content and localization of fmn, fe-s clusters and nickel in the nad-linked hydrogenase of nocardia...

Eur. J. Biochem. 142, 75 - 84 (1 984) I( FEBS 1984

Content and localization of FMN, Fe-S clusters and nickel in the NAD-linked hydrogenase of Nocardia opaca l b

Klaus SCHNEIDER, Richard CAMMACK, and Hans G. SCHLEGEL Institut fur Mikrobiologie der Universitat Gottingen; and Department of Plant Sciences, King’s College, London

(Received January 20iMarch 7, 1984) ~ EJB 84 0069

By preparative polyacrylamide gel electrophoresis at pH 8.5, and in the absence of nickel ions, two types of subunit dimers of the NAD-linked hydrogenase from Nocardia opaca Ib were separated and isolated, and their properties were compared with each other as well as with the properties of the native enzyme.

The intact hydrogenase contained 14.3+0.4 labile sulphur, 13.6*1.1 iron and 3.8A0.1 nickel atoms and approximately 1 FMN molecule per enzyme molecule. The oxidized hydrogenase showed an absorption spectrum with maxima (shoulders) at 380nm and 420 nm and an electron spin resonance (ESR) spectrum with a signal at g = 2.01. The midpoint redox potential of the Fe-S cluster giving rise to this signal was + 25 mV. In the reduced state, hydrogenase gave characteristic low-temperature (10 - 20 K) and high-temperature ( > 40 K) ESR spectra which were interpreted as due to [4Fe - 4S] and [2Fe - 2S] clusters, respectively. The midpoint redox potentials of these clusters were determined to be - 420 mV and - 285 mV, respectively.

The large hydrogenase dimer, consisting of subunits with relative molecular masses M,, of 64000 and 31 000, contained 9.9 0.5 iron atoms per protein molecule. This dimer contained the FMN molecule, but no nickel. The absorption and ESR spectra of the large dimer were qualitatively similar to the spectra of the whole enzyme. This dimer did not show any hydrogenase activity, but reduced several electron acceptors with NADH as electron donor (diaphorase activity).

The small hydrogenase dimer, consisting of subunits with M , of 56000 and 27000, was demonstrated to have substantially different properties. For iron and labile sulphur average values of 3.9 and 4.3 atoms/dimer molecule have been determined, respectively. The dimer contained, in addition, about 2 atoms of nickel and was free of flavins. In the oxidized state this dimer showed an absorption spectrum with a broad band in the 400-nm region and a characteristic ESR signal at g= 2.01. The reduced form of the dimer was ESR-silent. The small dimer alone was diaphorase-inactive and did not reduce NAD with H,, but it displayed high H,-uptake activities with viologen dyes, methylene blue and FMN, and H,-evolving activity with reduced methyl viologen. Hydrogen-dependent NAD reduction was fully restored by recombining both subunit dimers, although the reconstituted enzyme differed from the original in its activity towards artificial acceptors and the ESR spectrum in the oxidized state.

0.4 S2 - and 9.3

Besides the soluble hydrogenases of Alcaligenes eutrophus [I - 31 and Alcaligenes ruhlandii [4], the hydrogenase isolated from Nocardia opaca 1 b has been described as being NAD- linked [5,6]. With respect to many catalytic and molecular properties (electron acceptor specificity, pH optima, affinity for NAD and H2, molecular mass, subunit structure), this enzyme closely resembles the soluble hydrogenase of A . eutrophus HI6 [6]. The most extraordinary property of both enzymes is that they are tetramers each composed of four non- identical subunits. The subunits of the Nocardia hydrogenase have M , of 64000, 56000, 31000 and 27000 [6]. A unique property of this enzyme is the strict dependence of the NAD- reducing activity on the presence of either nickel ions, or high concentrations of moderately chaotropic salts, or pH values lower than 7. If none of these activating conditions are applied, the hydrogenase dissociates into two different subunit dimers with M , of 64000/31000 and 56000/27000 [6]. Whereas the larger dimer was found to be completely inactive, the smaller

Abbreviations. ESR, electron-spin resanance; SDS, sodium do-

Enzyme. Hydrogenase or H, :NAD+ oxidoreductase (EC 1.12.1.2). decyl sulfate; Me,SO, dimethylsulfoxide.

one was inactive with NAD but still active with methyl viologen. These results suggested that the diverse components (substrate binding sites, nickel, Fe-S clusters, flavin) which are probably involved in NAD reduction, are distributed on different subunits. To find out where each of the components is definitely localized, which component is involved in which reaction and thus to understand better the complexity of the enzyme structure and the mechanisms of the hydrogenase- catalyzed reactions, we investigated the content of iron, labile sulfide, nickel and flavin, the reactivity with electron acceptors and the spectral properties of the native hydrogenase as well as of both separated and isolated enzyme dimers. On the basis of the results obtained, a model of the substructure of the NAD- linked hydrogenases is discussed.

MATERIALS AND METHODS

Muter ials

The source of chemicals and biochemicals was as in [6]. Apoflavodoxin and purified FMN were gifts from Dr S. G. Mayhew (University College, Dublin).

I b

Purification of hydrogenase and isolation of subunit dimers

Hydrogenase isolated from Nocardia opaca 1 b was purified as described by Schneider et al. [6]. Separation and isolation of the subunit dimers of hydrogenase was performed by a preparative polyacrylamide gel electrophoresis procedure [6].

Enzyme assays

Hydrogenase activity (reduction of electron acceptors with H,) was measured photometrically [6].

Diaphorase activity with various electron acceptors was measured under analogous conditions with NADH (0.6 mM) instead of H, as electron donor. The reaction rates were determined anaerobically under N, .

Protein determination

Table 1. Metul content in natiae hydrogenuse and subunit prepurutions The homogeneous protein preparations were adjusted to a protein content of 2 -3 mgiml (in 50 mM potassium phosphate, pH 7.0) and analyzed for metal content by X-ray fluorescence. The values represent the average of three or four determinations

Metal Metal content of

native large small hydro- subunit subunit genase dimer dimer ( M , 178000) ( M , 95000) ( M , 83000)

______

~ ~ ~ _ _ _ _ _ _ ~ ~

mol/mol - ~

Magnesium 0 0 0 Iron 13.6 9.6 3.9 Nickel 3.8 0.18 1.8 Cobalt 0 0 0 Copper 0.3 0.37 0.63 Zinc 0.14 0.23 0.36

The protein content of preparations obtained by prepara- Selenium 0 0 0 tive electrophoresis was determined by the method of Brad- Rubidium 0.15 0.26 0.16 ford [7], the protein of all other samples was determined by the Lead 0 0.06 0.13 biuret method [8].

Anulysis of constituents of hydrogenase

Iron was determined by the o-phenanthroline method of Massey [9] as well as by energy-dispersive X-ray fluorescence measurements using a new type of spectrometer with totally reflecting sample support [lo]. The content of nickel and other metals in hydrogenase preparations was analyzed by the latter method.

Determination of acid-labile sulfide was conducted by the method of Brumby et al. [ll]. The flavin component of hydrogenase was extracted with trichloroacetic acid [ I 21 and

approximately equivalent amounts of both dimers (1.5 -2 mg each) could be isolated. The effectiveness of the separation procedures was controlled by analytical polyacrylamide gel electrophoresis in the presence as well as in the absence of sodium dodecyl sulfate. Whereas the two dimer preparations used for ESR spectra were slightly ( 5 - 10 %) contaminated with each other, the preparations used for physico-chemical analyses, absorption and fluorescence spectra and for activity measurements were demonstrated to be homogeneous.

qualitatively and quantitatively determined as described by Schneider and Schlegel [ I 31. Content of metals and labile sulfide in preparations

of native hydrogenase and its subunit dimers

Measurement of ESR spectra

ESR spectra were recorded on a Varian E4 ESR spectrometer (Varian Associates, Palo Alto, Ca, USA) with an Oxford Instruments ESR 9 liquid helium flow cryostat (Oxford Instruments, Osney Mead, Oxon, UK).

Redon titrutions

Oxidation-reduction potential titrations were carried out in an apparatus similar to that of Dutton [14] under experi- mental conditions as outlined by Schneider et al. [15].

RESULTS

Preparation of subunit din2er.s of native hydrogenase f rom Nocardia opaca Ib

To isolate the two types of heterodimers which together comprise the NAD-linked hydrogenase of N. opaca 1 b, the whole enzyme was first purified to a homogeneous state and then subjected to preparative polyacrylamide gel electrophoresis at pH 8.5 and in the absence of NiC& or any other metal ion. As already described in the preceding paper [6], under these conditions the two subunit dimers into which hydrogenase dissociates can be completely separated from each other. From 15 mg protein subjected to electrophoresis,

The content of metals in the homogeneous protein preparations (see Table 1) was analyzed by energy dispersive X-ray fluorescence [lo]. The native hydrogenase contained, based on an M , of 178000, 12.4'14.7 iron atoms. Control determinations of iron using the phenanthroline method [9] yielded values in the same region. For preparations of the NAD-linked hydrogenase of Alcaligenes eutrophus HI 6 with very high specific activity (compare [16]) 16 iron atoms/enzyme molecule have been determined by the X-ray fluorescence method [ 171. However, this value was calculated on the basis of an M , of 205000. If we refer the iron content of the A . eutrophus hydrogenase to the M , as estimated from subunit composition (176000) [6] then we obtain, as for the Nocardia enzyme, a value of approximately 14 atoms/enzyme molecule.

The nickel content of the native hydrogenase of N . opaca was determined to be 3.8 & 0.1 atoms/enzyme molecule. This is about twice the amount ofthat determined for the A . eutrophus enzyme. It has however to be mentioned that, throughout the purification procedure, the hydrogenase of N. opaca was dissolved in buffer containing 0.5 mM NiC1, or bound to columns equilibrated with the same buffer [6]. Although all samples used for metal analysis were pretreated with Chelex 100 (I-h incubation in a 0.9 x 10-cm column before elution with 50 mM potassium phosphate, pH 7.0) to remove nickel and nonspecifically bound metals, it cannot be excluded that hydrogenase still contained an excess of non-functional nickel. A treatment of hydrogenase with Chelex 100 longer than 1 h at

77

room temperature led to a decrease of the specific activity. A neutral, rather than an alkaline pH value was chosen to prevent dissociation of the enzyme.

No evidence was obtained for the presence of functional metal other than iron and nickel in active hydrogenase. Of copper, zinc and rubidium only low quantities (0.1 -0.3 atom/enzyme molecule) were detected indicating a nonspecific binding to the enzyme (Table 1). Magnesium, cobalt and selenium were not detectable at all in the hydrogenase preparations.

Metal analyses of the two subunit dimers revealed that the larger dimer (total MI 95000) contained, in accordance with the more intense yellow-brownish colour, the greater proportion of iron, namely 9.6 atoms/dimer molecule (Table 1). For the smaller dimer (total MI 83000) an average value of only 3.9 was determined. As the sum of these amounts almost exactly coincided with the iron content determined for the whole enzyme, it can be concluded that the separation and isolation of the subunit dimers was not accompanied with denaturation or partial denaturation of hydrogenase.

The analysis of the nickel content indicated that nickel is exclusively localized in the smaller dimer. Whereas this dimer contained 1.8 nickel atoms, in the preparation of the larger dimer only a tenth of that amount was found, which is in the conccntration range of the contaminating metals, zinc and rubidium.

The total nickel content of 2 atoms referred to both dimers is in discrepancy with the value of 4 analyzed for the native hydrogenase of N . opaca but is in good agreement with the nickel content of the analogous enzyme of A . eutrophus.

Particularly in the preparation of the small dimer, the content of copper and also of zinc has distinctly increased (0.63 and 0.36 atom/molecule, respectively) compared to the whole enzyme. This is assumed to be due to contaminations resulting from preparative electrophoresis and is therefore not of relevance. The same is true with detectable trace amounts of lead which has not been found in the native hydrogenase.

The labile sulfide contents in the diverse protein preparations were determined to be approximately equivalent to the contents of iron : whole enzyme, 14.3 -t 0.6 S2- ; large dimer, 9.9 0.4 S2 ; small dimer, 4.3 0.4 S2 -.

Quantitative determination and localization of FMN

The existence of a flavin component in the nativc hydrogenase was demonstrated by its characteristic fluorescence emission spectrum with a maximum at 525nm (Fig. 1, curve 1). The flavin extracted from the protein with trichloroacetic acid was identified as FMN by the specific reaction with apoflavodoxin from Megasphaera e$denii. This reaction was accompanied by the quenching of 95 A of the fluorescence (for the theory of this method see [18]).

The quantitative determination of FMN was performed by fluorimetric titration with apoflavodoxin from M . elsdenii [13,19]. The FMN content of homogeneous hydrogenase preparations was somewhat lower than 1 (0.9-tO.5) molecule/molecule. The activity of these preparations was stimulated not more than 10 % by added FMN. In an earlier study of the NAD-linked hydrogenase of A . eutrophus H16, values between 1.1 and 1.4 FMN/enzyme molecule were determined [13]. Because these enzyme preparations were activated by exogenous FMN to 50-80/0, Schneider and Schlegel [I31 discussed the possibility that the intact enzyme contains 2 FMN molecules but that a certain portion of FMN which is

I I I I I

670 620 570 520 L7 Wavelength (nml

Fig. 1. Fluorescence emission spectru of' native hydrogenase and its subunit dimers. The spectra were taken in a Hitachi fluorescence spectrophotometer 204. The excitation wavelength was 430 nm. The proteins were dissolved in 50 mM potassium phosphate, pH 6.5. In the case of the native hydrogenase, the buffer additionally contained 2 mM NiCI,. Curve 1, native enzyme (2.23 mg/ml); curve 2, large dimer (0.89 mg/ml); curve 3, small dimer (1.10 mg/ml)

needed for maximal activity gets lost during preparation. For direct comparison with the Nocardia enzyme and because a maximally active preparation of the A . eutrophus hydrogenase was available, the FMN analysis with this enzyme was repeated in a parallel procedure. Although the specific activity (120 pmol NAD reduced min-' mg protein-') of the A . eutrophus hydrogenase, purified by affinity chromatography [16], was about three times higher than that of preparations used in earlier FMN determinations and although the attivity was not significantly stimulated by added FMN (z 15 /.) we obtained values of the same magnitude as previously. These were 1.1 - 1.2 FMN/hydrogenase molecule when based on an M, of 205000 and 0.95 - 1.03 FMN/hydrogenase molecule when based on an M, of 176000, respectively.

From these results we conclude that the NAD-linked hydrogenases of N . opaca I b and of A . eutrophus H16, each contain only 1 molecule of enzyme-bound FMN. We consider that the activation of the A . eutrophus enzyme by exogenous FMN, whose degree was dependent on the preparation but not strictly on the specific activity, is unspecific and not of functional relevance.

To find out where the FMN is localized, the fluorescence properties of the subunits and subunit dimers was studied. Whereas the smaller dimer was not visibly fluorescent (Fig. 1, curve 3), the larger one with the highcr iron content showed a fluorescence peak at 525 nm coinciding with the peak of the native enzyme (Fig. 1, curve 2). If the latter dimer was subjected to SDS gel electrophoresis and the gel was, after the electrophoresis run, kept under ultraviolet light (366 nm), unambiguously the smaller subunit ( M , 31 000) was the one which exhibited a marked fluorescence and was therefore suggested to contain the bound FMN.

Absorption spectra

The absorption spectrum of the native oxidized hydrogenase (Fig. 2A, curve 1) exhibited a striking similarity

78

1.0

0.8

~ 0.6

n

u C 0

0 in D

L

0.L 4

0.2

0 3 L20 500 580

~

A

3

B

~

I L20 500 580

380 C I 120

I I I

3LO L20 500 580

Wavelength (nm)

Fig. 2. Absorption spectra of'hydrogenusepreparations. The spectra were taken in a Lambda 3 spectrophotometer (Perkin-Elmer). The buffers used were as described for Fig. 1. (A) Spectra of oxidized hydrogenases of N . opacu 1 b (curve 1 ) and A . eutrophus H16 (curve 2). The protein concentration of both enzymes was adjusted to 2.5 mg/ml. (B) Spectra of the Nocardia hydrogenase at different redox states; curve 1 , oxidized enzyme (as prepared under air) ; the protein concentration was 4 mg/ml ; curve 2, partly reduced enzyme, treated with 150 pM dithionite; curve 3, partly reduced enzyme, treated with 210 pM dithionite, curve 4, fully reduced enzyme, treated with 300 pM dithionite. (C) Spectra of the native Nocardia hydrogenase and its subunit dimers; curve 1, native enzyme (2.23 mg/ml); curve 2, large dimer (0.89 mgjml); curve 3, small dimer (1.10 mgjml)

with the spectrum of the soluble A . eutrophus hydrogenase (Fig. 2A, curve 2) with shoulders at 380 nm and 420 nm. Absorption coefficients of 224 mM-' cm-' (28Onm), 61 mM-' cm-' (380 nm) and 56 mM-l cm-' (420 nm) have been determined.

Fig. 2B shows the spectral changes during reduction with dithionite. Whereas the shoulder at 380nm, which is ap- parently due to FMN, disappeared on reduction, the band at 420 nm remained but became broader and was accompanied by a general absorption decrease which, wit! respect to the fully reduced enzyme, amounted to about 20 A. The enzyme was reducible to the same extent with H2 alone, if it was incubated for 1 h under hydrogen gas which was purified by passing through a copper column.

In Fig. 2C the absorption spectra of the single subunit dimers and of the native hydrogenase are compared. The spectrum of the larger dimer was only slightly different from that of the native enzyme. The band at 420nm was more pronounced and an additional weak shoulder appeared at 450 nm (Fig. 2C, curve 2). It is assumed that these maxima arise from a [2Fe-2S] cluster (420 nm) and from FMN (450 nm); both components are, after separation of the second dimer, less masked by additional iron absorption than is the case in the native enzyme. The absorption spectrum of the smaller dimer showed qualitatively a quite diverse profile. It did not have a distinct maximum but was characterized by a very broad absorption band ranging over 360 -460 nm. This spectrum is typical of proteins which contain 4Fe-4S chromo- phores and looks like the absorption spectra described for most of the hydrogenases [20].

ESR properties of native hydrogenase and its subunit dimers

The most characteristic ESR spectra of the native hydrogenase of N . opaca I b are summarized in Fig. 3. The enzyme, as prepared under aerobic conditions gave a low-temperature spectrum (10-20K) with a signal at g=2.01 and a broad downward feature at g = 1.94 (Fig. 3A). The integrated signal intensity corresponded to approximately 1 spin per protein molecule. With the soluble hydrogenase of A. eutrophus a spectrum with similar lineshape has been observed, however with much lower intensity (0.08 spin/protein molecule) [21]. If the Nocardia enzyme was partly reduced by mediator titration down to a potential of + 180 mV, or treated with mercapto- ethanol (6 mM for 2 h), the g = 1.94 feature disappeared, whereas simultaneously the signal amplitude at g = 2.01 increased 2-3-fold (Fig. 3B). A similar conversion of a complex ESR spectrum to a simpler narrow signal at g = 2.01 has recently been also described for the membrane-bound hydrogenases of Chromatium [22] and Alcaligenes eutrophus H16 [15]. A possible interpretation of this phenemenon is that the more complex spectrum is due to the interaction of an iron- sulphur cluster ([4Fe-4SI3+ o r [3Fe-3SI3+) with another paramagnetic species, possibly nickel. If this interaction is removed by reduction of the unidentified component, the Fe-S cluster signal at g=2.01 becomes more prominent. As for the soluble A . eutrophus hydrogenase [21] a specific nickel signal at g values of2.30,2.23 and 2.02 (compare [15,22,23]) was not detected, even if very high concentrated enzyme samples (30 mg/ml) were used. Obviously, in the active state of

79

U ._ - - a 5 - -

9 2.2 2.1 2.0 1.9 1.8 --

!?T>\& !

I I I I I

0.30 0.32 0.3L 0.36 0.38 Magnetic field (TI

Fig. 3. First deriautiae ESR spectra of intact hydrogenuse o f " . opaca. (A) Oxidized enzyme (3.2 mgiml), as prepared, spectrum recorded at 13 K ; (B) enzyme treated with 6 mM mercagtoethanol for 2 hat 20 "C before freezing, recorded at 13 K; (C) enayme reduced with 2 mM dithionite recorded at 40K; (D) reduced eqzyme, recorded at 13K. Other conditions of measurement: microwave power 20 mW, fre- quency 9.18 GHz, modulation amplitude 0.5 mT

hydrogenase, nickel is present in an ESR-silent form and it needs specific conditions, namely the absence of strong magnetic interactions with other paramagnetic species and a certain redox state, to be converted into a paramagnetically detectable form.

In the reduced state the Nocurdiu hydrogenase displayed, dependent on the temperature, two types of ESR spectra (Fig. 3C, D) which had almost identical lineshape and g values to the equivalent spectra of the A . eutrophus enzyme [21]. The high-temperature spectrum (> 40 K) at g = 1.95 and 2.04 was typical of a single iron-sulphur cluster (Fig. 3C). At lower temperatures, the spectrum was morc complex. Additional lines appeared at g=2.004, 1.93 and 1.86 and indicated the presence of a second iron-sulphur cluster (Fig. 3D). From the redox titrations (Fig. 4) it is clear that the component detectable at 40 K, which we presume to be a [2Fe-2S]lf cluster, makes only a small contribution to the signal amplitude at 12 K. The complexity of the spectrum at 12 K cannot be explained in terms of a simple paramagnetic species. Most probably it is due to a weak interaction between two iron- sulphur clusters [21]. From the shape of the spectrum of the reduced protein at low temperatures, the distance between the interacting clusters can be estimated :t 1.2 - 1.5 nm. From ESR spectra of samples treated with 80 A Me,SO (not shown) the presence of both [4Fe-4SI2+ and [2Fe-2SI2+ clusters could be identified. They were differentiated from each other by the characteristic diverse lineshape of their signals (compare Fig. 5 in [21]) and by the temperature dependence which was even more pronounced than that of the signals in the native protein. Double integrations of spin intensity indicated a 2:l relation

L . J

f I J[l

. -100 0 100 200

Redox potential (mV)

Fig. 4. Redox titration curues for the ESR-detectable iron-sulphur clusters in N. opaca hydrogenuse. Points represent the amplitudes of theg=2.01 signal, recorded at 13K (0); g = 1.95 signal, recorded at 53 K (A); g = 1.93 signal, recorded at 15 K (M)

l - v

I I I

0.30 0.32 0.3L 0.36 Magnetic field ( T i

Fig. 5 . ESR spectra o f " . opaca hydrogenase and itssubfractions, in the oxidized state. (A) Small dimer (1.24mg/ml); (B) large dimer (1.20 mg/ml); (C) equimolar mixture of dimers + 2 mM NiCI,, (D) native enzyme (2.50 mglml). The buffers used were as for Fig. 1 and the conditions of measurement were as for Fig. 3

between [4Fe-4S] (measured at 12 K) and [2Fe-2S] (measured at 70 K) clusters.

To determine the midpoint redox potentials of the Fe-S clusters, a redox titration of the described ESR signals was performed using dithionite as reductant and K,Fe(CN), as oxidant (Fig. 4). The midpoint potential of the Fe-S cluster(s) giving rise to the oxidized g = 2.01 signals, was + 25 mV. For the Fe-S clusters which were paramagnetic in the reduced state, midpoint potentials of -285 mV for the [2Fe-2S] cluster, measured at 53 K and -420 mV for the [4Fe-4S] cluster, measured at 15 K were determined.

The ESR study of the subunit preparations revealed interesting differences between the two dimers. High-potential components givingg = 2.01 signals were present in both dimers

80

(Fig. 5A,B) but the signals were remarkably different with respect to signal lineshape, the value of g, the temperature dependence and also the power saturation behaviour. The smaller, light-yellow dimer exhibited a sharp, narrow signal centred at g = 2.024 (Fig. 5A). The signal (measured at 20mW power) was strongest at 12 - 15 K, and the microwave power for 50% saturation, measured at 15 K, was 204 mW. The signal of the larger, dark-yellow dimer was at g = 2.008 and it was of smaller amplitude but broader (Fig. 5B). The temperature optimum of this signal was at 20 -25 K and the power for 50% saturation was only 3 mW. If the two dimers were mixed and 2 mM NiC12 added the resulting signal (Fig. 5C) showed intermediate properties. The broad feature at g = < 2.0, which was not seen in the spectra of the single dimer preparations, reappeared but with much less intensity than with the original native enzyme (Fig. 5D). Since the recombined dimers were completely active (Table 2), this signal is not necessary for enzyme activity.

Of the reduced samples only the dark-yellow dimer showed significant ESR spectra (Fig. 6). These spectra, recorded at low (13 K) and higher temperature (40 K) were qualitatively as

2 2.1 2.0 1.9 1.8 I I I A

-1

x

m L a,

c

a, >

0 (u K

c .-

c .- .- ... -

0.30 0.32 0.3L 0.36

9 2.2 2.1 2.0 1.9 1.8

I I I I I 6

-1

I I I

0.30 0.32 0.3L 0.36 0.38 Magnetic field IT)

Fig. 6. ESR spectra of' N. opaca hydrogenase und its subfructions, reduced with dithionite. ( A ) recorded at 40 K, ( B ) at 13 K. Spectra (1) were of the small dimer, (2) of the large dimer, (3) of the whole enzyme. Protein concentrations and the buffers used were as for Fig. 5. Conditions of measurement were as for Fig. 3C and D

well as quantitatively very similar to the spectra of the native hydrogenase (Fig. 6A,B; curves 2,3 each). This shows that all the Fe-S clusters which are paramagnetic in the reduced state are localized in the larger dimer, and the protein conformation around them is very similar to that in the native enzyme. The ESR spectra of the light-yellow dimer in the reduced state were of low intensity (Fig. 6A, B; curve 1 each). As these spectra, if they were recorded at a higher gain, resembled the spectra of the dark-yellow dimer qualitatively, it is suggested that they are due to a small contamination by dark-yellow dimer molecules. This is consistent with densitometric analyses of polyacrylamide gels showing that the light-yellow dimer preparation used for ESR spectra was contaminoated by the dark-yellow dimer, but by not more than by 10A.

Reactivity of the subunit dimers

To find out which of the partial reactions of the enzyme (reduction of electron acceptors by H2, reduction of acceptors by NADH, H, evolution) takes place in which subunit dimer and thus which of the cofactors and electron carriers are required for which reaction, the reactivities of the native hydrogenase, the single dimers and the reconstituted enzyme were examined and compared with each other (Tables 2 and 3) .

With hydrogen as electron donor, and the specific physio- logical acceptor NAD, the separate dimers were completely inactive. If samples of both dimer preparations were mixed together, before adding to the test cuvette or directly in the cuvette, a hydrogenase activity was achieved which was as high as that of the native enzyme (Table 2). Obviously, the two dimers remain intact during dissociation and isolation, and the whole enzyme can be fully reconstituted spontaneously with- out preincubation or any special treatment. A similar re- activation of hydrogenase activity was observed with ferricyanide. With all other acceptors tested (methylene blue, FMN, benzyl and methyl viologen) hydrogenase behaved differently. The larger dimer alone was totally inactive, but the smaller, nickel-containing dimer reduced the acceptors with high activity. The reaction rates even exceeded the rates of the native enzyme. The most extreme, namely a 48-fold increase of total activity, was obtained with methylene blue. The highest specific activity measured with this acceptor was 3070 U/mg of the small dimer (NAD reduction: 40 U/mg of the native protein). The simultaneous addition of both dimers to the reaction mixture led to a further increase of total activity, the magnitude of which depended on the acceptor (see Table 2).

Table 2. Hydrogenase uctiuity qf the subunit dimers with different electron ucceptors The activity of hydrogenase with electron acceptors was measured photometrically at 365 nm (NAD), 405 nm (ferricyanide), 445 nm (FMN) and 578 nm (methylene blue and viologen dyes). As test buffer 0.5 M potassium phosphate. pH 8.0, was used. The protein concentration of the native enzyme was 2.2 mg/ml, the dimer preparations were adjusted to a concentration of 1.1 mg protein each/ml

Electron acceptor Native enzyme Large dimer Small dimer Large + small dimer ~~ ~~ ~~

total specific total specific total specific total specific activity activity activity activity activity activity activity activity

U U/mg U U/mg U U/mg U U/mg NAD 132 40 0 0 0 0 135 41 Fet ricyanide 287 87 0 0 0 0 264 80 Methylene blue 105 32 0 0 5067 3070 5088 1542 FMN 80 24 0 0 143 86 254 77 Benzyl viologen 377 114 0 0 49 8 302 1710 518 Methyl viologen 113 34 0 0 141 85 894 271

81

Table 3. Diuphoruse activity of the subunit dimers with different electron acceptors The diaphorase activity with electron acceptors was measured photometrically at wavelengths as for hydrogenase activity (see Table 2). Instead of H,, NADH (0.6 mM) was used as electron donor. The atmosphere was N,

Electron acceptor Native enzyme Larger dimer Small dimer Large + small dimer

total specific total specific total specific total specific activity activity activity activity activity activity activity activity

U U/mg U U/mg U U/mg U Uimg Ferricyanide 31 6 96 1040 630 5.3 3.2 1168 354 Methylene blue 93 28 459 278 < 1.0 < 1.0 432 131 FMN 23 I 99 60 < 1.0 < 1.0 82 25 Benzyl viologen 113 34 603 365 0 0 41 6 134 Methyl viologen 17 5 21 16 0 0 79 24

The H,-evolving activity with reduced methyl viologen was, like the H,-uptake activity with various acceptors, exclusively localized in the smaller subunit dimer. The relative activities measured with both dimers, the native and the reconstituted enzyme were found to be in the same ratios as with the reverse reaction, the reduction of methyl viologen (Table 2) . The highest specific activity of H, evolution was reached with the reconstituted hydrogenase and amounted to 300 Uimg protein.

When we examined the capacity of the subunit dimers to reduce the acceptors using NADH instead of H2 as electron donor (diaphorase activity), their reactivity turned out to be just the reverse. Whereas in this case the small dimer showed no, or only traces of activity (ferricyanide, methylene blue, FMN), the large, flavin-containing dimer was highly active (Table 3). By analogy with the H,-dependent reactions, the activities of the single dimer were significantly, for most of the acceptors 3 -6 times, higher than the activities of the native enzyme itself. The highest specific diaphorase activity (630 U/mg of the large dimer) was achieved with ferricyanide. Reconstitution of hydrogenase did not lead to clearly enhanced total activity, which remained roughly on the same level. Generally, the diaphorase activities, except the activity which concerns ferricyanide reduction, were distinctly lower than the hydrogenase activities (compare Tables 2 and 3).

DISCUSSION

Organization and composition of the dimers

The fundamental observation, that the NAD-linked hydrogenase of Nocardia opaca lb , in the absence of NiCl,, at low ionic strength and at alkaline pK values, dissociates into two different types of subunit dimers 161, enabled us to isolate these dimers and to study them individually. Based on the results presented we have developed the following enzyme model (Fig. 7): the hydrogenase of N . opaca is a tetramer which can be considered to be composed of two substantially different dimers.

The small dimer, consisting of subunits with M, of 56000 and 27000, appeared to be in its spectral properties, relatively inconspicious. I t was not fluorescent, it showed a relatively featureless absorption spectrum but I-ihich was characteristic for other hydrogenases (broad band around 400 nm) and it was, like many of the oxygen-stable hydrogenases [23], ESR- silent in the reduced state. The only relevant spectral property of this dimer, and this again seems to be a general characteristic

NATIVE ENZYME

6 4 000 56 000

+N8CI2 I -NiC12

LARGE DlMER SMALL DlMER

C O N S T I T U E N T S C O N S T I T U E N T S

1 FMN 2 NI 1 [2Fe-2S]2* 1 [4Fe-4S13+/ [3Fe-xSI3* 2 [4Fe-4S12’

R E A C T I V I T Y REACTIVITY

D i a p h o r a r e act iv i ty Hydrogenare a c t i v ~ t y

Fig. 1. Enzyme model of the soluble NAD-linked hydrogenase of Nocardia opaca Ib. The dissociation/association process of the two subunit dimers is most specifically affected by NiC12. Other conditions which also influence this process and which are not indicated in the figure, concern the pH value, the presence of metal ions (Co”, Mg” and Mn2 +) and the concentration of moderately chaotropic salts (compare [6])

of most hydrogenases [20,23], was the distinct narrow g=2.01 ESR signal of the oxidized protein which is due to a high- potential three-iron or four-iron cluster. The other component of the small dimer, and perhaps the most significant, is nickel. The presence of two atoms of this metal has been demonstrated per dimer molecule.

The larger dimer consists of subunits with M , of 64000 and 31 000 [6]. With respect to the dark-yellow colour, to absorption and fluorescence properties and to ESR spectra in the reduced state, this dimer strongly resembles the native hydrogenase. This similarity results from a specific combination of chromophoric electron carriers, i.e. of FMN, [2Fe-2S] and

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low-potential [4Fe-4S] clusters, all of which are localized exclusively in the larger dimer (Fig. 7).

Function of the dimers

If we now consider the reactivity of the two hydrogenase dimers we can suggest a plausible relationship between the localization of cofactors and electron carriers on the one hand and of certain activities on the other hand. The smaller, light- yellow dimer showed H,-evolution activity with reduced methyl viologen, and H,-uptake activity with several acceptors, but no NAD reduction and no diaphorase activity. This means that this dimer is the one which contains those components (nickel and an iron-sulphur cluster) which are essential for basic hydrogenase-specific reactions. The larger dimer was not active as a hydrogenase but as a diaphorase. Finally, NAD reduction with H, was only catalyzed if both dimers were combined. These results allow the following conclusions : the binding sites and redox components involved in H, activation and the electron carriers (FMN, [2Fe-2S]), which obviously play a specific role in NAD reduction/NADH oxidation, are distributed on different dimers. As the H2- dependent NAD reduction needs both sets of components, the dissociation/separation of the dimers lead to an interruption of the intramolecular electron transport from H, to NAD which can only be restored by recombining the subunit dimers. From the fact that the two dimers are completely different with respect to their cofactor composition and that they have the capability of catalyzing, independently from each other, quite diverse reactions, the dimers can be considered as two single enzymes. One enzyme, an iron-sulphur nickel protein (small dimer) behaves like a simple hydrogenase, the second enzyme, an iron-sulphur flavo-protein (large dimer) behaves like an NADH dehydrogenase and shows some similarity with the 10w-M~ NADH dehydrogenase isolated from complex I of bovine heart mitochondria [24]. Both enzymes when associated form the complex and multifunctional hydrogenase with its unique property to be able to reduce NAD directly with H,.

Possible functions of the redox centres in the protein

Nickel has already been demonstrated and discussed as the redox-active site of H, activation in several other hydrogenases (for references see [6]). In the Nocardiu enzyme, nick1 appears to have two functions. Two atoms of nickel are loosely bound and are required to hold the two dimers together. Two more are firmly bound to the small subunit dimer and may have a function in the activation of hydrogen. Unlike the nickel atoms in some other hydrogenases [15,25 -281, these are not detectable by ESR.

The iron-sulphur cluster in the small dimer undergoes a reversible redox process, with the g = 2.01 signal disappearing on reduction and reappearing on reoxidation. The cluster giving rise to this signal does not show any ESR signal in the fully reduced state. It might be a [3Fe-xSI3+ cluster, as found in Desuljovibvio desulJuricun.7 hydrogenase [27], or possibly a [4Fe-4SI3 + cluster.

A g=2.01 signal was also obtained in the large dimer. The low intensity of this signal points to the probability that it is due to oxidative damage of a small proportion of the [4Fe-4S] cluster(s). It has recently been reported that such damage, which might be caused by aerobic enzyme preparation, can lead to a conversion of [4Fe-4S] to [3Fe-xS] clusters [29,30].

The midpoint potential of the g = 2.01 signal in the whole enzyme ( f 2 5 mV) is too high for the component to be

considered as an electron carrier to and from hydrogen. We have already considered possible functions for Fe-S clusters giving such a g=2.01 signal in hydrogenases, including a possible regulatory function [23]. If a control mechanism really exists, then an interconversion between the four-iron and the three-iron cluster might be involved as has been described for aconitase [31,32] and discussed for the membrane-bound hydrogenase of Alcaligenes eutrophus HI6 [15]. In aconitase it has been suggested that the Fe-S cluster regulates the activity of the enzyme, whereby [4Fe-4S] represents the active and [3Fe-xS] represents the inactive form. For the NAD-linked hydrogenases of A. eutrophus [2] and N . opacu [6] it has been found, as for most of the hydrogenases studied so far [20,33], that these enzymes are present in an inactive form after aerobic isolation and that their hydrogen- dependent reactions have to pass through a lag phase during which the enzyme is reductively activated. Albracht et al. [28] postulated for the Chromatium hydrogenase that only inactive enzyme molecules contain a [3Fe-xS] cluster ; active molecules, on the contrary, contain a [4Fe-4S] cluster which is not detectable by ESR in some preparations, and gave a complex spectrum in the oxidized state in others. A possible physiologi- cal significance of the interconversion between the cluster types has, however, not been discussed by the authors.

It can be concluded that the larger dimer contains two [4Fe-4SI2 + and one [2Fe-2SI2 + cluster, since a relation between [4Fe-4S] and [2Fe-2S] cluster of 2:l was found from spin-intensity measurements, and for iron and labile sulfur 10 atoms each per dimer molecule have been determined.

In an earlier paper [21], for the NAD-linked hydrogenase of A . eutrophus the [4Fe-4SI2+ clusters, because of their low redox potentials (A . eutrophus hydrogenase, -445 mV; N . opaca hydrogenase, -420 mV), have been postulated to be involved in H, activation. This has now been excluded since the clusters of this type are localized in the dimer which functions as NAD reductase/NADH dehydrogenase but not in the dimer where the H, activation takes place. A plausible scheme of electron transfer from H, to NAD is that electrons obtained from oxidation of hydrogen, possibly at a site containing nickel, are transferred via [4Fe-4S] clusters and then to [2Fe-2S], FMN and finally to NAD. The electron carrier sequence, [2Fe-2S] -+flavin+external acceptor is com- monly found in electron transfer systems that react with NAD or NADP [21,34]. In the case of the soluble A . eutrophus hydrogenase, inhibition of NAD reduction by dicoumarol confirmed that NAD is reduced at the flavin site 1351. Viologen dyes probably react at another site, because their reduction is not inhibited by dicoumarol (K. Schneider, unpublished re- sult). Moreover, the observation that the reduction of viologen dyes by hydrogen (Table 2) is much more rapid than that by NADH (Table 3) suggests that they couple with the [4Fe-4S] cluster(s) rather than with the higher-potential carriers [2Fe- 2SI2+ and FMN. During reduction with NADH, the proportion of reduced [4Fe-4S] clusters will be much lower than the proportion of reduced [2Fe-2S] clusters.

It was a surprising effect that the NAD-independent hydrogenase activities, as well as the diaphorase activities, measured with the separated dimers were higher, in some cases considerably higher, than with the native enzyme itself. One explanation for this phenomenon could be that in the native enzyme the acceptor binding sites are partly masked by close subunit binding and only after dissociation of the dimers are these sites freely accessible. When the whole enzyme was reconstituted, the expected decline of activity did not occur: the diaphorase activities remained at the same level and the

hydrogenase activities were enhanced even further. Therefore the reconstituted protein differed from the original enzyme, although it had equivalent H,: NAD oxidoreductase activity. The difference between the reconstituted and native enLyme was also seen in the ESR spectra of the oxidized proteins (Fig. 5C and D). The change in acceptor specifity could have two reasons. (a) The accessibility of the acceptor sites is not or not only improved by the dissociation of the dimers itself but rather by a conformrtional change which accompanies this dissociation process and which is not reversed after reassociation of the dimers. The change of protein conformation might also lead to more convenient positions of the electron transferring components involved, thus ensuring a more rapid electron flow. (b) The reactions, which the two subunit dimers are able to catalyze, have proved that binding sites for most of the acceptors are present in both dimers. Obviously the hydrogenase, in the native and reconstituted state, can use the intramolecular electron transport chains of both dimers simultaneously to reduce the nonspecific acceptors with H,. Hence, the final transfer of electrons to these acceptors takes place at two sites at least, which of course results in an increase of the reaction rates.

Comparison with other hydrogenases

The majority of the described types of hydrogenases contain two (e.g. the Miyazaki straim of Desuljovibrio vul- garis), three (e. g. Eschevichia coli, Clostridium pasteuri- anum, Desulfovibrio gigus) or more (Proteus mirabilis) Fe-S clusters (compare [20]). The hydrogenase-active dimer of the A . eutrophus enzyme, if regarded as single enzyme, rather belongs to the group of hydrogenases with the most simple structure and the content of only one Fe-S cluster. This raises the possibility that some of the simple hydrogenases that have been isolated are subunits of more complex enzymes. The best investigated representative of this group of enzymes is the hydrogenase of Chromatium vinosum which was recently reported to contain 3.8 atoms of nickel and one [4Fe-4S] ([3Fe-3S]) cluster per 60-kDa polypeptide [28]. From subunit structure the smaller subunit dimer of the Nocardiu hydrogenase resembles the membrane-bound hydrogenase of A . eutrophus. This enzyme is also a heterodimer ( M I = 1 x 67000, 1 x 31 000) [36] but contains, in addition to nickel at least two iron-sulphur clusters [ 151. By immunological comparison it was demonstrated that the isolated dimer of the soluble hydrogenase did not cross-react with the antibodies of the native membrane-bound enzyme indicating that these proteins are not related. Attempts to couple the membrane-bound hydrogenase to the larger, FMN-containing dimer of the soluble hydrogenase in order to achieve NAD reduction, were unsuccessful (K. Schneider, unpublished results).

The F420 (8-hydroxy-5-deazaflavin)-reducing hydrogenase of Methanobacterium thermoautotrophicum strain A H resembles the NAD-linked hydrogenases with respect to many molecular properties. It has a similar complex subunit structure (three distinct kinds of subunits with M , = 40000, 31 000 and 26000, present in a ratio of 2:2:l i and contain also flavin (2 mol FAD/mol of MI= 170000) and approximately two nickel atoms [38]. The question of why some hydrogenases only contain one or less than one nickel atom [15,26,27,37,39] and others two nickel atoms per enzyme molecule has not yet been solved.

The enzyme model developed for the hydrogenase of N . opaca in principle hold true also for the NAD-linked hydrogenase of A . eutrophus. Of course, there are slight

83

structural differences between these two enzymes, which was particularly shown by immunological comparison [6] ; however, the subunit structure, the content of cofactors and electron carriers as well as the spectral properties of both hydrogenases are basically identical.

This work was supported by grants from the Deutsche For- schzingsgemeinschuft and the UK Science and Engineering Research Council. We gratefully acknowledge the cooperation of J . Knoth and H. Schwenke in performing the metal analyses by X-ray fluorescence measurements and of Dr R. Brinkmann in providing cells of N . opacu 1 b. We further acknowledge the skilful technical assistance of K. Jochim, the assistance of Dr D. S. Patil and the assistance of Miss B. Dodemont with the ESR and redox potential measurements.

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K. Schneider and H. G. Schlegel, Institut fur Mikrobiologie der Georg-August-Universitat zu Gottingen, Grisebachstrane 8, D-3400 Gottingen, Federal Republic of Germany

Dr. R. Cammack, Department of Plant Sciences, School of Biological Sciences, King’s College London, 68 Half-Moon Lane, London, England SE24 9JF

content and localization of fmn, fe-s clusters and nickel in the nad-linked hydrogenase of nocardia...

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