimental procedure Materials
Methyl viologen (Serva, Switzerland), 2.6-dichlorophenol indophenol (Sigma, USA), NAD, N A D H (Reanal, Hungary), trypsin (Spopha, Czechoslovakia), urea, KH2PO4, K3Fe(CN)6, K O H (analytical grade, U.S.S.R.) were used without further purification. Phenyl-Sepharose was from Pharmacia (Sweden); Toyopearl TSK HW 55F, from Toyo Soda (Japan). The biomass of A. eutrophus Z-1 was kindly supplied by Dr. Ya.V. Fedorova (Institute of Biophysics of the Siberean Branch of the U.S.S.R. Academy of Science, Krasnoyarsk). Methods
Bacterial cells were destroyed by sonication as previously described [6]. Hydrogenase was purified by double hydrophobic chromatography on phenyl-Sepharose and by gel-filtration on Toyopearl HW-55F. The purified hydrogenase preparations were characterized by a specific activity of 15-20 /~mol/min per mg determined with H 2 and NAD as acceptor. Judging by gradient polyacrylamide gel electrophoresis the purity of the enzyme preparations after gel filtration was not less than 80%. They were used for the most of experiments. The similar results were always obtained for homogeneous enzyme preparations. The hydrogenase activity was assayed by spectrophotometric and gas chromatographic methods as described in Ref. 5. The NADH-dehydrogenase activity was measured spectrophotometrically at 340 nm at 37 °C. N A D H (0.2 mM), electron acceptors (methyl viologen (10 mM), dichlorophenol indophenol (DCPIP) (0.15 mM), K3Fe(CN)6 (0.2 mM)) and phosphate buffer were placed in a spectrophotometric cell. The reaction was initiated by adding the enzyme up to the total volume of 2 ml. All measurements were made in 0.05 M potassium phosphate buffer (pH 7.8). The activity was measured with Hitachi 557 spectrophotometer
(Japan) and an L H M - 8 M D gas chromatograph (U.S.S.R.). The molecular weights of the hydrogenase fragments were determined by gel-filtration through Toyopearl TSK HW-55F (1.6 × 90 cm) column or through TSK-G 3000 S.W. (7.5 × 600 mm) column with HPLC instrument (LKB, Sweden). Gradient polyacrylamide gel electrophoresis was performed as described in Ref. 7. Gels were stained for hydrogenase and NADH-dehydrogenase activities according to Ref. 8.
Results and Discussion Hydrogenase inactivation in urea
Figs. 1 and 2 show the inactivation profiles of the enzyme hydrogenase and NADH-dehydrogenase activities in urea solutions. The NADH-dehydrogenase activity measured with methyl viologen and ferricyanide as substrates is stable at low temperatures up to 2 M urea concentrations. Furthermore, the significant activation of the NADH-dehydrogenase activity at the initial stage is observed. Both the magnitude and the duration of the activation period depend on the urea concentration, temperature and presence of NAD. The NADH-dehydrogenase activity towards methyl viologen is readily lost in urea at ambient temperatures (Fig. 2). The descending parts of the inactivation profiles (25 °C) follow the pseudofirst-order reaction kinetics. In this case, the presence of N A D does not significantly stabilize the enzyme activity. In the absence of urea, the enzyme activity is completely stable under experimental conditions. The action of urea on NADH-dehydrogenase activity depends on the origin of the substrate. In particular, the NADH-dehydrogenase activity towards dichlorophenol indophenol rapidly decreases in 2 M urea (Fig. 1), but NAD completely protects the enzyme from inactivation. The hydrogenase activity of the enzyme is the most labile in urea (Fig. 1). The effect of N A D on this activity is insufficient. It should be emphasized that the hydrogenase activity towards both N A D and methyl viologen is simultaneously lost in urea solutions of different concentrations (data not shown). This indicates that the inactivation process occurs on the same stage of both reactions
212
Dissociation does not affect the enzyme NADH-dehydrogenase activity towards one-electron acceptors (methyl viologen, ferricyanide) but results in the inactivation of both the NADH-dehydrogenase activity towards dichlorophenol indophenol and the hydrogenase activity. This is in accord with our earlier observations concerning the role of the enzyme quarternary structure in its catalytic activities [5]. The proposed inactivation mechanism was validated by the results of electrophoresis and HPLC experiments. The electrophoregrams of the hydrogenase inactivated in the presence of 2 M urea reveal the dissociation fragments with molecular weights of 110 000 and 60 000. The staining of the gels for the NADH-dehydrogenase activity towards benzyl viologen reveals not less than three
7
[]
125
,-Ol, 100
75
? 9
50
<
"-,o~ 6 25
5
i 200
1 0
rain
studied. Contrary to the experiments with methyl viologen the inactivation profiles of both the NADH-dehydrogenase activity towards dichlorophenol indophenol and the hydrogenase activity towards N A D cannot be fitted by the simple exponential dependences. The results obtained can be rationalized within the following tentative scheme which assumes the dissociation of the enzyme tetramer to be an important part of the inactivation mechanism:
[-
dis] SOClatlon /oenase/ urea/ f r a g m e n t s / urea/ _ [hydro] /b
/--~ /
kmer
j
/ tetra- /
/
/
.
r
d i. s - 1
. . / /soctaUon/
/ ~
[ rathe /
|fragments/
/
I presence I
"\L-/"
H 2 + NAD +
z+
( k 1, k 2, k 3 -
I00~
~
~
•
,
5
? >
5C
"
•
+
-
_
+ +
+ +
+
NADH + DCPIP NADH+MV
15C
~
~Lof~J
thermo- ~ inactivation products
F i g . 1. Inactivation of the enzyme catalytic a c t i v i t i e s in 2 M urea at 4 ° C in the presence (2, 4, 6) and absence (1, 3, 5) o f 1 m M N A D . NADH-dehydrogenase activity with methyl v i o l o g e n , I , El. NADH-dehydrogenase activity with dichlorophenol indophenol, A, zx. Hydrogenase activity with N A D , O; O . NADH-dehydrogenase activity with methyl viologen in 3 M urea. @ . 0.05 M potassium phosphate buffer ( p H 7.8).
thermoinactivation constants).
I 0
200
•
3
300 min
F i g . 2. Inactivation of the enzyme NADH-dehydrogenase activ-
ity towards methyl viologen in urea solutions at 25 ° C . (1) C3, 1.5 M u r e a ; (2) L 1,5 M u r e a i n the presence of 1 m M N A D ; (3) A, 2 M u r e a ; (4) /,, 3 M u r e a ; (5) O, without urea. 0.05 M potassium phosphate buffer ( p H 7.8).
213
J
15
160
80
40
I
I
I
117
19
M r
2~I
(x
10 3)
VoI. (ml)
Fig. 3. Elution profile patterns of the enzyme on gel filtration on a calibrated HPLC column (0.7 x 60 cm) of TSK-3000 SW in the presence of 2 M urea (0.1 M potassium phosphate buffer (pH 7.2), 25 o C, 0.25 m l / m i n ) . The enzyme was incubated with elution buffer prior to the experiment for 2 h. A 2~0; e, NADH-dehydrogenase activity with methyl viologen; El, NADH-dehydrogenase activity with dichlorophenol indophenol; O, hydrogenase activity with NAD.
active enzyme fragments with molecular weights of 180 000 (native hydrogenase), 110 000 and 60 000. A similar inactivation pattern was obtained in H P L C experiments (Fig. 3). Only the native enzyme tetramer (160 000) preserves the hydrogenase activity towards NAD. The NADH-dehydrogenase activity towards dichlorophenol indophenol is observed in two elution fractions (160000 and 75 000), while the NADH-dehydrogenase activity towards methyl viologen is associated with three elution fractions (160 000, 75 000 and 55 000). The studies on NADH-dehydrogenase inactivation at 4 and 25 o C (Fig. 1 and 2) revealed that the products of enzyme dissociation are more thermolabile than the native tetramer (k2, 3 > kl). The postulated scheme also explains a complex two-exponential form of the enzyme inactivation profiles in the case of the hydrogenase activity as well as NADH-dehydrogenase activity towards dichlorophenol indophenol. The activation effect of urea in the N A D H - d e hydrogenase reaction with one-electron acceptors can emerge from a higher specific activity of the fragments of the native enzyme due to an increase
in the accessibility of certain prosthetic groups during dissociation. The similar effects are known for the N A D H - d e h y d r o g e n a s e of the mitochondrial electron transport chain, which is active towards a number of substrates [9]. During its dissociation, activities towards some of the substrates are decreased, while those towards others are dramatically increased. Meanwhile, the stabilizing effect of the coenzyme on the N A D H - d e h y drogenase activity towards dichlorophenol indophenol may result from the stabilization of intermediates of dissociation, which do not possess the hydrogenase activity but are competent in the NADH-dehydrogenase reaction. The complicated nature of the hydrogenase quarternary structure, its ability to catalyze various enzyme activities, and the presence of different prosthetic groups allows us to assume that the substrate binding sites are spatially independent in the hydrogenase molecule. The effect of urea on different types of hydrogenase catalytic activity provides some indirect evidence supporting this idea. The sorption sites for N A D ( H ) and one-electron carriers (methyl viologen, ferricyanide) appear to be in proximity and may be on the same subunit of the enzyme molecule. This conforms to the assumption that there is a special N A D H - d e hydrogenase fragment in the enzyme molecule [5]. The different type of inactivation of the enzyme hydrogenase and NADH-dehydrogenase activities indicates that the processes of the hydrogen activation and acceptor binding take place at the different sites of the enzyme. The correlation between the hydrogenase inactivation with N A D and methyl viologen as substrates supports an idea of a common inactivation mechanism for both types of the catalytic activity. One of the possible reasons for the enzyme inactivation is a breakdown in the enzyme intramolecule electron transport chain, e.g., as a result of a destruction of the subunits contacts, or the inactivation of the hydrogen binding site of the enzyme. The results obtained indicate that the dichlorophenol indophenol binding site does not coincide with that for N A D or methyl viologen, and is probably located between the binding sites for the coenzyme and hydrogen in the enzyme intramolecule electron transport chain.
214
Inactivation of hydrogenase by #mited proteolysis The hydrogenase and NADH-dehydrogenase activities of the enzyme differ in their stability towards limited proteolysis by trypsin. The NADH-dehydrogenase activity towards methyl viologen is completely stable under experimental conditions, while the hydrogenase activity rapidly falls in the presence of trypsin (Fig. 4). As in the case of the inactivation by urea, the NADH-dehydrogenase activity towards dichlorophenol indophenol is characterized by the intermediate stability (Fig. 4). The results obtained additionally verify the assumption that the binding sites for methyl viologen and dichlorophenol indophenol are different. Fig. 5 presents the results of gel-filtration experiments with the native enzyme and hydrogenase preparations at the late stages of limited proteolysis. The constant elution volume and the shape of the elution profile for the hydrogenase activity in the partially inactivated enzyme preparations indicate the fact that even minor defects in the enzyme structure can lead to the complete loss of the hydrogenase activity. Meanwhile, during the enzyme inactivation the broadening of NADH-dehydrogenase elution profile occurs. The partially hydrolyzed enzyme preparations contain
4
Too
->' 5c
L
TOO
_ _
_ _ _ _
I
200
rain
Fig. 4. Inactivation of the enzyme catalytic activities by trypsin. (1) NADH-dehydrogenase activity with methyl viologen (O); (2) NADH-dehydrogenase activity with dichlorophenol indophenol ([]); (3) Hydrogenase activity with N A D (A); (4). Control - hydrogenase activity without trypsin. 0.05 M potassium phosphate buffer (pH 7.8), 25 o C. Enzyme: trypsin = 40 : 1 (w/w).
140105 I
16
4.5
I
I
18
20
32 I
Mr
( x l O "3)
Vol. ( m l )
Fig. 5. Elution profile patterns of the enzyme on gel-filtration on a calibrated HPLC column ( 0 . 7 × 6 0 cm) TSK-3000 SW after limited proteolysis by trypsin (0.1 M potassium phosphate buffer (pH 7.2) 2 5 ° C , 0.5 m l / m i n ) . - - , A2~o; O, NADH-dehydrogenase activity with methyl viologen: (2), hydrogenase activity with NAD. The enzyme was incubated with trypsin prior to the experiment for 45 rain. Enzyme : trypsin =
80 : 1 (w/w). fragments with the molecular weight of about 100000, which preserve substantial NADH-dehydrogenase activity. The results obtained afford additional evidence for the existence of a special enzyme fragment which is responsible for the NADH-dehydrogenase activity of the enzyme. Thus, the study of the inactivation of the hydrogenase from A. eutrophus Z-1 by urea and limited proteolysis allows us to make several assumptions about the enzyme structure and propose a probable mechanism of its inactivation. It has been shown that the hydrogenase binds substrates at different, probably spatially independent, sites. On the basis of the present study, three sites can be distinguished: the site for hydrogen binding and activation, the site for dichlorophenol indophenol binding and the site for methyl viologen and ferricyanide binding. It is worth mentioning that the existence of several spatially independent subsites for acceptor binding was postulated for the hydrogenase from Clostridium pasteurianum as well [10]. The special attention should be paid to the location of the NADH-binding site. On the basis of
215
the inactivation experiments, one can assume that it is located near the binding sites for one electron acceptors, such as methyl viologen and ferricyanide. Meanwhile, the results of inhibition of hydrogenase from A. eutrophus H16 by dicoumarol give evidence that N A D and methyl viologen bind to the separate sorption sites [11]. We have found that N A D H is a non-competitive inhibitor versus methyl viologen in the reaction of hydrogen evolution from sodium dithionite reduced methyl viologen for the hydrogenase from A. eutrophus Z-1 [5]. This observation also accords with an assumption of the existence of separate sites for acceptor and N A D binding. On the basis of the results obtained, a hypothetical scheme of the arrangement of the binding sites in the molecule of hydrogenase from A. eutrophus Z-1 can be proposed: H2 H+
DCPIPox DCPIPred /,~ NADH [Ni, Fe-S clustersl ~ [fiavln] ~ NAD
\
MVox MVred
The mode suggests a close location of the binding sites for methyl viologen (MV), ferricyanide and NAD, probably on the same N A D H - d e h y d r o genase fragment, while the sorption site for dichlorophenol indophenol is located on another subunit or in the area of subunit contact.
Acknowledgements We are grateful to Dr. Ya.V. Fedorova for supplying with ample amounts of A. eutrophus Z-1 cells. We thank Prof. H.G. Schlegel for providing us with information on the hydrogenase from A. eutrophus H16 prior to publication.
References 1 Schneider, K., Schlegel, H.G., Cammack, R. and Hall, D.O. (1979) Biochim. Biophys. Acta 578, 445-461 2 Friedrich, C.G., Schneider, K. and Friedrich, B. (1982) J. Bacteriol. 152, 42-48 3 Pinchukova, E.V., Varfolomeev, S.D. and Kondratjeva, E.N. (1979) Biochimiya (USSR) 44, 605-614 4 Schneider, K. and Schlegel, H.G. (1976) Biochim. Biophys. Acta 452, 66-80 5 Popov, V.O., Berezin, I.V., Zaks, A.M., Gazaryan, I.G., Utkin, I.B. and Egorov, A.M. (1983) Biochim. Biophys. Acta 744, 298-303 6 Gazaryan, I.G., Zaks, A.M., Egorov, A.M., Popov, V.O. (1983) Prikl. Biokhim. Mikrobiol. (USSR) 19, 751-757 7 Pharmacia Fine Chemicals (1980 Polyacrylamide Gel Electrophoresis, Laboratory Techniques, pp. 7-19, Pharmacia Fine Chemicals, Rahms; Lund 8 Gazaryan, I.G., Avilova, T.V., Semenova, Ya.V., Egorov, A.M. and Popov, V.O. (1981) Prikl. Biochim. Microbiol. USSR 17, 545-554 9 Singer, T.P. and Gutman, M. (1971) Adv. Enzymol. 34, 79-153 10 Khan, S.M., Klibanov, A.M., Kaplan, N.O. and Kamen, M.D. (1981) Biochim. Biophys. Acta 659, 457-465 11 Egerer, P. and Simon, H. (1982) Biochim. Biophys. Acta 703, 158-170