Nickel and iron-sulphur centres in Desulfovibrio gigas hydrogenase: ESR spectra, redox properties and interactions

98

Biochimica et Biophvsica Acta 912 (1987) 98-109 Elsevier

BBA 32665

N i c k e l a n d i r o n - s u l p h u r c e n t r e s in

Desulfovibrio gigas

hydrogenase:

ESR spectra, redox properties and interactions Richard Cammack

÷, D a u l a t S. P a t i l *, E. C l a u d e H a t c h i k i a n ** a n d V i c t o r M . F e r n f i n d e z ***

King's College, Department of Plan t Scwnces, London (U. K. ) (Received 23 June 1986)

Key words: Hydrogenase: Nickel center: Iron-sulfur cluster: ESR: ( D. gigas)

Some properties of a nickel species (Ni-C) in Desulfovibrio gigas hydrogenase (ferredoxin:H + oxidoreductase, EC 1.18.99.1), which is associated with the activated state, are described. At temperatures above 20 K this species yields an ESR spectrum at g--2.19, 2.16, 2.01, but at lower temperatures the spectrum changed into a fast-relaxing species with a complex lineshape. Substitution of the enzyme with 61Ni shows that the complex spectrum is associated with nickel. The splitting was correlated with the presence of a broad ESR signal which probably originates from the reduced [4Fe-4S] clusters. It did not correlate with the reduced 13Fe-xS] cluster. These results indicate that the complex spectrum is due to splitting of the Ni-C spectrum by spin-spin interaction with the [4Fe-4S] cluster or clusters. The Ni-C species was light-sensitive in frozen samples, and underwent a change in spectrum which was reversed in the dark at temperatures above 200 K. Treatment of the enzyme with carbon monoxide in the presence of hydrogen induced a new type of ESR signal, which disappeared on removal of either hydrogen or carbon monoxide. The Ni-C species represents an intermediate oxidation state of the enzyme. The midpoint redox potentials, estimated by mediator titrations under controlled hydrogen/argon gas mixtures, were shown to be strongly pH-dependent. The values at pH 7.0 were estimated to be - 2 7 0 mV (-120 m V / p H unit) for the appearance of the Ni-C ESR signal and - 3 9 0 mV ( - 6 0 m V / p H unit) for its disappearance. The midpoint potential of the broad ESR signal was estimated to be -350 ( - 60 m V / p H unit). Possible schemes for redox states of the various nickel species are discussed.

+ Department of Biochemistry, King's College (KQC), Campden Hill Road, London W8 7AH, U.K. * Physics Dept, Emory University, Atlanta, GA30322, U.S.A. ** Laboraloire de Chimie Bactdrienne, CNRS, BP 71, 13277 Marseille, Cedex 9, France. *** Instituto de Catfilisis del CSIC, Serrano 119, Madrid, Spain. Correspondence: R. Cammack, Department of Biochemistry, King's College (KQC), Campden Hill Road, London W8 7AH, U.K.

Introduction Hydrogenase from Desulfovibrio gigas has a high content of iron and labile sulphide (approx. 12 atoms of each) as well as approx. 1 nickel atom per molecule of 89 kDa [1-3]. The iron and sulphide appear to be arranged as two [4Fe-4S] and one [3Fe-xS] clusters [4]. In the enzyme as isolated, two types of ESR signals have been observed: a narrow signal centred at g = 2.02, and

0167-4838/87/$03.50 J~'J~1987 Elsevier Science Publishers B.V. (Biomedical Division)

99

detected only at low temperatures, which is probably due to an oxidized [3Fe-xS] cluster [4]; and signals which have been identified by isotopic substitution as due to nickel [5]. Of these the most prominent is Ni-A, at g = 2.32, 2.23, 2.01 (Fig. la). A second signal, Ni-B, at g = 2.34, 2.16, 2.01 is present as a minor species (Fig. lb). The relative amounts of these nickel signals can be altered by reduction and reoxidation [6,7]. Similar ESR signals due to nickel have been observed in some, but not all, nickel-containing hydrogenases [8]. A compilation of different types of ESR signal derived from nickel in D. gigas hydrogenase is presented in Fig. 1. MOssbauer spectroscopic studies of D. gigas hydrogenase indicate that at least one of the two [4Fe-4S] centres is reducible by treatment with hydrogen [6]. A corresponding decrease in optical absorption is also observed [7,9]. However, ESR signals from these clusters are difficult to observe, in either their oxidized or reduced states. A complex spectrum between g = 2.1 and g = 1.84 was observed [6] in the reduced protein, but only after very prolonged reduction with hydrogen or treatment with dithionite. We have presented evidence for an even broader ESR signal [7,10] which correlates with optical absorption of the clusters. Both the [3Fe-xS] cluster and the Ni-A centre are reversibly reducible, but since their midpoint redox potentials ( - 3 5 mV and - 1 5 0 mV, at pH 7.0 [2], or - 7 0 mV and - 2 2 0 mV at pH 8.5 [11], respectively) are considerably less negative than the hydrogen potential ( - 420 mV), their involvement in reactions such as hydrogen production is not clear. It may be significant, however, that the midpoint potential of the Ni-A species is pH-dependent [2], changing by - 6 0 m V / p H unit, indicating that the reduction is associated with protonation of the 6nzyme. A third type of nickel signal, termed Ni-C, at g = 2.19, 2.16, 2.01 was reported in D. gigas hydrogenase by LeGall and coworkers [3] (Fig. lc). It was produced by prolonged treatment with hydrogen or other strong reducing agents. Under these conditions the Ni-A and Ni-B signals disappear. The presence of this spectrum has been found to correlate with the active state of the enzyme [7]. Spectra with similar g values have been observed in hydrogenases from Methanobac-

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Magnetic field (mT) Fig. 1. ESR spectra of D. gigas hydrogenase samples, prepared under various conditions. (a) As prepared in air (Ni-A signal), recorded at 105 K; (b) after activation and reoxidation with dichloroindophenol, in the Ready state, showing the Ni-B signal [18], 105 K; (c) activated under hydrogen for 4 h, (Ni-C), 30 K; (d) as (c), recorded at 8 K; (e) as (c), illuminated in the ESR cavity at 30 K; (f) treated with H 2 and carbon monoxide as described in the text, 105 K. Other conditions of measurement: microwave power 20 roW, frequency 9.19 GHz, modulation amplitude 1 mT.

100 terium thermoautotrophicum [12] and Chromatium vinosum [13] after treatment with hydrogen. The intensity of the spectrum in all three proteins has been found to be relatively small on full reduction by hydrogen, and to reach a maximum at an intermediate redox potential, produced for example by decreasing the hydrogen tension under argon. Redox titrations of the signal in D. gigas hydrogenase at pH 8.5 [6] showed that it developed at - 2 7 0 mV, reached maximum intensity at - 3 5 0 mV, and disappeared below - 4 0 0 mV. Therefore the spectrum represents an intermediate oxidation state of the enzyme. A particularly interesting observation made by Van der Zwaan et al. [13] on the C. vinosum enzyme was that the Ni-C species is photosensitive. On illumination at temperatures below 77 K, the ESR spectrum changed drastically. The rate of photochemical conversion was nearly 6-times slower in 2H20 than in H20, indicating that the nickel was coordinated to an exchangeable hydrogen atom. This observation supports the view that the nickel centre is involved in the reaction of the enzyme with hydrogen. On decreasing the temperature below 20 K, the ESR spectrum of Ni-C changes gradually [6,10] to a more complex shape (Fig. ld). A prominent feature of this spectrum appears at a g value (at X-band) of 2.21. It was suggested that this change is due either to the presence of a superimposed spectrum of an iron-sulphur cluster species, or to a splitting of the spectrum of Ni-C by spin-spin interaction with iron-sulphur clusters [6,10]. In this paper we describe further observations of the ESR spectra of D. gigas hydrogenase, which relate to the redox equilibria of Ni-C and its interaction with other paramagnetic species. Materials and Methods

Hydrogenase was purified as described previously [1]. Hydrogenase enriched in 61Ni was isolated from cells grown on Starkey's medium [14] to which 550 /~g per litre of 89.4% enriched 61Ni (Bureau des Isotopes Stables, CEA, Saclay, France) were added. This corresponds to a nickel concentration of 9 /tM. The residual natural nickel isotopes in the lactate, yeast extract and potassium phosphate in the culture medium were estimated

to be approx. 1 /~M, so the enrichment in the culture medium was estimated to be 80%. The 61Ni content was estimated from the gz feature of the ESR spectrum of the oxidized protein, which shows a clearly defined hyperfine splitting into four lines [5,8]. The amplitude of the split signal from unenriched nickel was estimated by computer subtraction to be 20% of the total, indicating a final enrichment of 80%. Samples of hydrogenase under various partial pressures of hydrogen were prepared on a vacuum line with various mixtures of purified hydrogen and argon as previously described [7]. The composition of these mixtures could be regulated by means of a manometer. Redox titrations under hydrogen-argon mixtures. A modified redox titration cell with a small minimum solution volume (less than 0.5 ml) was made by an adaptation of the design of Dutton [15]. The redox potential was measured by a platinum electrode on the base of the cell, the circuit being completed by the calomel reference of a small Radiometer combined pH electrode (Type GK2301B). Before redox titrations the enzyme was activated by incubation under hydrogen at 30°C for 1 h. Methyl viologen, benzyl viologen and indigodisulphonate (all 50 ~M) were added as mediators. The cell was flushed continuously with a slow flow of a water-saturated mixture of hydrogen and argon, controlled by a precision gas blender (Signal Instruments Ltd, Camberley, Surrey, U.K.). At low redox potentials (below - 3 0 0 mV at pH 7) the potential was adjusted by varying the argon-hydrogen ratio. Stable potentials could be achieved within 5-10 min after each adjustment. At less negative potentials a pure argon atmosphere was used and the potential was adjusted with dithionite or K3Fe(CN)6 solutions. Samples were withdrawn with a 250-/~1 syringe (Hamilton Co, Bonaduz, Switzerland) through a small chamber (see Fig. 1 of Ref. 16) into quartz tubes and frozen for ESR spectroscopy. The chamber and quartz tubes were all flushed with the same hydrogen-argon mixture as the titration vessel. ESR spectra were recorded on a Varian E4 spectrometer with E102E Bridge and an Oxford Instruments ESR9 helium flow cryostat.

10l g-value

Results 220

Spin-spin interactions between Ni-C and ironsulphur clusters The Ni-C signal has been observed [6,10] to show a splitting at low temperatures (below 20 K). This signal shows enhanced electron spin relaxation, so that it is much less readily saturated by microwave power than the Ni-A and Ni-B signals. These results suggest a dipolar interaction with a rapidly relaxing species, which must presumably be an iron-sulphur cluster. Possibilities are a reduced [3Fe-xS] cluster or a reduced [4Fe-4S] cluster. These can be distinguished by preparing samples of protein in which the other redox centres are either oxidized or reduced. Various oxidation states of D. gigas hydrogenase can be obtained by activation of the Unready enzyme under hydrogen, and subsequent treatment with hydrogenargon mixtures on a vacuum line [7]. The enzyme, as prepared in air, is mostly in the Unready state, which requires prolonged treatment with reducing agents to become active [17]. During this activation process, the reduction of the [3Fe-xS] cluster and the appearance of the broad ESR signal occur rapidly, while the Ni-C signal takes more than an hour (at 20°C) to reach m a x i m u m size. Thus, by a short treatment with hydrogen, it was possible to prepare samples in which the Ni-C signal was almost absent but the broad signal was fully reduced [7]. During a longer treatment with hydrogen, the enzyme activity increased in parallel with a steady increase in the amplitude of Ni-C. If the samples were adequately stirred to maintain equilibrium with the gas phase, there was no subsequent decrease in Ni-C signal intensity, but the final amplitude of the Ni-C signal was small. Thus it was possible to obtain a sample in which the Ni-C signal (measured at 30 K or above) was present, together with a maxim u m amount of the broad signal (cf. Fig. 2 of Ref. 7). After activation the hydrogen was flushed out from the sample by argon, whereupon the protein was partially reoxidized by protons from water. In this way, at a sufficiently low p H (below 7), it was possible to obtain a sample in which the Ni-C was quite intense but the broad signal was absent. During these changes, the amplitude of the broad

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ESR signal correlated with the absorption spectrum between 400 and 600 nm corresponding to iron-sulphur clusters [7]. Fig. 2 shows spectra of Ni-C in samples of D. gigas hydrogenase in these samples. Fig. 2a shows the spectrum of Ni-C in the presence of a maxim u m amplitude of the broad signal (cf. Fig. 2c in Ref. 7). When measured at low temperatures, the X-band spectrum showed the splitting, with a derivative peak at an apparent g value of 2.21. The spectrum was the same at high and low microwave power (Fig. 2a and b). The power for half-saturation of this signal at 7.5 K was 8 dB (31 roW). (The feature at g = 2.00 is a signal from a

102

small amount of free radical, which was present to a variable extent in different hydrogenase preparations.) Fig. 2c shows the spectrum of another sample, treated with hydrogen and then flushed with argon, which had a substantial Ni-C signal but only a weak broad signal (cf. Fig. 2d of Ref. 7). At high microwave power (10 dB) the spectrum was similar to that of Fig. 2a, showing the 'split' Ni-C signal, with prominent features at g = 2.21 and 2.11 (Fig. ld). However, at low power (50 dB), additional features at g = 2.19 and 2.16 can be seen (Fig. 2c), similar to those seen in the spectra of Ni-B at higher temperatures (Fig. 2d). These latter features were much more readily saturated with microwave power; the power for half-saturation of this signal at 7.5 K was 35 dB (0.063 mW), and hence it was saturated at the higher power. This ' u n s p l i r signal of Ni-C can be interpreted as arising from a fraction of hydrogenase molecules in which the Ni-C was not interacting with a fast-relaxing paramagnetic species. In these experiments, and in the redox titrations described later, the amplitude of this signal was greatest under conditions where the Ni-C signal was present but the broad signal was small. The Ni-A and Ni-B signals were also examined, at temperatures down to 4.2 K, for evidence of splitting due to electron spin-spin interactions with other paramagnetic centres, but none was observed. Such an interaction has been inferred in oxidized C. vinosum hydrogenase [18] from a splitting in the nickel signal (Signal 4). In the latter case the splitting was correlated with a a complex signal (Signal 2) derived from an iron-sulphur cluster, presumed to be an oxidized [4Fe-4S] 3÷ cluster. In D. gigas hydrogenase the [4Fe-4S] clusters are in the diamagnetic [4Fe-4S] 2÷ state at the potentials where the Ni-A and Ni-B signals appear [7], and the [3Fe-xS] cluster appears to be too distant from the nickel to interact significantly [2]. Effects of 61Ni substitution on the 'split Ni-C' signal In order to determine whether the spectrum containing the g = 2.21 signal arises from a modification of the nickel spectrum, or the superimposition of the spectrum of an additional species, such as an iron-sulphur cluster, we examined the spectrum in samples that were enriched in 61Ni

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and should therefore show hyperfine splitting or broadening if the species involves nickel. The spectrum shows a small but significant broadening (Fig. 3b) compared with the native enzyme (Fig. 3a). It is extremely difficult to simulate such spectra where there may be dipolar and exchange interactions, and both the g and A tensors are anisotropic, because of the large number of unknown parameters to be fitted. Therefore in order to estimate the extent of hyperfine broadening the spectrum of the unenriched enzyme was shifted in the computer, by an amount corresponding to the hyperfine splitting. For this empirical simulation four equally-separated species, as expected for 6] Ni ( I = ~), were added together with an equal amount of the unsplit signal, to allow for the 80% enrich-

103 T

ment of 61Ni, Fig. 3c. The best fit to the g = 2.21 region was obtained with a hyperfine interaction of 0.6 mT. This is to be compared with 0.8 m T in the g = 2.32 feature of the Ni-A signal (R. Cammack and E.C. Hatchikian, unpublished data; cf. [191). These results indicate that the complex spectrum of Fig. l d is entirely derived from the Ni-C species which is split by interaction with a fast-relaxing species. The occurrence of the species is correlated with the appearance of the broad ESR spectrum.

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Measurements of midpoint potentials of the iron-sulphur and nickel centres by the mediatortitration method, as used in previous titrations of the enzyme [2,6,11], are complicated, when extended to low redox potentials (e.g. below - 3 0 0 mV at p H 7), by the fact that the redox centres in hydrogenase will rapidly come to equilibrium with the H + / H 2 couple in solution. Therefore if the hydrogen is swept away by an inert gas stream, the redox potential and p H of the solution will be unstable. The midpoint potentials of the lowpotential centres were determined by titrations in which the redox potential was measured by means of mediators and a platinum electrode, and the solution was kept under controlled hydrogen-argon mixtures as described in the Methods section. In Fig. 4a and b the amplitudes of the broad ESR spectrum and Ni-C signal are plotted against applied redox potential, and fitted to curves calculated from the Nernst equation, assuming single-electron redox processes. It can be seen that the amplitude of the broad signal reached a maxim u m level at low potentials, while the Ni-C reached a maximum at an intermediate potential then decreased again, consistent with previous observations [6,12,13]. It is also obvious that the amplitudes of the signals are pH-dependent. The two E m values for the Ni-C signal, estimated by fitting the titration data, showed different pH-dependence (Fig. 5a). The lower En, for appearance of the signal varied by approx. - 6 0 m V / p H unit, while the more positive E m for disappearance of the signal varied by more than this; the line in Fig. 5a is for - 1 2 0 m V / p H unit, corresponding to two protons per electron. As a

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Fig. 4. (a) and (b) amplitudes of the broad ESR signal (squares) and the Ni-C spectrum (triangles), plotted against applied redox potential. Filled points were from a titration in 0.1 M 4-morpholineethanesulphonate buffer, pH 5.7: open points in 0.1 M N,N-bis(2-hydroxyethyl)glycine buffer, pH 8.1. (c) The amplitudes of the split (filled circles) and unsplit (open circles) Ni-C species, as described in the text, plotted for the titration at pH 5.7. Curves were calculated from the Nernst equation assuming one-electron redox processes.

consequence, the Ni-C signal reached a higher intensity, and was detected over a wider range of potential, at more acid pH. The E m of the broad signal varied by approx. - 6 0 m V / p H unit (Fig. 5b). The E m values at p H 7.0 were estimated by interpolation to be - 2 7 0 mV and - 3 9 0 mV for the appearance and disappearance of Ni-C, respectively, and - 350 mV for the broad signal. In Fig. 4c the amplitudes of the signals due to the split and unsplit nickel, estimated from spectra similar to Fig. 2c, are plotted. The curves fitted to the data were calculated from the potentials for Ni-C and the broad ESR signal, assuming that the

104

splitting of the Ni-C signal was only observed under conditions where the reduced iron-sulphur cluster was present in the same molecule. The results are reasonably in agreement with this hypothesis, considering the signal : noise ratio of the data.

Effects of illumination on the Ni-C spectrum Van der Z w a a n et al. [13] have reported that the ' g = 2.19' nickel species in C. vinosum hydrogenase is sensitive to light at low temperatures, and converts to a new signal with apparently inverted rhombic symmetry. As shown in Fig. le, the same is true of the Ni-C species in D. gigas hydrogenase. The illuminated sample had a signal at g = 2.30, 2.13, 2.05, plus a small free radical at g = 2.00, possibly arising from photo-oxidation product. Sometimes, depending on the temperature of illumination, a variable a m o u n t of a second signal was observed, at g = 2.28, as seen in C. uinosum hydrogenase [13]. This effect is probably due to the enzyme being frozen in different .conformations which differ in coordination of the nickel centre. On raising the temperature to 200 K, the spectrum reverted to the Ni-C signal. The broad signal was unaffected by illumination treatments. The effect of illumination therefore appears to be to alter the coordination state of the nickel. Treatment of the enzyme with carbon monoxide C a r b o n monoxide is an inhibitor of hydroI

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Fig. 5. P l o t s o f t h e m i d p o i n t p o t e n t i a l s u s e d to fit t h e r e d o x d a t a o f Fig. 4, as a f u n c t i o n o f p H . F i l l e d a n d c l o s e d c i r c l e s c o r r e s p o n d to the p o t e n t i a l s o f a p p e a r a n c e a n d d i s a p p e a r a n c e , r e s p e c t i v e l y , o f N i - C d u r i n g r e d u c t i o n ; s q u a r e s to t h e b r o a d signal.

genase, competitive with hydrogen [20]. This indicates that it binds to the same site as hydrogen. A sample of D. gigas hydrogenase was activated under hydrogen for 4 h (Fig. 6a), then treated with approx. 20% carbon monoxide by volume. A new spectrum appeared at g = 2.12, 2.07, 2.02 (Fig. 6b). This can be better observed after subtraction of the Ni-C signal (Fig. 6c). The spectrum of this sample was reasonably stable if it was kept at r o o m temperature for 19 h. However, the spectrum was very sensitive to the ratio of gases present. If the hydrogen-treated enzyme was flushed with argon to optimize the amplitude of the Ni-C ESR spectrum, the ESR signal completely disappeared on treatment with carbon monoxide. This

105 spectrum is similar to one observed in C. vinosum hydrogenase during treatment with carbon monoxide [21]. Discussion

The activity of the D. gigas and related hydrogenases varies with the type of preparation and the history of the sample [22,23]. To explain this behaviour we have proposed [17] that any particular preparation of the enzyme can be considered as a mixture of an active form, and two forms that are both unreactive towards hydrogen. These two inactive forms are termed the Unready state, which is only reactivated slowly by strong reductants, and the Ready state, which can be rapidly reactivated. After cycling between the hydrogentreated, active form and the oxidized, inactive forms, a correlation has been noticed between the proportion of the Ready state and the amplitude of a second species of Ni(III) ESR signal, Ni-B (Fig. lb) [6,7]. It has been suggested that the change from the Unready to' the Ready state is accompanied by a change in the coordination state of nickel, reflected in the ESR lineshape of the Ni(III) species. The ESR signals from nickel in D. gigas hydrogenase (Fig. 1) have their counterparts in other nickel-containing hydrogenases. Signals with g values very similar to those of Ni-A (Fig. la) are observed in hydrogenase from M. thermoautotrophicum (strains Marburg [19] and AH [12]) and both Ni-A and Ni-B in C. vinosum [18], where they were named Signals 3a and 3b, and hydrogenases from other Desulfovibrio spp. such as D. salexigens [24]. Signals equivalent to Ni-C, which reached maximum amplitude at low partial pressures of hydrogen, have also been observed in these hydrogenases [12,13]. In these enzymes at least, the nickel site appears to exist in several states which are highly conserved. In C. vinosum hydrogenase, however, no evidence was found to relate them to states of activation of the enzyme. It should be emphasized that not all nickel-containing hydrogenases give rise to ESR signals of this type. Some give very weak or no ESR signals in their oxidized or reduced states [25]. The observation that in C. vinosurn hydrogenase the signal was light-sensitive (cf. Fig. le),

and that the rate of photochemical conversion was slower in 2H20 than in H 2 0 [13], provides evidence for a function of this form of nickel in catalysis. The finding that the redox potentials of the Ni-C species are pH-dependent is further evidence for the involvement of nickel in the active site of the enzyme for hydrogen binding. There is considerable uncertainty about the chemical nature of the Ni-C species, and species giving rise to similar g = 2.19 spectra in other hydrogenases. The oxidation state of nickel has been variously proposed to be Ni(III) [6] or Ni(I) [10,13]. In a previous study of spectroscopic changes of the enzyme during activation [7] we have favoured the latter view. The effects of deuterium substitution on the kinetics of photolysis indicate an exchangeable hydrogen atom, possibly hydride, in the first coordination sphere [13]. However, the hyperfine splitting due to exchangeable ~H is very small, about 0.5 mT [13,26]. As to the nature of the species giving the complex spectrum of Fig. ld, with the feature at g = 2.21, a number of observations indicate that it is derived from a dipolar interaction of the Ni-C species, with one or both reduced [4Fe-4S] clusters in the reduced, paramagnetic [4Fe-4S] 1÷ state. (a) The spectrum at low temperatures was broadenen by 61Ni-substitution. (b) The proportion of the nickel species giving the complex spectrum was correlated, in various redox states, with the presence of the broad ESR signal which in turn has been correlated with the decrease in optical absorption between 400 and 600 nm [7]. It is acknowledged that such a correlation does not necessarily imply a causal relationship. However some other possible interactions are thereby excluded. For example, the complex spectrum was not correlated with the presence of [3Fe-xS] clusters in the paramagnetic reduced state. (c) The complex spectrum showed unusually rapid electron-spin relaxation, and changed into the normal Ni-C signal at temperatures above 20 K. At the same temperature the broad ESR signal disappeared. Both observations are explained if the relaxation rate of the [4Fe-4S] clusters becomes rapid, on the ESR timescale, at temperatures above 20 K, so that the effects on the Ni-C species are averaged out. If the splitting is due to a spin-spin interaction,

106

it should be possible to place a limit on the distance between the nickel and iron-sulphur centres. The splittings observed are characteristic of rather weak interactions. Distances of 1.0-1.4 m m have been estimated for a similar interaction between and iron-sulphur cluster and Mo(V) in xanthine oxidase [27]. The extent of the splitting depends critically on the angles between the gtensor axes of the centres, and on the relative extent of exchange and dipolar interactions. However, it is unlikely that the ESR-detectable nickel and iron-sulphur clusters are close enough to be considered part of a single centre. This does not preclude the possibility that they become closer when the protein is in some other oxidation state. The Ni-A and Ni-B signals were also examined for splitting at low temperatures (down to 4.2 K), and none was observed. This is not surprising, as the [4Fe-4S] clusters are fully oxidized to the diamagnetic [4Fe-4S] 2+ state at the potentials where these signals appear. This is in contrast to oxidized C. vinosum hydrogenase, where a splitting in the nickel signals has been observed [28], and attributed to interaction with an ESR-detectable oxidized [4Fe-4S] 3+ cluster. The midpoint potentials of the various activities and ESR signals of D. gigas hydrogenase are TABLE I REDOX

PROPERTIES

O F DESULFO VIBRIO GIGAS H Y -

DROGENASE R e d o x event

E S R of [ 3 F e - x S ] " + b E S R of N i - A E S R of N i - C : a p p e a r a n c e of the signal d i s a p p e a r a n c e of the signal d E S R o f [4Fe-4S] 1 ~ Reductive activation Oxidative deactivation C a t a l y t i c activity of activated enzyme (hydrogen evolution)

E., ( p H 7.0) (mV)

Em/pH (mV)

Ref.

- 35 150

0 60

2 2 this w o r k

- 270

120

390 350

60 60

this w o r k

- 310 133

60 60

22 34

- 360

60

23

Signal at g = 2.01: h signal at g = 2.24: " signal at g = 2.19; d b r o a d signal o b s e r v e d at t e m p e r a t u r e l o w e r t h a n 11 K.

summarized in Table I. The pH-dependence of the E m values is a striking feature of the nickel species in D. gigas hydrogenase. A pH-dependence of - 6 0 m V / p H unit implies that, during reduction, one proton is taken up per electron. From the pH-dependence of the redox potentials of the ESR-detectable components, it appears that complete reduction of D. gigas hydrogenase entails the uptake of 4 - 5 electrons and 4 - 5 protons. One electron and one proton are taken up for reduction of Ni-A [2], a process which we have interpreted as the reduction of Ni(III) to Ni(II) [7]. The Ni-A signal is associated with the inactive, Unready state of the enzyme [17], and a conversion to the Active state must take place before further ESR-detectable redox states are observed. The rate of the activation process appears to be pH-independent [17]. The reduction of the Ni-C species appears to require as many as three protons: two during the appearance of the signal and one during its disappearance (Fig. 3). The reduction of the broad ESR signal required one proton per electron. This signal has been associated with one or both [4Fe-4S] clusters [7]. The reason for the broadness of the signal is not clear. If it were due to spin-spin interaction between centres, one would expect to see narrower spectra of single clusters at intermediate stages of reduction, but these were not observed in our preparations (cf. Fig. 2 of Ref. 7). If there was strong cooperativity between the reduction of the two clusters, one would expect the reduction curve to fit an n = 2 redox process; instead it appears to be n = 1 (Fig. 3). On this basis, the most likely interpretation is a single [4Fe-4S] cluster. So far it has not been possible to determine the midpoint potential of the second Ni(III) species, Ni-B, because of lack of reversibility of the redox process. We have interpreted the Ni-B signal as the Ready state, i.e. the Ni(III) state of the enzyme in its active conformation. Of the protons taken up during reduction of the hydrogenase, two can be assigned to the production of H 2. The others are presumably associated with the protonation of other sites on the enzyme. These need not necessarily be directly involved in the catalytic mechanism. Such pH-dependence has been observed in other metalloenzymes such as nitrogenase [29] and xanthine

107 oxidase [30], and has been interpreted as due to redox-dependent changes in the pK a of basic groups.

Mechanism of the hydrogenase reaction On the basis of the present results it is possible to draw up a number of possible schemes for the activation of hydrogenase, and the reaction of the activated enzyme with hydrogen, which are based on the proposition that the nickel centre is involved. A mechanism which is consistent with the present results is as follows: The reductive activation of the Unready state appears to involve reduction of Ni-A, Ni(III), to an ESR-silent state which we proposed to be Ni(II) [7]. The activation then occurs slowly, apparently by some intramolecular transformation. After activation the nickel can be reduced to give the Ni-C form, though this is not necessary for activation to occur. The activation appears to be independent of the reduction state of the ironsulphur clusters; it can be done under mild reducing conditions where the broad signal is not induced and only the [3Fe-xS] cluster is reduced, or alternatively the iron-sulphur clusters in the Unready enzyme may be reduced prior to activation. The Ready state, correlated with the Ni-B signal, presents the Ni(III) oxidation state in the activated conformation [7]. The ESR signals due to nickel in activated D. gig,as hydrogenase undergo significant changes during activation and in various redox states that are relevant to the catalytic cycle. It should also be emphasized that experiments on the redox potentials, being equilibrium measurements, cannot define the kinetic mechanism of the enzyme reaction. Because hydrogenases readily catalyse the hydrogen-deuterium exchange reaction even under conditions in which there is no electron transfer, it is generally assumed that the mechanism of catalysis involves a heterolytic cleavage of the hydrogen molecule, as suggested by Krasna and Rittenberg [31]. The general scheme for the reaction, considered as going in the direction towards hydrogen production, involves the binding of a proton, followed by a two-electron reduction step to produce a hydride ion, H . To stabilize the hydride intermediate, it is assumed to be formed at a

metal centre, such as an iron-sulphur cluster or nickel centre. The hydride then combines with a second proton to produce H R. If this second proton is bound to another base, the release and binding of hydrogen to the enzyme does not require electron transfer from or to other acceptors. Teixeira et al. [6] have presented a type of mechanism for D. gigas hydrogenase, in which the Ni-A, Ni-B and Ni-C species are all in oxidation state Ni(III); the disappearance of Ni-A on reduction was interpreted as due to spin-coupling to an iron-sulphur cluster. From considerations of the changes in ESR signals during activation of the enzyme [7] we have suggested instead that the Ni-C species is Ni(I), in agreement with Van der Zwaan et al. [13]. In this scheme we consider the reaction of the enzyme in the Ready state with hydrogen. This state will not react with hydrogen directly (for example, it will not catalyse hydrogen-tritium or hydrogen-deuterium exchange [33]) until it is reduced by strong reductants such as methyl viologen or cytochrome c3. The first stage of the reaction would be the reduction of the Ready enzyme to give the Active state. This process appears to be associated with the reduction of Ni-B: Ni(III) + e - = Ni(II)

(1)

The catalytic cycle would progress with the heterolytic cleavage of hydrogen: Ni(II)+H 2=Ni(II)-H +H +

(2)

The proton released is probably transferred initially to a base on the enzyme, though this need not necessarily be an iron-sulphur cluster. Ni(II). H + [4Fe-4S]~+ = Ni(I)-H + + [4Fe-4S]~+

(3)

Here we have proposed that a proton remains attached to the coordination sphere of the Ni(I), to explain the kinetic isotope effect of deuterium substitution on the rate of photolysis of the Ni-C species. [4Fe-4S]k+ + [4Fe-4S]~+ = [4Fe-4S]2A+ + [4Fe-4S]~+

(4)

Ni(I). H + + [4Fe-4S]ZA+ = Ni(II) + [4Fe-4S]IA+ + H +

(5)

This mechanism is consistent with most of the

108

ESR spectroscopic observations of the nickel centre in D. gigas hydrogenase. The disappearance of the Ni-C signal at very redox potentials may be interpreted as the reduction of Ni(I) to Ni(II)- H - [10]. It also explains why the splitting of the Ni-C signal by exchangeable protons is very small [13,26]. Hydrides of this type have been proposed as intermediates in the reactions of nickel catalysts of olefin hydrogenation [32]. Moreover, if we assign the g = 2.19 signal to the Ni(I) species, the above mechanism explains why the signal disappears at both low and high partial pressures of hydrogen. At low pressures the applied redox potential of the H ÷ / H 2 is insufficient to reduce Ni(II) to Ni(I), while at high pressures the [4Fe-4S] clusters will become fully reduced, leading to an accumulation of Ni(II) • H - . The above hypothesis implies that the important role of nickel in hydrogenase would be played by Ni(II), which is ESR-silent, and Ni(I). Since Ni(III) is not essential to the mechanism this would explain why in a number of nickel-containing hydrogenases no signals are seen in the oxidized state [25]. Signals of Ni(I) in the reduced state would be more significant. The species seen on brief treatment with hydrogen and carbon monoxide is presumably an Ni(I)-carbonyl. After prolonged treatment with inhibitory concentrations of carbon monoxide, the nickel becomes ESR-silent. The species might be oxidized by protons, or traces of oxygen, to an ESR-undetectable Ni(II) carbonyl. Since Ni(II) is ESR-silent, other methods would be required to investigate its function in the hydrogenase reaction cycle. Acknowledgements This work was supported by grants from the U.K. Science and Engineering Research Council, PIRSEM (ATP No. 329), CNRS (ATP No. 376), the Nuffield Foundation, the University of London Central Research Fund, and an Anglo-Spanish Joint Action grant. V.M.F. is the recipient of a Fellowship from the Royal Society under the European Science Exchange Programme. We thank Mr A. Austin for assistance with some of the ESR measurements, and Dr. S.P.J. Albracht for sending information on Co vinosum hydrogenase prior to publication.

References 1 Hatchikian, E.C., Bruschi, M. and LeGall, J. (1978) Biochem. Biophys. Res. Commun. 82, 451-461 2 Cammack, R., Patil, D., Aguirre, R. and Hatchikian, E.C. (1982) FEBS Lett. 142, 289-292 3 LeGall, J., Ljungdahl, P.O., Moura, I., Peck, H.D., Xavier, A., Moura, J.J.G., Teixeira, M., Huynh, B.H. and DerVartanian, D.V. (1982) Biochem. Biophys. Res. Commun. 106, 610-615 4 Kruger, H.-J., Huynh, B.H., Ljungdahl, P.O., Xavier, A.V., DerVartanian, D.V., Moura, I., Peck, H.D., Jr., Teixeira, M., Moura, J.J.G. and LeGall, J. (1982) J. Biol. Chem. 257, 14620-14623 5 Moura, J.J.G., Moura, I., Huynh, B.H., Kriager, H.-J., Teixeira, M., DuVarney, R.C., DerVartanian, D.V., Xavier, A.V., Peck, H.D., Jr. and LeGall, J. (1982) Biochem. Biophys. Res. Commun. 108, 1388-1393 6 Teixeira, M., Moura, I., Xavier, A.V., Huynh, B.H., DerVartanian, D.V., Peck, H.D., Jr., LeGall, J. and Moura, J.J.G. (1985) J. Biol. Chem. 260, 8942-8950 7 Fernandez, V.M., Hatchikian, E.C., Patil, D.S. and Cammack, R. (1986) Biochim. Biophys. Acta 883, 145-154 8 Cammack, R., Hall, D.O. and Rao, K.K. (1984) in Microbial Gas Metabolism (Poole, R.K., ed.), pp. 75-102, Academic Press, New York 9 Berlier, Y.M., Fauque, G., Lespinat, P.A. and LeGall, J. (1982) FEBS Lett. 140, 185-188 10 Cammack, R., Patil, D. and Fernandez, V.M. (1985) Biochem. Soc. Trans. 13,572-578 11 Teixeira, M., Moura, I., Xavier, A.V., DerVartanian, D.V., LeGall, J., Peck, H.D., Huynh, B.H. and Moura, J.J.G. (1983) Eur. J. Biochem. 130, 481-484 12 Kojima, N., Fox, J.A., Hausinger, R.P., Daniels, L., OrmeJohnson, W.H. and Walsh, C. (1983) Proc. Natl. Acad. Sci. USA 80, 378-382 13 Van der Zwaan, J.W., Albracht, S.P.J., Fontijn, R.D. and Slater, E.C. (1985) FEBS Lett. 179, 271-277 14 Starkey, R.L. (1938) Arch. Microbiol. 9, 268-304 15 Dutton, P.L. (1978) Methods Enzymol. 54, 411-434 16 Chamorovsky, S.K. and Cammack, R. (1982) Photobiochem. Photobiophys. 4, 195-200 17 Fernandez, V.M., Hatchikian, E.C. and Cammack, R. (1985) Biochim. Biophys. Acta 832, 69-79 18 Albracht, S.P.J., Kalkman, M.L. and Slater, E.C. (1983) Biochim. Biophys. Acta 724, 309-316 19 Albracht, S.P.J., Graf, E.-G. and Thauer, R.K. (1982) FEBS Lett. 140, 311-313 20 Hallahan, D.L., Fernandez, V.M., Hatchikian, E.C. and Hall, D.O. (1986) Biochimie 68, 49-54 21 Van der Zwaan, J.W., Albracht, S.P.J., Fontijn, R.D. and Roelofs, Y.B.M. (1986) Biochim. Biophys. Acta 872, 208-215 22 Lissolo, T., Pulvin, S. and Thomas, D. (1984) J. Biol. Chem. 259, 11725-11730 23 Fernandez, V.M., Aguirre, R. and Hatchikian, E.C. (1984) Biochim. Biophys. Acta 790, 1-7 24 Teixeira, M., Moura, I., Fauque, G., Czechowski, M.,

109

25 26 27 28 29

Berlier, Y., Lespinat, P.A., LeGall, J., Xavier, A.V. and Moura, J.J.G. (1986) Biochimie 68, 75-84 Cammack, R., Fernandez, V.M. and Schneider, K. (1986) Biochimie 68, 85-91 DerVartanian, D.V., Kruger, H.J., Peck, H.D., Jr. and LeGall, J. (1985) Rev. Port. Quim. 27, 70-73 Coffman, R.E. and Buettner, G.R. (1979) J. Phys. Chem. 83, 2392-2400 Albracht, S.P.J., Van Der Zwaan, J.W. and Fontijn, R.D. (1984) Biochim. Biophys. Acta 766, 245-258 O'Donnell, M.J. and Smith, B.E. (1978) Biochem. J. 173, 831-839

30 Hille, R. and Massey, V. (1986) in Molybdenum Enzymes (Spiro, T., ed.), pp. 443-518, Wiley, New York 31 Krasna, A.I. and Rittenberg, D. (1954) J. Am. Chem. Soc. 76, 3015-3020 32 Tolman, C.A. (1972) J. Am. Chem. Soc. 94, 2994-2999 33 Hallahan, D.L., Fernandez, V.M., Hatchikian, E.C. and Cammack, R. (1986) Biochim. Biophys. Acta 874, 72-75 34 Mege, R.-M. and Bourdillon, C. (1985) J. Biol. Chem. 260, 14701-14706