Eur. J. Biochem. 138, 533-541 (1984) (j FEBS 1984

Effect of nickel on activity and subunit composition of purified hydrogenase from Nocardia opaca 1 b

Klaus SCHNEIDER, Hans G. SCHLEGEL, and Karla JOCHIM Institut fur Mikrobiologie der Universitat Gottingen

(Received July 25/0ctober 31, 1983) - EJB 83 0796

The NAD-reducing hydrogeiiase of Nocardia opaca Ib was found to be a soluble, cytoplasmic enzyme. N. opaca I b does not contain an additional membrane-bound hydrogenase. The soluble enzyme was purified to homogeneity with a yield of 19 % and a final specific activity of 45 lmol H, oxidized min- mg protein- ’.

NAD reduction with H, was completely dependent on the presence of divalent metal ions (Ni2+, CO”, Mg2+, Mn2 ’) or of high salt concentrations (0.5 - 1.5 M). The most specific effect was caused by NiCl,, whose optimal concentration turned out to be 1 mM. The stimulation of activity by salts was the greater the less chaotrophic the anion. Maximal activity was achieved in 0.5 M potassium phosphate. Hydrogenase was also activated by protons. The pH optimum in 50 mM triethanolamine/HCl buffer containing 1 mM NiC1, was 7.8 - 8.0. In the absence of Ni2’, hydrogenase was only active at pH values below 7.0. The reduction of other electron acceptors was not dependent on metal ions or salts, even though an approximately 1 .Sfold stimulation of the reactions by 0.1 - 10 pM NiCI, was observed. With the most effective electron acceptor, benzyl viologen, a 50-fold higher specific activity was determined than with NAD.

The total molecular weight of hydrogenase has been estimated to be 200000 (gel filtration) and 178 000 (sucrose density gradient centrifugation, and sodium dodecyl sulfate electrophoresis) respectively. The enzyme is a tetramer consisting of non-identical subunits with molecular weights of 64000,56000,31000 and 27 000. It was demonstrated by electrophoretic analyses that in the absence of NiC1, and at alkaline pH values the native hydrogenase dissociates into two subunit dimers. The first dimer was dark yellow coloured, completely inactive and composed of subunits with molecular weights of 64000 and 31 000. The second dimer was light yellow, inactive with NAD but still active with methyl viologen. It was composed of subunits with molecular weights of 56000 and 27000.

Immunological comparison of the hydrogenase of N. opuca 1 b and the soluble hydrogenase of Alcaligenes eutrophus H16 revealed that these two NAD-linked hydrogenases are partially identical proteins.

Two basic types of hydrogenases have been found in aerobic hydrogen bacteria so far: a cytoplasmic, NAD-specific hydrogenase and a membrane-bound enzyme, which is coupled to the respiratory chain and completely unable to react with NAD(P) [l]. The great majority of hydrogen bacteria, including organisms as taxonomically diverse as Alcaligenes lutus [2], Paracoccus denitrijicans [ 1, 31, Xanthobacter autotrophicus [4], Pseudom onas pseudoflu va [ 51, A quasp irillurn auto tr op h icum [l, 61 and Bacillus schlegelii [7], contain one single hydrogenase which is exclusively bound to membranes. Only in a few hydrogen bacteria, i.e. in Alcaligenes (Pseudomonas) ruhlandii (81 and in different strains of Alcaligenes eutrophus [l] in addition to the membrane enzyme, a soluble NAD-reducing hydrogenase was detected.

Nocardia opuca 1 b, a gram-positive microorganism, charac- terized by filamentous growth 191, takes an exceptional position insofar as it is the only hydrogen bacterium described, for which a clearly defined membrane-bound hydrogenase could not be demonstrated. On the other hand, it has been reported that after fractional centrifugation of crude extracts the activity of the NAD-specific hydrogenase, which is definitely present in this organism, was localized in both the soluble and the

Enzyme. Hydrogenase or H, : NAD’ oxidoreductase (EC 1.12.1.2).

sediment fractions [9]. The enzyme isolated from the soluble extract fraction has been partially purified and characterized [lo, 111. The most significant property of this hydrogenase is that its activity can be drastically stimulated by nickel ions. The analogous enzyme, the NAD-specific hydrogenase of A. eu- trophus is not activated by nickel: however, it was the first hydrogenase for whose formation a specific nickel requirement was demonstrated [12]. Only recently it has been confirmed that nickel is, in fact, a constituent of the soluble A. eutrophus hydrogenase [I 31. Until now, several further hydrogenases, isolated from different strains of Methanobacterium ther- moautotrophicum [14- 261, Desuljovibria gigas [17-201, Desulfovibrio desulfuricans [21,22], Chromatium [23] and Vibrio succinogenes [24] had also been shown to be nickel-containing enzymes. For none of these hydrogenases, however, has a direct effect ofexogenous nickel on the activity of the isolated enzyme been described. The observed behaviour of the Nocardia hydrogenase towards nickel appeared, therefore, to be unique. This fact and the possibility that this enzyme, in contrast to other hydrogenases, might contain a dissociable nickel cofac- tor, prompted us to study the effect of nickel ions and the role of nickel in this NAD-specific hydrogenase more closely. We also present a purification procedure for the Nocardiu hydrogenase and compare the catalytic, structural and immunological properties of this enzyme with the properties of the well- described soluble hydrogenase of A . eutrophus.

534

MATERIALS AND METHODS

Materials

Phenyl-Sepharose was obtained from Pharmacia, agarose from Serva and ethyleneglycol from Merck. The sources of all other chemicals used were as listed in [25].

Organisms, growth conditions and harvesting of Celts

Nocardia opaca I b (DSM 427) was grown autotrophically in a mineral medium [26] in 10-1 fermentors as described by Aggag and Schlegel [9]. During the stationary growth phase, when the culture reached an absorbance (A436) of about 20, cells were harvested, washed with 50 mM potassium phosphate buffer, pH6.5, containing 0.5mM NiCl, and 5mM MgSO, and finally resuspended in the same buffer up to an absorbance of 300 - 350. Cells of Alcaligenes eutrophus H16 (ATCC 17699) were grown heterotrophically with 0.5 % fructose and 0.2 % glycerol as carbon sources [I21 and harvested as described previously [25].

Enzyme purification

All purification steps were carried out between 0 and 4 "C under aerobic conditions. Each buffer used throughout the purification procedure routinely contained 0.5 mM NiCl, and 5 mM MgS04. After disruption of cells by sonication [25] cell debris and larger particles were removed by centrifugation at 100 000 x g for 60 min. The supernatant was referred to as crude extract. Protamine sulfate treatment and ammonium sulfate fractionation were performed as outlined in [lo]. The protein precipitate from ammonium sulfate treatment (35 - 55 % saturation) was dissolved in 200 mM potassium phosphate (pH 6.5) and directly applied to a phenyl-Sepharose column (2.5 x 40 cm) pre-equilibrated with the same buffer. After successive washings of the column with 200mM potassium phosphate, pH 6.5 (300 ml) and 50 mM potassium phosphate, pH 6.5 (500 ml) to remove protein which was, respectively, not bound or only weakly bound to the gel, hydrogenase was eluted with a 20 mM solution ofthe same buffer. More strongly bound proteins were eluted with 80 % poly(ethyleneglyco1) at a flow rate of 40 ml/h. Fractions with a volume of 4 ml were collected. The most active fractions were combined and the volume concentrated to 5 ml by ultrafiltration in an Amicon diaflo cell.

The protein solution was then layered on the top of a Sephadex G-200 column ( 5 x 100 cm), which was equilibrated and eluted with 50 mM potassium phosphate buffer, pH 6.5. Fractions of 4 nil were collected at a flow rate of 24ml/h. The active fractions were combined, concentrated to about 2.5 mg protein/ml and stored at - 20 "C.

The soluble hydrogenase of A. eutrophus, which was used for comparative studies, was purified following the procedure described by Schneider et al. [27].

Eizzyme assays

Hydrogenase activity was routinely assayed by measuring the reduction of NAD spectrophotometrically in I-cm cuvettes at 365 nm at 30 "C. The reaction mixture contained 0.5 M H,-saturated potassium phosphate buffer (pH 8.0), 0.8 mM NAD and an appropriate amount of enzyme. The unit of enzyme activity was defined as the reduction of lpmol NAD/min.

The reduction of several other electron acceptors with H, was also measured spectrophotometrically. The acceptor con-

centration and the wavelengths used were: FMN, 0.3mM, 445 nm; FAD, 0.3 mM, 450 nm; methylene blue, 0.2 mM, 578nm; methyl and benzyl viologen, 3mM, 578nm; ferri- cyanide; 1.2mM, 405 nm. Ifnot stated otherwise, H,-saturated triethanolamine/HCl (50 mM, pH 8.0) was used as assay buf- fer. Before the reaction was started with the electron acceptor, the enzyme was reductively activated by the addition of either 50pM dithionite or 25pM NADH.

H, uptake activity with various electron acceptors was measured manometrically as described previously [25]. Instead of Tris/HCl, triethanolamine/HCl buffer (50 mM; pH 8.0) was used.

H, evolution from reduced electron carriers was also measured manometrically. The reaction mixture contained 100 mM potassium phosphate (pH 7.0) 15 mM dithionite and the following concentrations of electron donors: 10 mM methyl viologen, 10mM benzyl viologen, 4mM NADH. The atmosphere was N,.

Protein determination

The protein content of preparations resulting from prepara- tive electrophoresis was determined by the method of Bradford [28], the protein of all other samples was determined by the Biuret method [29].

Polyacrylamide gel electrophoresis

Electrophoresis experiments were conducted in a flat gel apparatus for vertical slab electrophoresis constructed by Stegemann [30]. For analytical gel electrophoresis in 7.5 % polyacrylamide 30 mM Tris/borate, pH 7.9, was used as gel and electrophoresis buffer [31]. Protein and activity staining was carried out according to Schneider and Schlegel [25].

To separate and to isolate the two subunit fractions, into which the Nocardia hydrogenase dissociates at alkaline pH values and in the absence of NiCl,, electrophoresis was performed on a preparative scale using a gel of 1 cm thickness, which contained 5 % polyacrylamide. The electrophoresis buffer used was 30mM Tris/borate, pH 8.5. Conditions of the electrophoresis run and of the procedure of protein isolation were the same as described for the native NAD-specific hydrogenase of A . eutrophus [25].

Polyacrylamide gel electrophoresis in the presence of so- dium dodecyl sulfate was done by the method of Weber and Osborn [32]. The gel routinely contained 7.5 polyacryl- amide, 0.2 % sodium dodecyl sulfate and 8 M urea.

Production and purification of antibodies

Antibodies against the soluble hydrogenase of A . eutrophus were produced as described by Schink and Schlegel [33] and purified according to Bowien and Mayer [34].

Immunodiffusion test

Immunodiffusion (Ouchterlony) tests were carried out in gels containing 1 % agarose in 50 mM diethylbarbiturate/ acetate buffer, pH 8.2, on microscopic slides [35]. To analyze the occurrence and the type of cross-reactions, highly purified hydrogenases of N. opaca and A . eutrophus and purified antibodies against the soluble hydrogenase of A. eutrophus were used.

535

Table 1. Purification of hydrogenase from Nocardia opaca Ib

Step Total Total protein activity

Specific activity Purification Yield

mg units Crude extract 3853 5879 Protamine sulfate treatment 2906 5033 Ammonium sulfate

fractionation (35 - 55 %) 1128 3859 Phenyl-Sepharose 68.7 1989 Sephadex G-200 24.7 1112

units/mg protein -fold % 1.53 1 .o 100 1.73 2.1 86

3.42 28.95 45.02

2.2 18.9 29.4

66 34 19

80% poly- ZOOrnM 50mM 2OmM (ethylen glycol1 12

I I

:: 0.2

C w 0.1

i

100 200 300 100 500 Fraction number

Fig. 1 . Elution profile of hydrogenuse f r o m the phenyl-Sepharose column. 1.1 g protein was applied to the column (2.5 x 40cm) and eluted with a discontinuous potassium phosphate (pH 7.0) gradient with concentrations as indicated in the figure. More strongly bound protein was eluted with 80 % poly(ethyleneglyco1). Fractions of 4ml were collected. (0) Protein; (0) hydrogenase activity

RESULTS

Localization of hydrogenase in Nocardia opaca Ib

If the crude extract of N. opaca with a protein content of 15 - 20 mg/ml was fractionated by a routine procedure (centri- fugation at 100000 x g for 1 h) to separate soluble and mem- brane proteins, the NAD-reducing hydrogenase activity was localized in the soluble fraction exclusively, regardless of whether the cells were disrupted by a French pressure cell or by sonication. This result is in contrast with observations reported by Aggag and Schlegel [9], who found the NAD-reducing activity to be proportionately distributed among supernatants and sediments. In our hands, in the particulate fractions there was no hydrogenase activity detectable at all, either with NAD or with any other electron acceptor. Thus, we conclude that N . opaca l b contains only one hydrogenase which is soluble and, like the soluble hydrogenase of Alcaligenes eutrophus, able to reduce NAD. The absence of a membrane-bound hydrogenase was confirmed by immunodiffusion assays. Neither the mem- brane proteins of N. opaca, treated with Triton X-100 (0.5 %) and deoxycholate (0.1 %), nor the soluble protein fraction, which might have contained solubilized hydrogenase, showed any cross-reaction with the antibodies against the membrane- bound hydrogenase of A. eutrophus H16. N. opaca l b can, therefore, be described as the first hydrogen bacterium which does not contain a membrane-bound, respiratory-chain- associated hydrogenase.

Purification of the NAD-linked hydrogenase

The soluble, NAD-linked hydrogenase from autotrophi- cally grown cells of N . opaca 1 b was purified to homogeneity including the following steps. Protamine sulfate precipitation, ammonium sulfate fractionation, phenyl-Sepharose chroma- tography and Sephadex G-200 gel filtration (Table 1). Column chromatography on phenyl-Sepharose is a technique in which proteins are separated on the basis ofthe differing strengths of their hydrophobic interactions. Since soluble enzymes are genqrally expected not to be strongly hydrophobic and hy- droqenases are described to exhibit a predominance of acidic amino acids [36], it was surprising that, with respect to the purilfication of the Nocardia hydrogenase, the phenyl- Sepharose step was the most effective one of the whole proaedure. Although the binding of hydrogenase to phenyl- Sepbarose was relatively weak, the elution of the enzyme with 20 mM potassium phosphate (pH 6.5) was of high selectivity (Fig. 1). It yielded an enzyme preparation that was, as analyzed by polyacrylamide gel electrophoresis, about 65 % pure and contained only one additional contaminating protein (not sho$vn). This impurity could easily be separated by subsequent gel filtration (Sephadex G-200). The resulting hydrogenase preparation, which had a specific activity of 45 units/mg protvin, proved to be electrophoretically homogeneous (Fig. 2, gel 1). The overall yield of hydrogenase activity was 19 %.

Dependence of the NAD reduction on metal ions and high salt concentrations

If the reaction mixture for NAD reduction contained the required substrates (H,, NAD) and a suitable buffer system (triethanolamine/HCl, pH 8.0) without any further addition, the enzyme activity was, independent of the degree of purity of hydrogenase, absolutely zero (Tables 2, 3). If, however, nickel ions (NiC1,) were added to the test system, hydrogenase was activated exhibiting a characteristic time-dependent reaction course, as described for the NAD-reducing hydrogenase of A. eutrophus [25]. The optimal NiC1, concentration for enzyme activation was 1 mM. Concentrations higher than 1 mM were inhibitory. As other chloride salts (KC1, NaC1, NH,Cl) at concentrations between 0.1 mM and 10mM had no effect on the NAD reduction rate at all, it was clear that the activation of hydrogenase by NiClz is due to Ni2+ and not to the chloride anion. To find out whether the requirement for nickel is specific, the influence of several other metals, salts and buffers were examined (Table 2). Nickel could be replaced by cobalt ; however, to achieve a similar high activity a higher metal concentration (2.5 mM) was required. Less activation at even higher concentrations was observed with MgC12 (25 mM) and MnC1, (100mM). Other divalent metal cations (Zn”, Cu2+, Ca2+) as well as FeCl, had no effect on enzyme activity. FeC1,

536

Table 2. N A D reduction rates of hydrogenase in the presence of' metal ions and high salt concentrations

Addition Optimal concentration Hydrogenase activity Relative activity

Without NiCI, COCI, MgC1, MnCI, NiCl, + MgS0, FeC1, FeCl, ZnC1, CUCI, CaC1, NaCl KF KC1 K Br KI NH4C1 (NH4)2S04 K m 4

KNO, Triethanolamine/HCl Tris/HCI

K,HPO4/KHZPO,

M

0.001 0.002s 0.02s 0.100 0,0005 + 0.005 0.005 0.005 0.010 0.010 0.010 1 .s 1 1 1 1 0.2 0.5 0.5 0.5 1 0.5 0.5

- A A 365/min 0 0.508 0.500 0.438 0.184 0.880 0.254 0 0 0 0 0.089 0.835 0.329 0.086 0 0.333 0.760 0.600 1.440 0 0 0

% 0

100 98 86 36

173 50 0 0 0 0

18 164 65 17 0

66 150 118 283

0 0 0

Fig. 2. Polyacrylamide gel electrophoresis ofpurified hydrogenase. Gel 1 contained 7.5 % acrylamide and was run for 3 h at 14 mA and 380 V. Gel and electrophoresis buffer used were 30mM Tris/borate (pH 8.0) containing 0.5 mM NiC1, and 5 mM MgSO,. Gel 2 and buffer did not contain NiC1, and MgSO,. Other conditions were as for gel 1. Gel 3 contained 5 % acrylamide, 0.1 %sodium dodecyl sulfate and 8 M urea. It was run for 1.5 h at 80 mA and 200 V. Gels 4 and 5 contained 7.5 % acrylamide, 0.2 % sodium dodecyl sulfate and 8 M urea. The gels were run for 2 h under the same conditions as gel 3. On gels 1-4 N . opaca hydrogenase and on gel 5 A . eutrophus hydrogenase was applied. Protein was stained with Coomassie brilliant blue

( 5 mM) only stimulated the initial rate of NAD reduction, after 30-40s the reaction ceased again. Among various com- binations of metal ions, the combination of NiC1, (0.5 mM) and MgS04 ( 5 mM) was the most efficient, yielding an activity

0.60 1 1

PH

Fig. 3. p H optimum of hydrogenase reactions. (0) NAD reduction in 100 mM Tris/Mes buffer ; amount of enzyme in the reaction mixture : 24 pg protein. (0) NAD reduction in 50 mM triethanolamine/HCl buffer in the presence of 3 mM NiCI,; amount of enzyme in the reaction mixture 2.4pg protein. (A) Benzyl viologen reduction in 50 mM glycine/KOH buffer in the presence of 1 pM NiCI, ; amount of enzyme in the reaction mixture: 0.03 pg protein

of about 170 "/,compared to the activity (I00 "/o) obtained in the presence of NiCl, alone. Almost as high or lower activities were detected at high concentrations (0.2- 1.5 M) of some salts [KF, KC1, K,S04, NH4C1, (NH4),S04]. A comparison of the effect of the halides indicates that the degree of stimulation increased in the sequence KI, KBr, KC1, K F (see Table 2); this means that the observed salt effect was the more pronounced the less chaotropic the anion. The maximum activity (283%) was measured in 0.5M potassium phosphate buffer. N o hy- drogenase activity at all was detectable in solutions of high concentrations of KI, KNO, and in triethanolamine/HCl and Tris/HCl buffers.

The fact that some metals can partially or completely substitute for nickel and that various salts, if provided at sufficiently high concentrations, even exceed the degree of enzyme activation caused by NiZ+ ions, led to the conclusions that nickel, because of its effectiveness at low concentrations, is

537

Table 3. Effect of NiCl, on the rates of reduction of dijferent electron acceptors The reduction of the electron acceptors was measured photometrically at 365 nm (NAD), 445 nm (FMN), and 578 nm (benzyl viologen, methylene blue). For reduction of NAD and FMN 1.8 pg protein, for reduction of benzyl viologen and methylene blue 0.3 pg protein was used

NiCl, concn Hydrogenase activity

NAD FMN benzyl viologen methylene blue

0 0.1 1

10 100 500

1000

0 0 0.01 5 0.066 0.279 0.357 0.315

0.380 0.590 0.603 0.486 0.354 0.203 0.116

0.960 1.292 1.380 1.380 1.020 0.555 0.276

1.320 2.100 1.804 3.800 0.975 0.278 0.160

Table 4. Electron acceptors of hydrogenase: comparison of activities and K, values The activity of hydrogenase with different electron acceptors was measured photometrically as described in Materials and Methods. The enzyme assays were done with a homogeneous hydrogenase preparation. As test buffers 0.5 M potassium phosphate, pH 8.0 (NAD) 50mM glycine/KOH, pH 8.0 and 9.4 (benzyl viologen) and 50mM triethanolamine/HCI, pH 8.0 (all other acceptors) were used

Electron acceptor NiCl, concentration Specific in the reaction activity mixture

Activity K m

NAD FAD FMN Methyl viologen Benzyl viologen, pH 8.0 Benzyl viologen, pH 9.4 Methylene blue Ferricyanide

PM a -

1 1 1 1 1 0.1

1000

units/mg protein 45.0 44.7 17.2

309.6 675.7

2257.0 476.0 91 .O

% 100 99 39

688 1501 501 3 1058 202

mM 0.15 0.38

0.90 0.05

0.44 4.00

-

-

a 0.5 M potassium phosphate was used without NiCl,

indeed the most specific activator but an involvement of this extra added nickel in enzyme catalysis can certainly be excluded.

Effect of proton concentration on hydrogenase activation

The pH optimum of hydrogenase activity (NAD reduction) in 0.5 M potassium phosphate as well as in triethanol- amine/HCl and Tris/HC1 (50mM each) in the presence of NiCI, was, like that of the soluble hydrogenase of A . eutrophus [25], pH7.8-8.0 (Fig. 3). To examine whether the activation/inactivation process of hydrogenase is a function of the proton concentration, the NAD-reducing activity was measured at low p H values using a 100 mM Tris/Mes buffer in the absence of nickel. Above pH7.0 hydrogenase was com- pletely inactive. However, below pH 7.0, on increasing the proton concentration the activity increased linearly to reach a maximum at pH 6.1 (Fig. 3). This maximum was relatively low and corresponded to about 8 % of the activity measured at pH 8.0 in the presence of nickel. At pH values lower than 6.1 activity slowly decreased. A comparison of the effect of added nickel (1 mM) at varied pH values showed that the activation by nickel was the lower the higher the proton concentration. Whereas at pH 8.0 the stimulation of activity was infinite and was 32-fold at pH 7.0, at pH 6.1 it was only 2-fold. This con- firms that nickel ions can, in fact, at least partly be replaced by protons.

Reuctivity of hydrogenase with other electron carriers and influence of nickel ions on the reaction rates

Like the soluble hydrogenase of A . eutrophus [25], the Nocardia hydrogenase was able to react directly with various physiological and artificial electron acceptors including flavins, quinones, cytochrome c (horse heart), O,, viologen dyes, methylene blue, 2,6-dichloroindophenol, phenazine methosul- fate and ferricyanide.

Surprisingly, all electron acceptors tested were reduced by hydrogenase at high rates also in the absence of nickel, except NAD. Closer studies on some of these acceptors (FMN, benzyl viologen, methylene blue) revealed that their reaction with hydrogenase was also stimulated by nickel; however, only to small extent ( z 1.5-fold) and at very low Ni2+ concentrations (0.1 - 20 pM) (Table 3). Higher concentrations (0.5 - 1 mM), which were optimal for NAD reduction, strongly inhibited the reactions. It should be noted that the activity of the NAD- linked A . eutrophus hydrogenase, which was examined in parallel assays, was not stimulated at all by nickel ions, other metal ions or any salts, neither with NAD nor with any other electron acceptor.

For the electron carriers listed in Table 4 photometric enzyme assays have been elaborated and optimized with respect to substrate concentration, nickel concentration, buffer system and pH value. Benzyl viologen turned out to be the most effective electron acceptor. At the pH optimum of 9.4 (see

538

Fig. 3) benzyl viologen was reduced with a specific activity of 2257 U/mg. This is the 50-fold activity of that measured with NAD. A direct comparison of the H, uptake rates of both NAD-specific hydrogenases, from A. eutrophus and N. opaca, showed that the Nocardia enzyme reacted with benzyl viologen about ten times faster. The K,,, values, determined at pH 8.0, were 0.05 mM for benzyl viologen (A. eutrophus hydrogenase: 9 mM) and 0.15 mM for NAD (A. eutrophus hydrogenase: 0.56mM). Very high activities, up to 7- 10-fold of the NAD reduction rate, were also obtained with methyl viologen and methylene blue as electron acceptors. In contrast, some elec- tron acceptors, such as dichloroindophenol and phenazine methosulfate, which were reduced by the A. eutrophus enzyme at an especially high rate, were reduced by the Nocardia enzyme at only one-tenth of that rate (data not shown). Also with ferricyanide the hydrogenase showed only moderate activity and low affinity (K, = 4mM, compare Table4).

A further difference between the hydrogenases of N . opaca and A . eutrophus concerns their reactivity in the oxidized state. The A . eutrophus hydrogenase has been described to be completely inactive with acceptors different from NAD, if the enzyme is not transformed into a reductively activated state prior to the start of reaction [25]. The enzyme of N. opaca, on the other hand, was demonstrated to be able to catalyze, although at a by 70 - 85 % diminished rate, the complete reduction of electron carriers without any pretreatment with reducing compounds (NADH, dithionite).

Double-reciprocal plots of H, saturation curves were biphasic consisting of two linear sections (data not shown). This might be explained by the presence of a mixture of high- affinity and low-affinity forms of the enzyme. The affinity of hydrogenase to H, was higher at low H, concentrations (apparent K,,, = 20pM) and decreased at H, concentrations above 30pM (apparent K,,, = 126pM). The Hill plot also showed a 'break' changing from a Hill coefficient of n = 1.07 to n = 0.58, which indicated a change from non-cooperativity to negative cooperativity at higher H, concentrations. Apparent negative cooperativity was also described for the soluble hydrogenase of A . eutrophus [25].

The reversibility of enzyme function, the evolution of H, from NADH and dithionite-reduced viologen dyes, was dem- onstrated manometrically. The pH optimum of H, evolution from NADH was 6.0. At this pH and in the presence of 4 mM NADH the rate of H, production corresponded to 18 % of the reverse reaction and was shown to be independent of nickel ions. The highest H, production rate, which was approximately as high as the H, uptake rate, was measured at pH7.0 with reduced methyl viologen. With benzyl viologen about 40 % of that rate was obtained.

Stability

Nickel ions or high salt concentrations were found to be essential not only for the NAD-reducing activity but also for stability of hydrogenase. In the presence of 0.5 mM NiCl, and 5 mM MgC1, the enzyme was stable (at 4 "C under air) over a range of pH values between 6 and 8 (Table 5). In the absence of metal ions hydrogenase stability was drastically decreased. The observed inactivation process was the more rapid the higher the pH value. The loss of activity (NAD reduction) during one day was 36 % at pH 6.0 and 86 % at pH 8.0. The most favourable buffer for storage of hydrogenase was 0.5 M potassium phos- phate pH 7.0. In this buffer at 4 "C the loss of enzyme activity after three days was only 10%.

Table 5. Stability of hydrogenase

Storage conditions Activity

PH presence of after 1 day after 3 days 0.5 mM NiCl, + 5 mM MgCI,

% - 6.0

6.0 + 7.0 -

7.0 + 8.0 8.0 + 7.0

-

a -

54 20 91 56 38 13

100 79 14 0

100 73 100 90

a 0.5 M potassium phosphate was used without NiCI,.

Like the soluble hydrogenase of A . eutrophus [37] the Nocardia enzyme also became irreversibly inactivated by self- produced superoxide radicals if reducing compounds and 0, were simultaneously present in the enzyme solution. However, this hydrogenase was distinctly more stable under these conditions than the Alcaligenes hydrogenase. Whereas this latter enzyme under oxyhydrogen (90 % HJI 0 0,) and in the presence of 25 pM NADH was almost completely inactivated (1 - 5 % residual activity) within 12 h, the Nocardia hydro- genase still exhibited 43 % of activity after one day and 6 % after one week.

Subunit structure of hydrogenase

The subunit structure of hydrogenase from N . opaca 1 b was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. This was done in a comparative study including the soluble hydrogenase of A . eutrophus H16, which has previously been described to consist of three types of subunits with molecular weights of 68000, 60000 and 29000 present in a 1 : 1 : 2 molar ratio [38]. Generally it can be stated that the native hydrogenases of N. opaca and A. eutrophus showed identical patterns of bands whatever conditions were used.

Because it was observed that, under standard conditions (7.5 % acrylamide, 0.1 % dodecyl sulfate), the band of the smallest subunit always appeared to be relatively broad and diffuse, we have, in this work, further optimized the experimen- tal conditions of electrophoresis. A sharpening of this band, occurred after decreasing the concentration of polyacrylamide from 7.5 % to 5 % in the presence of 0.1 % dodecyl sulfate and 8 M urea (Fig. 2, gel 3). If higher gel concentrations (7.5 - 10 %) were maintained but the dodecyl sulfate concentration was increased to 0.2%, this single band was split into two clearly separated weaker bands (Fig. 2, gels 4, 5). We, therefore, concluded from the electrophoretic analyses that the NAD- linked hydrogenases are tetramers composed of four non- identical subunits with molecular weights of 64000, 56000, 31 000 and 27000 (Nocardia enzyme) and 63000,56000,30000 and 26 000 ( A . eutrophus enzyme) respectively. Several control experiments (denaturation of the enzymes at different tempera- tures, in the presence of various concentrations of dodecyl sulfate and mercaptoethanol, in the presence of 8 M guanidine hydrochloride without sodium dodecyl sulfate, in the presence of single and combined added protease inhibitors (p-amino-

539

6LOOO

Gel length

Fig. 4. Densitometer tracings of dissociated hydrogenase, the dark yellow coloured and the light yellow coloured subunit dimer on dodecyl sulfate/polyacrylamide gels. The conditions of the electrophoresis runs were as described for gels 4 and 5 of Fig. 2. The gels were stained with Coomassie brilliant blue and scanned at 609nm with a Vitatron TLD 100 densitometer. Curve 1, whole enzyme; curve 2, dark yellow subunit dimer; curve 3, light yellow subunit dimer

benzamidine . HCl, p-toluene sulfonic acid, phenylmethyl- sulfonyl fluoride, N-a-tosyl-1-lysine chloromethyl ketone, aprotinin) ascertained that none of the bands was due to an artifact or the result of protease activity.

Effect of nickel ions on the subunit composition of the Nocardia hydrogenase

If polyacrylamide gel electrophoresis of the hydrogenase from N. opaca was performed in the absence of dodecyl sulfate, either 2 mM NiC1, or 0.5 mM NiC1, + 5 mM MgCl, had to be added to the gel and to the electrophoresis buffer (30mM Tris/borate, pH8.0) in order to guarantee stability of hy- drogenase. Under these conditions purified hydrogenase mig- rated as a single homogeneous band (Fig. 2, gel 1). This band was hydrogenase-active and had an R, value of 0.11 (A . eutrophus hydrogenase: 0.12). In the absence of the metal ions the hydrogenase band dissociated into two bands of approx- imately the same intensity (protein staining) but of different colour and electrophoretic mobility (Fig. 2, gel 2). One band, with an R, value of 0.1 8, was dark yellow, looked like the native enzyme, but lacked any hydrogenase activity. The second band had an RF value of 0.44, was light yellow and was inactive with NAD but reduced methyl viologen.

These results posed the question: is the occurrence of two protein bands due to the presence of a contaminant, to the dissociation ofhydrogenase into subunit complexes of different composition, or is it due to the separation of two different fractions of the same undissociated but modified and in- activated enzyme?

To answer this question the two protein bands were separated and isolated on a preparative scale using polyacryl- amide gel slabs of 1 cm thickness, from which the bands were cut and eluted (see Materials and Methods). The two isolated protein components were subjected to dodecyl sulfate gel electrophoresis and directly compared with the whole enzyme. The patterns of polypeptide bands revealed that both com- ponents are dimers composed of two different subunits, all of which were unambiguously identified as subunits of hy- drogenase (Fig. 4). The dark yellow component consisted of

Fig. 5. Immunodiffusion of soluhle hydrogenases from N. opaca l b and A. eutrophus H16. (A) The agarose gel was prepared in the absence of NiCI,; the centre well contained 40 pg purified hydrogenase of N . opaca, wells 2 and 5 contained 1.3mg each of anti-hydrogenase ( A . eutrophus) immunoglobulins, wells 1, 3, 4 and 6 remained empty; (B) the agarose gel was prepared in the presence of 2 mM NiCl,; the additions of enzyme and antibodies were as for gel (A); (C) the centre well contained 436 pg anti-hydrogenase ( A . eutrophus) immuno- globulins, wells 1 and 6 contained 19pg each of purified soluble hydrogenase of A . eutrophus, wells 3 and 4 contained 30pg each of purified hydrogenase of N . opaca, wells 2 and 5 remained empty; (D) the centre well contained 436 pg anti-hydrogenase ( A . eutrophus) immunoglobulins, wells 1 and 3 contained 19pg each of purified soluble hydrogenase of A . eutrophus, wells 4 and 6 contained 30 pg each of purified hydrogenase of N . opaca, wells 2 and 5 remained empty. Gels (C) and (D) contained, as gel (B) 2mM NiCI,. Immunodiffusion was performed in a humid atmosphere at room temperature for 24 h. The gel slabs were washed for 24h in physiological saline and subsequently stained with Coomassie brilliant blue

subunits with molecular weights of 64000 and 31 000, and the light yellow component consisted of subunits with molecular weights of 56 000 and 27000. Isolation and re-electrophoresis of the single bands reproduced only single bands without any tendency to form dissociation or association products. This conclusively confirmed that the larger subunits (64000,56000) were not dimers of the smaller ones.

Based on the molecular weights determined for the sub- units, the total molecular weight of hydrogenase was 178 000. This coincided exactly with the value obtained by sucrose density gradient centrifugation but was not in full accordance with the value of 200000 5000 as determined by gel filtration (Sephadex G-200).

Immunological comparison of the NAD-linked hydrogenases of N. opaca l b and A. eutrophus HI6

By the Ouchterlony double-immunodiffusion technique it was demonstrated that the Nocardia hydrogenase and the purified antibodies raised against the soluble hydrogenase of A . eutrophus showed a significant cross-reaction (Fig. 5). To obtain precipitin bands of intensity similar to the homologous antigen ( A . eutrophus hydrogenase)/antibody reaction, an approximately twofold amount of enzyme and a threefold to fourfold amount of antibodies had to be used. If the agarose gels (in diethylbarbiturate/acetate buffer, pH 8.2) were pre- pared without the addition of NiCl, one major band and a second very fine band were formed (Fig. 5A) indicating that a small proportion of the enzyme dissociated during diffusion. In the presence of 2mM NiCl, only a single precipitin band

540

appeared (Fig. 5 B). To characterize the relationship between the two NAD-linked hydrogenases more specifically immu- nodiffusion was performed with a gel that contained both enzymes in two neighbouring wells and the antibodies in the centre well. The resulting precipitin lines were partly fused with the formation of a single spur (Fig. 5 D) suggesting that the two hydrogenases are partially identical proteins. In Fig. 5 C the characteristic patterns of spur-free and completely fused pre- cipitin lines of fully identical enzymes are shown for comparison.

DISCUSSION

A specific nickel requirement for chemolithoautotrophic growth of strain of Alcaligenes eutrophus (Hydrogenomonas) was discovered in 1965 [39]. The effect of nickel on the activity of the hydrogenase isolated from N . opaca 1 b was reported for the first timein 1974[10]. Inspite oftheseearlyobservations the possibility that nickel might be involved in hydrogenase function was not pursued until the nickel requirement for the synthesis of the catalytically active hydrogenases in A. eutro- phus was discovered [12]. Since that time the research on nickel in hydrogenases was strongly intensified. The dependence of hydrogenase synthesis on nickel ions has recently been confirmed for the enzymes of various bacterial species [40 - 421. By chemical and physical analysis nickel has convincingly been demonstrated to be a constituent in a number of hydrogenases [13-241. In the case of the hydrogenases from Methano- bacterium autotrophicum strains [15, I61 and Desulfovibrio species [I7 - 221, as well as the membrane-bound hydrogenase of A. eutrophus HI6 [43], additional evidence for the presence of nickel was presented by characteristic Ni(II1) electron spin resonance signals. Although some authors [15- 17, 19-21] described the nickel component as redox-sensitive and, there- fore, discussed it as the site possibly involved in H, activation, the actual role of nickel in enzyme catalysis is still unknown. A promising approach to elucidate the function of nickel is to study nickel-free hydrogenases, which lack this metal due either to mutation or (ir)reversible dissociation. The description of the NAD-specific hydrogenase of Nocardia opaca I b as being dependent on nickel ions in the isolated and purified state [lo] implied that this enzyme might be a representative of such an nickel-free hydrogenase and therefore an ideal subject to study.

In fact, we confirmed that the Nocardia hydrogenase in its isolated form was completely inactive and could be converted to a highly active form by the addition of Ni2+ ions. However, the observed inactivity of hydrogenase was not caused by the loss of a catalytically functional nickel cofactor, which can be replaced by excessively added nickel ions. This conclusion was drawn from the following results: (a) the lack of enzyme activity was limited exclusively to the reaction with NAD; all other electron acceptors reacted rapidly also in the absence of nickel; (b) the activating effect of nickel was not specific; the activation of hydrogenase was also effected by other metal ions, by high salt concentrations and by lowering the H + con- centrations to less than pH 7.0; (c) as demonstrated by electrophoretic analyses, under non-activating conditions hy- drogenase dissociated into two subunit dimers. It is our conclusion that this dissociation is the real reason for the inactivation of hydrogenase. Probably the H,-activating and NAD-binding site(s) and all the electron-transferring com- ponents (Ni, Fe-S clusters, flavin) involved in NAD reduction are not located in only one subunit dimer but are distributed on both dimers. The dissociation of these dimers apparently leads

to an interruption of the intramolecular electron transport from H, to NAD.

Our results unequi\ocally suggest that the role of metal ions, salts and protons, in the activation process of hy- drogenase, is to bring the subunit dimers to association or to prevent dissociation. The compound which achieved this effect at the lowest concentration (1-2mM), was NiC12. The possibility, therefore, can not be excluded that nickel plays a specific role in the binding of the subunits of this hydrogenase. Less specific but even stronger activation effects were reached by about 1 M solutions of salts [K,HPO,/KH,PO,, (NH4),S04, KF], which are known to be particularly effective in dehydrating proteins and thus give rise to increased hy- drophobic interactions and stronger aggregation of enzyme and subunit molecules (‘salting-out’ effect). Obviously the NAD-reducing activity of hydrogenase is the more stimulated the closer the contacts between the subunits.

The reaction rates of the hydrogenases from Megasphaera elsdenii [44,45] and Chlamydomonas reinhardtii (H, production from methyl viologen) [46] were also reported to be greatly enhanced by salts. However, these salt effects are quite the opposite of that effect observed with the Nocardia hydro- genase: whereas in the case of the latter enzyme the stimulation effect increases with decreasing chaotropicity of the anion (e.g. no effect with KI, strong effect with KF), with the hy- drogenases of M . elsdenii and C. reinhardtii it is just the reverse (no effect with KF, strongest effect with KI). Roessler and Lien [46] discussed several possible mechanisms by which the effect of chaotropic anions could be explained. Recently the deazaflavin( F,,,)-reducing hydrogenase of Methanobacterium thermoautotrophicum has been noted to require 1 M KC1 for optimal reductive activation [16].

Both NAD-linked hydrogenases of N. opaca I b and A , eutrophus HI6 are composed of four non-identical subunits, which are present in a 1 : l : l : l molar ratio. This subunit structure is very unusual and among hydrogenases certainly the most complex one, but it has convincingly been demonstrated to be real by several lines of evidence: (a) by several control analyses which showed that none of the detected polypeptides is an artifact; (b) by successful separation and differentiation of two different types of heterodimers, which are similar in size but differ strongly with respect to colour (cofactor com- position) and reactivity and do not show immunological identity (K. Schneider, unpublished result); (c) by the fact that the dodecyl sulfate electrophoretic patterns of bands of the two purified hydrogenases are identical, although they were iso- lated from organisms of completely different taxonomic groups and from cells which were each cultivated under different conditions ( N . opaca: heterotrophically ; A. eutrophus: auto- trophically) and which are, therefore, expected to contain a quite diverse composition of proteins. Moreover, we have now obtained evidence that the purified soluble hydrogenases from other A. eutrophus strains (type strain, CH34) also have the same subunit structure as the enzymes of strain HI6 and N. opaca with only slight differences in the molecular weights of the subunits (K. Schneider and G. Schulze, unpublished results). This structural conformity among hydrogenases of different origin makes it unlikely that one of the polypeptides described might be due to any protein contaminant and suggests that the composition of four non-identical subunits IS characteristic for the type of NAD-specific hydrogenase in aerobic hydrogen bacteria. It should, however, be mentioned that the respective enzyme of strain Z1 of A. eutrophus has also been described to have a tetrameric structure, but to contain only two types of subunits (2 x 30000; 2 x 65 000) [47]. Whether

54 1

this enzyme is really different from the others or whether the resolving power of the electrophoresis conditions used was not sufficient to separate the two similar large subunits each remains t o be elucidated.

In a recent paper Popov et al. 1481 reported that the Z1 hydrogenase became inactivated and dissociated into various fragments upon dilution. The molecular weights of the main components were 90000 and 60000. The 60000-M, fragment was hydrogenase-inactive but showed diaphorase (NAD dehy- drogenase) activity with methyl viologen. Whether these en- zyme fragments represented, as speculated by the authors, defined subunits and subunit complexes, and thus a dissocia- tion phenomenon similar t o that described for the Nocardia hydrogenase, must await further subunit analyses of the separated fragments.

This work was supported by grants from the Deutsche Forschungsgerneinscha~t. The cooperation of Dr R. Brinkmann in providing cells of N . opaca I b is gratefully acknowledged.

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K. Schneider, H. G. Schlegel, and K. Jochim, Institut fur Mikrobiologie der Georg-August-Universitit LU Giittingen, GrisebachstraDe 8, D-3400 Gottingen, Federal Republic of Germany