445

Biochimica et Biophysica Acta, 578 (1979) 445--461 © Elsevier/North-Holland Biomedical Press

BBA 38187

THE IRON-SULPHUR CENTRES OF SOLUBLE HYDROGENASE FROM ALCALIGENES EUTROPHUS

KLAUS SCHNEIDER a, RICHARD CAMMACK b, HANS G. SCHLEGEL a and DAVID O. HALL b

a Inst i tut fiir Mikrobiologie der Gesellschaft fiir Strahlen- und Umweltforschung mbH and Inst i tut flit Mikrobiologie der Universit~'t G6ttingen, Grisebachstr. 8, D-3400 G6ttingen (F.R.G.) and b Department o f Plant Sciences, King's College, 68 Half Moon Lane, London SE24 9JF (U.K.)

(Received October 9th, 1978)

Key words: Iron-sulfur center; Hydrogenase; ESR; (Alcaligenes eutrophus)

Summary

The soluble hydrogenase (hydrogen:NAD ÷ oxidoreductase, EC 1.12.1.2) from Alcaligenes eutrophus has been purified to homogeneity by an improved procedure, which includes preparative electrophoresis as final step. The specific activity of 57 gmol H2 oxidized/min per mg protein was achieved and the yield of pure enzyme from 200 g cells (wet weight) was about 16 mg/purifica- tion. After removal of non-functional iron, analysis of iron and acid-labile sulphur yielded average values of 11.5 and 12.9 atoms/molecule of enzyme, respectively, p-Chloromercuribenzoate was a strong inhibitor of hydrogenase and apparently competed with NAD not with H2. Chelating agents, CO and 02 failed to inhibit enzyme activity. The oxidized hydrogenase showed an EPR spectrum with a small signal at g = 2.02. On reduction the appearance of a high temperature (50--77 K) signal at g = 2.04, 1.95 and a more complex low temperature (<30 K) spectrum at g = 2.04, 2.0, 1.95, 1.93, 1.86 was observed. The pronounced temperature dependence and characteristic lineshape of the signals obtained with hydr0genase in 80--85% dimethylsulphoxide demon- strated that iron-sulphur centres of both the [2Fe-2S] and [4Fe-4S] types are present in the enzyme. Quantitation of the EPR signals indicated the existence of two identical centres each of the [4Fe-4S] and of the [2Fe-2S] type. The midpoint redox potentials of the [4Fe-4S] and the [2Fe-2S] centres were determined to be --445 mV and --325 mV, respectively. Spin coupling between two centres, indicated by the split feature of the low temperature spectrum of the native hydrogenase around g = 1.95, 1.93, has been established by power saturation studies. On reduction of the [4Fe-4S] centres, the electronspin

446

relaxation rate of the [2Fe-2S] centres was considerably increased. Treatment of hydrogenase with CO caused no change in EPR spectra.

Introduction

Early studies on the enzyme hydrogenase, which is capable of activating and evolving molecular hydrogen, suggested that iron is involved in enzyme catalysis [ 1]. From spectra properties and iron and acid-labile sulphide content of the enzyme from Clostridium pasteurianum, Nakos and Mortenson [2] first identified hydrogenase as an iron-sulphur protein. This has since been con- firmed for hydrogenases of several other microorganisms [3--10]. However, only limited information is available on the chemical nature and number of iron-sulphur centres in hydrogenase. Electron paramagnetic resonance (EPR) spectra were published for hydrogenase from Desulfovibrio vulgaris [3] and Chromatium [6] but not further analyzed. Quantitative extrusion of the iron- sulphur cores from hydrogenases of Desulfovibrio gigas [10] and Cl. pasteurianum [11] indicated that both enzymes probably contain three [4Fe-4S] centres. A more detailed characterization of the iron-sulphur centres by EPR has so far only been presented for Clostridium hydrogenase [11--13]. Under reducing conditions, EPR spectra showed only the equivalent of two centres [12]. At redox potentials more positive than --330 mV a rhombic 'Hipip type ' EPR signal was observed. The site for H2 binding and activation, and the role of the individual iron-sulphur centres in the hydrogenase reaction are still unknown.

The soluble hydrogenase (hydrogen:NAD ÷ oxidoreductase, EC 1.12.1.2) from Alcaligenes eutrophus is the only hydrogenase described which contains a flavin as additional electron-transferring component [7]. Recently Schneider and Schlegel [14] identified the flavin as FMN and provided evidence that hydrogenase contains two FMN/molecule. In this paper we present, in addition to an improved procedure of enzyme purification, a characterization of soluble hydrogenase from A. eutrophus as an iron-sulphur protein including the description of EPR properties and a differentiation of the iron-sulphur centres detected in this enzyme.

Methods

Enzyme purification. Small scale purification was performed according to Schneider and Schlegel [7] with the modification, that the last two steps, Sephadex G-200 and hydroxyapat i te chromatography were exchanged and a second G-200 column has been added as the final step.

Larger scale purifications (starting material: 28--34 g protein extracted from 200 g cells, wet weight) were modified more extensively. After disruption of cells by sonication [7] cell debris was removed by centrifugation at 10 000 X g for 20 min. The supernatant is referred to as crude extract. Depending on the redox state of hydrogenase, 1--5 mM ferricyanide was added to the crude extract to stabilize the enzyme. The crude extract was then centrifuged at 100 000 X g for 1 h and the sediment was discarded. The two following purifi-

447

cation steps (cetavlon treatment and ammonium sulphate fractionation) remained unchanged [7]. The protein precipitate from ammonium sulphate treatment (40--60% saturation) was dissolved in 50 mM potassium phosphate buffer (pH 7.0), dialyzed against the same buffer and then applied to a DEAE- cellulose (Whatman DE 52) column (5 X 60 cm) equilibrated with the dialysis buffer. The protein was eluted with a linear KC1 gradient (0.05--0.35 M) at a flow rate of 90 ml/h. Fractions with a volume of 10 ml were collected. The active fractions were combined and the volume concentrated to 5 ml by ultra- filtration in an Amicon diaflo cell.

The protein solution was then layered on the top of a Sephadex G-200 column (5 X 100 cm), equilibrated with 50 mM potassium phosphate buffer (pH 7.0). After elution of 4-ml fractions the most active fractions were com- bined and concentrated to about 25 mg protein/ml.

Final purification to homogeneity of hydrogenase was achieved by prepara- tive gel electrophoresis in a flat gel apparatus for vertical slab electrophoresis (Panto-Phor, Fa. Mi~ller, Hann.-Miinden), which has been constructed and described by Stegemann [15]. Before use, commercial acrylamide and N,N- methylenebisacrylamide were purified by recrystallization in chloroform and acetone, respectively. The optimal electrophoresis buffer system was found to be Tris/borate, pH 7.9 [16]. For effective separation of proteins not more than 30 mg protein (1--1.5 ml) should be applied to electrophoresis. Since the protein yield after gel filtration was about 80--90 mg, three parallel runs of electrophoresis were carried out. The protein samples were mixed with sucrose (10%), faintly stained with bromophenol blue and layered on the surface of the gel slab. 23 mA and 240 V were then applied to each gel. After 6 h electro- phoresis was stopped, the intensively yellow-brown coloured hydrogenase bands were cut out of the gel and crushed by pressing them through a syringe. The gel particles were suspended in about 100 ml 50 mM potassium phosphate buffer (pH 7.0) and the hydrogenase eluted from the gel by stirring the suspension for 3 h. The hydrogenase solution was separated from gel particles by filtration, then concentrated to about 4 mg protein/ml, dialyzed against 50 mM potassium phosphate buffer (pH 7.0) and stored at --20°C.

Hydrogenase assay. Hydrogenase activity was measured spectrophoto- metrically by following the reduction of NAD [7].

Protein determination. After preparation of hydrogenase by preparative electrophoresis, small amounts of substances in the enzyme solution were found, which originate from polyacrylamide gel [17] and gave positive reac- tions with the Folin as well as with the Biuret reagent, but not with the Coomassie blue reagent. We, therefore, determined protein by the method of Bradford, which uses the latter reagent [18]. It was shown by control experi- ments with protein samples free of acrylamide substances, that the values obtained from the Bradford method were in agreement with those obtained from the method of Lowry et al. [19].

Analysis of constituents of hydrogenase. Iron was determined by the o-phenanthroline method of Massey [20].

Chelex 100, used for removal of nonspecific iron from the protein, was prepared as described by Willard et al. [21].

Acid-labile sulphide was measured by a modification [22] of the method of Fogo and Popowsky [23].

448

Measurement of EPR signals. EPR spectra were recorded on a Varian E4 EPR spectrometer (Varian Associates, Palo Alto, CA, U.S.A.) with an Oxford Instruments ESR 9 liquid helium flow cryostat (Oxford Instruments, Osney Mead, Oxon, U.K.). For numerical analysis, spectra were accumulated on a Nicolet 1020A signal averager (Nicolet Instrument Corp, Madison, WI, U.S.A.) interfaced to a Hewlett-Packard 9830A calculator (Hewlett Packard Corp., Palo Alto, CA, U.S.A.).

Redox titrations. Oxidation-reduction potential titrations were carried ou t as described by Cammack et al. [24]. The method involves titrating the protein, in an apparatus similar to that of Dut ton [25] with Na2S:O4 as reductant and K3Fe(CN)6 as oxidant, in the presence of the following media- tots: Safranine T, methyl viologen, benzyl viologen and triquat, all at 40 pM concentration. The buffer used was 0.1 M Tris-HC1, pH 8.0. After equilibration for 1 min at 25°C, samples were taken and frozen for EPR spectroscopy. The intensities of the various signals were plotted as a function of redox potential, and fitted to a curve calculated from the Nernst equation.

Me2SO treatment of hydrogenase. Treatment of the proteins with dimethyl- sulphoxide was carried out by the procedure of Cammack and Evans [26].

Results

Purification of hydrogenase By following the previously described procedure of purification of soluble

hydrogenase from A. eutrophus (Hydrogenomonas H 16) [7] the average yield of homogeneous enzyme was only 1.5 mg. For the purpose of detailed studies on the content of iron and labile sulfide and on EPR properties of hydrogenase, larger protein amounts were required. However, the intention to reproduce the previous purification procedure on a larger scale, failed. The separation of hydrogenase from two contaminating proteins was not sufficiently effective and in addition, the recovery of total enzyme activity was decreased con- siderably during the first purification steps. We, therefore, elaborated a new improved procedure of hydrogenase preparation which is summarized in Table I.

The earlier assumption, the instability of hydrogenase in crude extracts is

T A B L E I

P U R I F I C A T I O N O F S O L U B L E H Y D R O G E N A S E F R O M A. eutrophus

S t e p T o t a l T o t a l Spec i f i c Purif i - Yie ld p r o t e i n ac t i v i t y ac t i v i t y c a t i o n (%) (mg) (un i t s ) ( u n i t s l m g ( - fo ld)

p r o t e i n )

C r u d e e x t r a c t 28 1 2 0 1 4 4 4 0 0 .51 1 . 0 : 1 0 0 1 0 0 0 0 0 X g s u p e r n a t a n t 1 6 0 1 0 13 2 3 0 0 . 8 3 1 .6 9 2 C e t a v l o n t r e a t m e n t 1 4 0 6 0 1 2 2 0 0 0 .87 1.7 8 4 A m m o n i u m s u l p h a t e f r a c t i o n a t i o n (40---60%) 5 3 7 0 11 0 7 0 2 .06 4 . 0 77 D E A E - c e l l u l o s e 6 2 0 5 9 9 0 9 .68 1 9 . 0 : 4 1 S e p h a d e x G - 2 0 0 8 5 2 9 6 8 3 4 . 9 2 6 8 . 6 21 P r e p a r a t i v e e l e c t r o p h o r e s i s 1 6 9 1 0 5 6 . 8 8 1 1 1 . 5 6

449

due to the presence of reducing compounds [7] was confirmed by the finding that hydrogenase can be stabilized by gassing of crude extracts with oxygen or by addition of ferricyanide [27]. Thus, before starting to purify hydrogenase, we routinely added ferricyanide to the crude extract. The optimal concn, of ferricyanide depended on the redox state of the enzyme and ranged between 1 and 5 mM. The most marked modification of the enzyme preparation was the application of preparative gel electrophoresis which enabled us to dispense with three column steps (DEAE-cellulose at pH 8.2, hydroxyapatite and a second gel filtration on Sephadex G-200). Since hydrogenase is yellow-brown coloured, it could be seen as a sharp single band in the gel slabs of 10 mm thickness. The band of enzyme was cut out and eluted from the gel (see Methods). Analytical electrophoresis in polyacrylamide gel confirmed that the hydrogenase prepara- tion obtained from preparative electrophoresis was homogeneous. The yield of pure enzyme was about 16 mg/purification, ten times as much as in earlier preparations.

Iron and acid-labile sulphide content o f hydrogenase Studies on the iron content of different preparations of soluble hydrogenase

from A. eutrophus confirmed the previously reported data [28] that hydro- genase as purified contains 16--20 iron atoms/molecule. However, after passage of enzyme solutions down a Chelex 100 column the iron content of the preparations decreased about 10% and after treatment with 1 mM o-phenanthroline plus 1 mM Tiron (incubation at 4°C for 20 h followed by dialysis against 50 mM potassium phosphate, pH 7.0) the iron content further decreased to 11.5 atoms/molecule of enzyme, although no loss of catalytic activity was observed and the content of acid-labile sulphide was the same before and after treatment with the chelating agents (Table II). From four different experiments an average value of 12.9 mol/mol of enzyme was calculated for acid-labile sulphide. These results suggest (1) that hydrogenase when freed from non-functional iron contains 12 iron as well as 12 labile sulphur atoms/ molecule and (2) that higher iron values estimated for the untreated enzyme, result from iron which is not essential for catalytic activity but might come from growth medium and buffers and bind nonspecifically to the protein. Very similar observations were recently made with hydrogenase preparations from D. vulgaris [5] and have been reported earlier also for other iron-sulphur proteins [29]. The iron atoms removed from inhomogeneous hydrogenase

T A B L E II

I R O N A N D A C I D - L A B I L E S U L P H I D E C O N T E N T O F H Y D R O G E N A S E P R E P A R A T I O N S

A h o m o g e n e o u s e n z y m e preparat ion ( 0 . 7 8 m g / m l ) obta ined after preparative e lectrophores i s was used. The specif ic act ivity w a s 51 U / r a g prote in .

E n z y m e preparat ion Iron c o n t e n t Sulfide conten t ( a t o m s / m o l ) ( m o l / m o l )

Untreated 1 8 . 5 Treated wi th Chelex 1 0 0 16 .7 Treated wi th o -phenanthro l ine + T i r o n (1 m M e a c h ) 1 1 . 5

12.6 13.1 12.5

450

preparations of C. pasteurianum by treatment with o-phenanthroline were first interpreted as 'extra iron' required for functions such as participation in structural features [30], however, were later found to be constituents of a con- taminant protein [31].

Inhibitors of hydrogenase Hydrogenase activity was strongly inhibited by the heavy metals mercury,

cadmium and copper and by organic mercurials such as p-chloromercuribenzo- ate (Table III), which are known to complex with sulphydryl groups, and also with sulfide groups releasing the iron of iron-sulphur centres. The percentage of inhibition by p-chloromercuribenzoate did not change with varying H2 con- centrations (Fig. la) but it was higher, the lower the NAD concentration (Fig. lb ) , suggesting that p-chloromercuribenzoate is competitive with NAD and non-competit ive with H2. However, as the degree of inhibition was time dependent and NAD only prevented inhibition bu t was not able to reverse it, this type of inhibition did not fit the definition of reversible competitive inhibition. At 0.8 mM NAD the p-chloromercuribenzoate required for 50% inhibition was 20 pM. The data obtained allow us to conclude (1) that at least one SH group is involved in the binding of NAD and (2) that iron-sulphur centres as sites of H2 activation and electron transfer are not affected by organic mercurials. The latter statement was manifested by the observation, that t reatment of hydrogenase with p-chloromercuribenzoate or mersalyl did not result in a loss of absorption in the iron-sulphur chomphore region.

CO and chelating agents failed to inhibit hydrogenase; Tiron, o-phenanthrol- ine and EDTA even stimulated activity 20--50%. Only preincubation of hydro- genase with the reagents at 4°C over a longer period than 20 h initiated the release of functional iron and thus the process of inactivation.

The influence of oxygen on enzyme activity was also tested. We were able to demonstrate that soluble hydrogenase is not only highly stable during storage un- der air [7] but is extraordinary 02 tolerant also under reaction conditions. Equi-

T A B L E I n

E F F E C T OF I N H I B I T O R S ON H Y D R O G E N A S E A C T I V I T Y

Inhib i tor C o n c e n t r a t i o n Inhib i t ion ( raM) (%)

Mercur ic ch lor ide 0 .0027 50 0 .001 100

Copper chlor ide 0 . 0 0 4 5 50 0.2 100

C a d m i u m su lpha te 0 .012 50 1 100

p - C h l o r o m e r c u r i b e n z o a t e 0 . 020 50 0 .15 100

S o d i u m i o d o a e e t a t e 1 10 T i ron 1 0 o -Phenan th ro l ine 1 0 E D T A 1 0 CO 0.54 0 (}2 0.7 18

4 5 1

80

6C g

~ 4c

2C

Q

io I I I 1 20 30 40 [p-CMB] (~a)

,po b

I I I I 5 10 15 20

[ p - C a B ] (IJM)

Fig. 1. I n h i b i t i o n o f h y d r o g e n a s e b y p - c h i o r o m e r c u r i b e n z o a t e . T h e r e a c t i o n m i x t u r e (3 ml) c o n t a i n e d 50 m M Tr is -HCl b u f f e r ( p H 8 .0 ) , 5 #g p r o t e i n a n d t h e f o l l o w i n g c o n c e n t r a t i o n s o f H 2 a n d N A D : (a) 0 . 1 1 3 m M H 2 , 0 . 8 m M N A D (4) ; 0 . 2 5 7 m M H 2 , 0 . 8 m M N A D (o) ; 0 . 4 5 m M H 2 , O.S m M N A D (e) ; (b ) 0 . 4 5 rnM H 2 , 0 . 2 6 m M N A D (~); 0 . 4 5 m M H 2 , 0 . 5 raM N A D (m); 0 . 4 5 raM H 2 , 0 .S m M N A D (o) . The c o n c e n t r a - t i o n o f p - c h l o r o m e r c u r i b e n z o a t e w a s va r i ed as i n d i c a t e d in t h e f igure .

libration of the reaction mixture with gas containing 80% H2 and 20% 02 caused no detectable inhibition of enzyme activity. Oxygen concentrations up to 0.7 mM, corresponding to a partial pressure of 60% diminished the activity by only 17--19%.

EPR spectra Of oxidized and reduced hydrogenase Five different preparations of hydrogenase were used for EPR measure-

ments, with puri ty between 25% and 100% including enzyme purified only by column chromatography and enzyme purified by a procedure that included preparative electrophoresis (see Table I). All preparations gave essentially the same EPR signals, which were commensurate with their enzymic activities. This was as expected because already after chromatography on DEAE-cellulose hydrogenase was the only visible coloured protein and thus even the less pure enzyme preparations were not contaminated with other iron-sulphur proteins, flavoproteins or cytochromes which might have EPR signals.

In the unreduced form of hydrogenase as obtained after aerobic preparation, a spectrum was observed at low temperatures {below 20 K), consisting of a sharp upward feature at g= 2.02 and a broad downward feature around g = 1.92 (Fig. 2a). The former feature was slightly increased by t reatment with K3Fe(CN)6 showing that it was an oxidized species, bu t quantitatively this spectrum w a s small, with an integrated intensity less than 8% of the signals observed in the reduced state. It is, therefore, clear that the resonance at g = 2.02 is not due to a high potential iron-sulphur centre bu t rather represents a higher oxidation state of a low potential centre, a phenomenon which is of ten seen in proteins with [4Fe-4S] centres [32].

To achieve optimal reduction of hydrogenase, the effects of different reducing agents were examined. Treatment with NADH (2 mM) induced a free radical signal at g = 2.003, apparently due to the flavin component (FMN) of hydrogenase, and a signal which is small but typical o f reduced iron-sulphur centres (Fig. 2b). After incubation of hydrogenase under molecular hydrogen, the physiological reducing substrate, the enzyme was not reduced and the EPR

452

dX '_~' dH

g -Value 2 2 2 0 it.~l ll,~

I

Q30 32 .34 0.36

Mognetic field (~T)

Fig. 2. E P R s p e c t r a of p u r e so lub l e h y d r o g e n a s e (3 .8 m g / m l ) f r o m A. eutrophus r e c o r d e d a t 1 2 K. (a) O x i d i z e d e n z y m e as p r e p a r e d ; (b) r e d u c e d w i t h 2 m M N A D H ; (c) r e d u c e d w i t h 20 /~M N A D H u n d e r h y d r o g e n . S p e c t r a w e r e r e c o r d e d w i t h the f o l l o w i n g i n s t r u m e n t settings: microwave p o w e r 20 m W , frequency 9 . 2 5 G H z ; m o d u l a t i o n a m p l i t u d e , 1 m T ; f r e q u e n c y 1 0 0 k H z .

spectrum was unchanged when compared to the spectrum of the oxidized enzyme, presumably because of the effect of reversibly bound oxygen. Or/ addition of a small amount of NADH (20 pM), however, the protein was reduced by H2 giving a complex signal centred at g = 1.95 (Fig. 2c).

Reduct ion by dithionite produced a similar spectrum to Fig. 2c. Although reduction was slow, requiring several minutes at 20°C, the intensity of signals was stronger probably because dithionite has a lower redox potential than hydrogen [33]. The dithionite-reduced enzyme was, therefore, used for further characterization of the spectrum.

The first indication for the existence of more than one iron-sulphur centre in hydrogenase was obtained by the finding that the lines]~ape of the EPR spectrum of the reduced protein and the number of g values were distinctly dependent on temperature. At relatively high temperature, 50--77 K, the spectrum was typical of a single reduced iron-sulphur centre, at g = 2.04, 1.95

2.1

g-Volue

2.0 I

1.9 1.8

453

JP (3.

(

i i I i I ' l~34 i I I , L 032 036

Magnetic field ( T )

Fig. 3. E P R s p e c t r a o f h y d r o g e n a s e , r e d u c e d w i t h 5 m M N a 2 S 2 0 4 f o r 2 m i n b e f o r e f reez ing . Spec tra w e r e r e c o r d e d (a) at 5 5 K ; (b) a t 1 2 K. Other c o n d i t i o n s as for Fig. 1.

(Fig. 3a). We will refer to this centre as Fe-S I. At lower temperatures, below 30 K, the spectrum became more complex as additional features at g = 2.004, 1.93 and 1.86 appeared. As the temperature was progressively lowered, the signals became saturated with microwave power, and the features of the new spectrum became more prominent (Figs. 2c and 3b). We will refer to the com- ponent giving rise to this signal as Fe-S II.

In order to obtain information on interaction of the centres with H2 and to find ou t whether the split spectrum at low temperature is due to an exchange- able proton, the protein was transferred to 98% deuterium oxide. However, in accordance with observations made with Clostridium hydrogenase in analogous experiments [ 13 ] the EPR spectra were not significantly altered.

In none of the EPR spectra measured were any signals detected which could be at tr ibuted to molybdenum.

Midpoint redox potentials of centres Fe-S I and Fe-S H The centres giving rise to signals Fe-S I and Fe-S II could be distinguished

not only by their temperature dependence but also by their different midpoint redox potentials. Hydrogenase was t i trated reductively with dithionite and retitrated oxidatively with ferricyanide. Fig. 4 shows the size of the EPR signals as a function of the redox potentials at which the samples were poised before freezing. At 50 K the sginal of Fe-S I behaved as a single one-electron acceptor

454

."z_

<

E <

co _

I I

- 6 0 0 - 5 0 0 - 4 0 0 - 3 0 0 - 2 0 0

R e d o x P o t e n t i a l (mY)

I i I I I

-~oo o ~;o

Fig. 4. Midpo in t r edox po ten t ia l s of the ixon-sulphur cen t res of hydrogenase . The r ed o x t i t r a t ion was p e r f o r m e d in 0.1 M Tris-HCl, pH 8.0, w i th N a 2 S 2 0 4 as r e d u c t a n t , K 3 F e ( C N ) 6 as o x id an t and saf ranin T, m e t h y l v io logen, b e n z y l v io logen and t r i qua t as med ia to r s . A peak- to-d ip a m p l i t u d e at g = 1.95, r e c o r d e d a t 55 K; o, the same signal, m e a s u r e d at 12 K.

with midpoint potential --325 mV at pH 8.0. At 12 K the signal at g = 1.95 consisted of a small contribution from Fe-S I (highly saturated) and of Fe-S II, which gave an apparent potential of about --445 mV. These titrations were carried out under argon atmosphere, and a complication at low potentials was caused by the expected production of hydrogen. Hence at pH 8 the potential could not be held steady below about --440 mV. To measure the maximum signal from fully reduced Fe-S II, a sample was taken and fully reduced with dithionite + bipyridyl dyes.

The EPR spectra of the protein samples from redox ti tration yielded no evidence for the existence of an additional Fe-S centre which differs from centre I and II. The diverse peaks of signals appeared and increased synchro- nously.

Chemical nature o f centres Fe-S I and Fe-S H Influences of protein conformation can cause considerable alterations in

lineshape and g values of EPR signals. Therefore, to determine whether the iron-sulphur centres of hydrogenase are of the [2Fe-2S] or [4Fe-4S] type, the enzyme was treated with dimethylsulphoxide as described by Cammack and Evans [26]. The principle of this method is, that the presence of high concen- trations of Me2SO unfolds the protein and removes the constraints on the centre, which then show spectra more characteristic for the type of centre. The ability of the Me2SO method, to be able to distinguish between the two types of iron-sulphur centres in one and the same protein and in the presence of a flavin chromophore, has now been demonstrated for the first time with the soluble hydrogenase of A. eutrophus.

When hydrogenase was reduced in 85% (v/v) Me2SO the type of a two-iron

455

~ X *f

d H

g - V a l u e

2.1 2 . 0 1.9 1 .8 i I i i

I J I I I I | I l t I I I 0 3 2 Q 3 4 0 3 6

Megnet ic field ( T )

F ig . 5. E P R s p e c t r a o f i z o n - s u l p h u r p r o t e i n s in m i x t u r e s o f M e 2 S O w i t h a q u e o u s 0 .1 M Tr is -HC1, p H 9 . 5 , r e d u c e d w i t h 2 m M d i t h i o n i t e . (a ) A. e u t r o p h u s h y d r o g e n a s e in 8 5 % M e 2 S O r e c o r d e d a t 55 K; ( b ) s p i n a c h f e r t e d o x i n in 8 0 % M e 2 S O , r e c o r d e d a t 50 K; (c ) A. e u t r o p h u s h y d r o g e n a s e in 8 5 % M e 2 S O , r e c o r d e d a t 1 6 K ; ( d ) C. p a s t e u r i a n u m t w o [ 4 F e 4 S ] f e ~ e d o x i n i n 8 0 % M e 2 S O , ~ e c o z d e d a t 1 6 K . O t h e r c o n d i t i o n s o f m e a s u r e m e n t as f o r Fig. 1.

centre (centre Fe-S I) and the type of a four-iron centre (centre Fe-S II) could be clearly differentiated by the typical symmetry and temperature dependence of their respective EPR signals. At 55 K a signal at g < 2.0 was observed (Fig. 5a), which is very similar to the spectra of simple [2Fe-2S] proteins in Me2SO. The spectrum of spinach ferredoxin in 80% Me2SO [26] is given in Fig. 5b for comparison. As the temperature of measurement was decreased, this first signal became saturated with microwave power, and a second axial signal at g = 2.04, 1.93 appeared. This spectrum was clearest at about 16 K, when the spectrum of the first species was saturated out (Fig. 5c). It strongly resembles the spectrum of [4Fe-4S] proteins in Me2SO, such as C. pasteurianum ferre- doxin (Fig. 5d) as well as the spectrum of reduced synthetic tetranuclear iron- sulphur analogues [34].

To find out the minimum Me2SO concentrat ion required for complete protein denaturation and to compare the different responses of centres Fe-S I and Fe-S II to protein unfolding, hydrogenase was t i trated at 0°C with

456

dH

g -Value

2.1 2i0 , l l9 , 1L~

032 Q34 036

Magnetic field (T)

I I I I I I Q32 034 0.36

Megnetic field ( T )

g -Value

2.1 2.0 1.9 1.8

dx -

dH

Fig. 6. M e 2 S O t i t r a t i o n o f h y d r o g e n a s e . E P R spectra were m e a s u r e d o f h y d t o g e n a s e in m i x t u r e s o f di f f e ren t c o n c e n t r a t i o n s of M e 2 S O w i t h a q u e o u s 0 .1 M Tris-HCl, p H 9 .5 , r e d u c e d w i t h 2 m M d i t h i o n i t e ; (a) spectra r e c o r d e d a t 2 0 K; (b) s p e c t r a o f t he s ame s a m p l e s r e c o r d e d a t 5 0 K.

increasing concentrations of Me2SO and reduced before preparing frozen samples. The samples have been measured for EPR at temperatures (20 and 50 K) where the spectra of the native enzyme were not dissimilar but where the spectra of the unfolded protein were substantially different (Fig. 6). The low temperature signal of denatured hydrogenase needed 70% Me2SO to be produced (Fig. 6a) whereas the spectrum of the two-iron centre changed to the unfolded type only in 80% Me2SO. Some lineshape changes were observed even at low (10%) Me2SO concentrations. Parallel tests of activity revealed, that in the presence of 10% Me2SO hydrogenase activity was not decreased bu t was actually stimulated by about 10%. These effects on EPR spectra and catalytic activity probably reflect 'subtle' changes in the environment of the iron-sulphur centres, as seen in other proteins [35]. At increased Me2SO concentration hydrogenase was rapidly inactivated. Complete inactivation of the reduced enzyme occurred within 2 min in 50% Me2SO.

Interaction between centres Fe,S I and Fe-S H If two paramagnetic centres are present in a protein, a magnetic interaction

457

may cause changes in the EPR spectrum. If the interaction is strong, the spectra may be broadened or split by exchange and dipolar interactions, producing a composite spectrum. An example of this is in the two [4Fe-4S] ferredoxins in which the iron-sulphur centres are 1.2 nm apart [36] which show a complex spectrum in the fully reduced state. A smaller degree of interaction may not produce significant lineshape changes, but cause an increase in the electron spin relaxation rate, which can be observed by power saturation studies.

In the case of soluble hydrogenase from A. eutrophus, there was little evidence of splitting of the spectrum of Fe-S I at high temperatures. At 50 K the lineshape of the signal was the same in samples poised at potentials such that Fe-S II was mostly oxidized or mostly reduced. However, the electron spin relaxation rate of Fe-S I was considerably increased on reduction of Fe-S II. Fig. 7 shows the microwave power saturation curves for Fe-S I plotted as log (Signal/x/Power) vs. log(Power) according to Beinert and Orme-Johnson [ 37 ]. The power for half-saturation P1/2, a measure of the electron spin relaxation rate, was calculated as described by Rupp et al. [38]. The g = 1.95 feature was measured at 40 K, a temperature at which the signal of Fe-S II is almost com- pletely broadened out. It can be seen that in the sample poised at --372 mV (Fe-S I reduced, Fe-S II oxidized) the signal was significantly saturated with PI/2 = 7 mW. In the sample reduced with mediators + dithionite (both Fe-S I and Fe-S II reduced), the signal of Fe-S I was much less saturated with P1/2 = 204 mW.

It is likely, therefore, that even at very low temperatures the signal of Fe-S I is not completely saturated out. This may account for the split feature of the

~L o

I I I 1 10 1 0 0

M i c r o w a v e p o w e r (mW)

Fig. 7. M i c r o w a v e p o w e r s a tura t i on o f t h e g = 1 .95 signal d u e to t h e Fe-S I , meatLred at 4 0 K. A e n z y m e f u l l y r e d u c e d w i t h Na2S 2 0 4 + m e d i a t o r s ; s , e n z y m e p o i s e d at - - 3 7 2 m V , so t h a t Fe-S I I was ap p ro x . 95% oxid ized . D a t a are p l o t t e d so t h a t a h o r i z o n t a l line r ep resen t s an u n s a t u r a t e d signal. S a t u r a t i o n is indi- c a t ed b y d o w n w a r d curvature o f t h e line.

458

complex signal at 12 K around g = 1.95, 1.93 (Figs. 2c and 3b). The possibility, however, that this signal represents two spin-coupled four-iron centres cannot be excluded.

Quantitation o f the EPR signals Double integrations of spin intensity were carried out at various tempera-

tures, using Cu(II)-EDTA as standard. Integration of the signal of Fe-S I at 50 K gave a value of 1.3 spins/molecule, while the complex signal at 12 K integrated to 3--4 spins/molecule. Since any spin coupling would be expected to decrease the apparent intensity it seems likely that in total four iron-sulphur centres exist in hydrogenase, i.e. two [2Fe-2S] and two [4Fe-4S] centres. As the two single centres of each type are not distinquishable by EPR and by their redox potentials they appear to be identical.

EPR spectra o f hydrogenase after CO treatment In the past, carbon monoxide has repeatedly been shown to inhibit hydro-

genase in many organisms and was, therefore, considered as a general inhibitor of hydrogenase [1]. Recently Erbes et al. [13] found that CO is a strong com- petitive inhibitor of hydrogenase from C. pasteurianum with respect to H2 and causes fundamental changes in the EPR spectra of the enzyme. By contrast, when soluble hydrogenase of A. eutrophus was treated with CO, either before or after reduction, there was no change in EPR spectra and no effect on enzyme activity (compare Table III).

Discussion

Soluble hydrogenase of A. eutrophus was earlier shown to be an iron-sulphur protein by typical absorption spectra [7] and the content of iron and acid- labile sulphide [28]. The enzyme has now been conclusively identified as an iron- sulphur protein of the ferredoxin type by the most reliable diagnostic criterion, the classical EPR signal around g = 1.94 [39]. As it has been recently demon- strated that hydrogenase contains, in addition to iron-sulphur centres, FMN as enzyme-bound electron carrier [14], this enzyme has to be assigned to the group of 'conjugated iron-sulphur proteins' which are defined as Fe-S proteins containing flavin, molybdenum or other electron-transferring groups [39]. Until now soluble hydrogenase from A. eutrophus is the only hydrogenase described to be conjugated with a flavin component . The reason apparently is the specific involvement of FMN in the reduction of NAD. Other iron-sulphur proteins, which also contain FMN as coenzyme, are, like hydrogenase ('H2 dehydrogenase') complex pyridine nucleotide-dependent dehydrogenases. These are NADH dehydrogenases from different organisms [40] and formate dehydrogenase from Pseudomonas oxalaticus [41]. Equimolar quantities o f FMN and FAD in the same iron-sulphur protein have been reported for dihydroorotate dehydrogenase from Zymobacterium oroticum [42] and for NADPH sulfite reductase of different origin [40].

With respect to the composit ion of iron-sulphur centres hydrogenase turned out to represent a new type of iron-sulphur protein containing the [2Fe-2S] as well as the low potential [4Fe-4S] type of iron-sulphur centre. The two

459

types of centres were distinguished unambiguously by their redox potentials and by the lineshape and temperature dependence of their ERP signals which were produced after unfolding of hydrogenase in 80--85% Me,SO. Of the com- plex iron-sulphur proteins so far analyzed succinate dehydrogenase is the only one for which the simultaneous existence of the [2Fe-2S] and the [4Fe-4S] type of centre has been postulated [43]. However, in the case of succinate dehydrogenase the [4Fe-4S] centre is of the 'Hipip type ' (Era = + 60 mV), only detectable by EPR in the oxidized form and, therefore, different from the [4Fe-4S] type of centre in hydrogenase.

From FMN analysis [ 14] present iron and labile sulphide determinations and calculation of the spin content of hydrogenase the ratio of the three different detected species of electron-transferring compoents , FMN, [4Fe-4S] and [2Fe-2S] is expected to be 2 : 2 : 2. Conclusive elucidation of the number and localization of these components will be achieved after separation and individual s tudy of the different subunits of hydrogenase. The enzyme consists of four subunits, three subunits of which are non-identical. They have molecular weights of 68 000, 60 000 and 29 000 and are present in a 1 : 1 : 2 molar ratio (Schneider, K., manuscript in preparation). Another open question is the sequence of the redox components in electron transfer. The four-iron centres have a midpoint redox potential (Era = - - 4 4 5 mV) which is at the potential level of hydrogen and it, therefore, might be that these centres are responsible for H2 activation and for acceptance of the hydrogen electrons, respectively. The two-iron centres with a redox potential of E m = - - 3 2 5 mV probably play a role in the mediation of electrons to NAD. Although the redox potential of the FMN and its position in electron transfer is not ye t known, the sequence [2Fe-2S] ~ flavin # NAD(P), which is employed in systems as diverse as chloroplast Photosystem I [44], steroid [45] and camphor hydroxylases [46] and xanthine dehydrogenase [47] seems to be the most probable one.

Relatively few and conflicting data exist on the content of iron and acid- labile sulphur and on EPR characteristics in hydrogenases of other organisms. For the enzyme of C. pasteurianum Erbes et al. [13] determined 4--5 atoms of iron and corresponding amounts of labile sulphur/molecule, Chen et al. [12] about 12 atoms for each element. The latter value can be considered to be the more reliable one because it is in agreement with the results of quantitative extrusion experiments [11]. It is no tewor thy that the hydrogenases of A. eutrophus and C. pasteurianum in spite of their corresponding iron-sulphur content , are completely different with respect to EPR properties and the com- position of their iron-sulphur centres. No temperature dependence has been described for the EPR spectrum of the Clostridium hydrogenase bu t with varying redox potentials it undergoes a series of changes [12]. The fully reduced enzyme exhibits a spectrum with g values at 2.08, 1.96, 1.89 and some additional fine features. Around --360 mV a complex spectrum of many weak signals is induced and at potentials more positive than --330 mV a typical rhombic signal of the Hipip type with g values greater than 2.0 appears. In con- trast, with the soluble hydrogenase from A. eutrophus we observed with varying temperature the sequential occurrence of two markedly different signals of the low potential Fe-S centre type, however, no significant changes in EPR spectra or the appearance of further distinguishable signals during redox titration. Con-

460

sequently, whereas the Alcaligenes hydrogenase appears to contain both four- iron and two-iron centres, most probably two of each, it is expected from EPR and established by extrusion studies, that in the Clostridium hydrogenase only four-iron centres are present, at least two, but rather three, one of which exists under reducing conditions in an EPR-silent form [11,12].

Recently 12 atoms/molecule for both iron and labile sulphur have been also reported for the hydrogenases of D. vulgaris [5] and D. gigas [10] and for the enzyme of the latter organism indication has been obtained from quantitative extrusion of the Fe-S centres, that three [4Fe-4S] centres are present in one molecule [10]. The results of EPR studies on Desulfovibrio hydrogenase are still of preliminary character. With a reduced D. vulgaris enzyme preparation which contained not more than 3.5 iron atoms/molecule LeGall et al. [3] observed a signal at g = 1.86. On the other hand with the 12-iron preparation of D. gigas only a Hipip signal at g ~ 2.0 was detected, however, the spin content calculated from this signal was lower than one spin/mol of hydrogenase (Hatchikian, C. and Cammack, R., unpublished results).

Besides the group of soluble hydrogenases containing 12 atoms each of iron and labile sulphur/molecule, membrane-bound hydrogenases from the photo- synthetic bacteria Chromatium [6], Rhodospirillum rubrum [8] and Thiocapsa roseopersicina [9] have been reported to contain four iron a toms/enzyme molecule. Gitlitz and Krasna [6] presented an atypical EPR spectrum of the reduced enzyme of Chromatium with a signal at g = 2.2 and 2.06. Adams and Hall [8] did not detect any EPR signal from the reduced form of hydrogenase from R. rubrum. The oxidized protein, however, gave a spectrum, which is characteristic of a Hipip-type [4Fe-4S] centre.

Acknowledgement

The cooperat ion of Dr. R. Brinkman in providing ample amounts of auto- trophically grown cells is gratefully acknowledged.

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