The three-iron cluster in a ferredoxin from Desulphovibrio gigas A low-temperature magnetic circular dichroism study

Biochimica et Biophysica Acta, 670 (1981) 93-100 Elsevier/North-Holland Biomedical Press

93

BBA 38714 THE THREE-IRON CLUSTER IN A FERREDOXIN FROM DESULPHOVIBRIO GIGAS A LOW-TEMPERATURE MAGNETIC CIRCULAR DICHROISM STUDY ANDREW J. THOMSON a, A. EDWARD ROBINSON a, MICHAEL K. JOHNSON a, JOSE J.G. MOURA b,c, ISOBEL MOURA b,c, ANTONIO V. XAVIER b,c and JEAN LEGALL d a School o f Chemical Sciences, University o f East Anglia, Norwich, NR4 7TJ (U.K.), b Centro de Quimica Estrutural des Universidades de Lisboa LS. T., 1000 Lisbon (Portugal), c Gray Freshwater Biological Institute, University of Minnesota, Box 100, Navarre, MN 55392 and d Department o f Microbiology and Biochemistry, University of Georgia, Athens, GA 30602 {U.S.A.)

(Received Februaxy 2nd, 1981)

Key words: Ferredoxin; Iron-sulphur cluster; Magnetic circular dichroism; [Desulphovibrio gigasJ

Ferredoxin H from Desulphovibrio gigas is a tetrameric protein containing a novel iron-sulphur cluster consisting of three iron atoms. The low-temperature magnetic circular dichroism (MCD) spectra of the oxidized and dithionite-redueed forms of ferredoxin H have been measured over the wavelength range approx. 3 0 0 - 8 0 0 nm. Both oxidation levels of the cluster are shown to be paramagnetie, although only the oxidized form gives an EPR signal. MCD magnetization curves have been constructed over the temperature range approx. 1.5-150 K and at fields between 0 and 5.1 Tesla. The curve for the oxidized protein can be fitted to a ground state of spin S = ½ with an isotropic g factor of 2.01. There is evidence for the thermal population of a low-lying electronic state above 50 K. The reduced protein gives a distinctive set of magnetization curves that are tentatively assigned to a ground state of S = 2, with a predominantly axial zero-field distortion that leaves the doublet M s -- +2 lowest in energy. The zero-field components have a maximum energy spread of approx. 15 cm -1. which places an upper limit of 4 cm -1 on the axial zero-field parameter D. The MCD spectra of the oxidized and reduced forms of the cluster are quite distinctive from one another. The spectra of the oxidized state are also different from those of oxidized high-potential iron protein from Chromatium and should provide a useful criterion for distinguishing between four- and three-iron clusters in their highest oxidation levels. Introduction Recently M6ssbauer spectroscopy has convincingly demonstrated the presence of a previously unrecognized iron-sulphur cluster containing three iron atoms and acid-labile sulfur [1]. The centre was first identified in a ferredoxin from A z o t o b a c t e r vinelandii, in which a high-potential cluster of the four-iron, Fe4S4, type and a low-potential centre of the novel threeiron type were shown to exist [1]. X-ray diffraction Abbreviations: MCD, magnetic circular dichroism; HipiPox, high-potential iron protein, oxidized; HipiPred , high-potential iron protein, reduced.

data on crystals of the same protein appear to be consistent with this interpretation and a 0.25 nm map, although not refined, suggests an Fe4S4 cluster and a smaller grouping of three iron atoms [2]. The details of the bridging ligands and the nature of the ligands attaching this latter group to the polypeptide chain have not been determined to date. Subsequently, the new three-Iron centre was shown, again by M6ssbauer spectroscopy, to be present in a ferredoxin, called ferredoxin II from Desulphovibrio gigas [3]. Ferredoxins from D. gigas have been isolated in different oligomeric forms. Ferredoxin II is a tetramer of identical polypeptide subunits, each monomer having 57 amino acids o f known sequence, which has a surpris-

0005-2795/81/0000-0000502.50 © Elsevier/North-Holland Biomedical Press

94 ing degree of homology with the ferredoxin from Clostn'dium pasteurianum [4]. Since ferredoxin II contains only one type of metal centre it provides an opportunity to examine in detail the electronic properties of the new three-iron cluster. The conclusions drawn from the studies of ferredoxin II are as follows [3]. As isolated, ferredoxin II exhibits an almost isotropic EPR signal centred at g = 2.0.1 [5]. The cluster consists of three ferric ions, probably in tetrahedral environments of predominantly sulphur atoms. The magnetic M6ssbauer spectrum of this oxidized form of the centre shows three iron sites, although two of the sites have very similar hyperfine parameters and one site is rather different. The reduced protein gives no EPR spectrum, but the M6ssbauer data reveal that the state is paramagnetic. It is suggested that the electron spin is integral with spin S/> 1. Two iron species, in the ratio 2 : 1, are found in the dithionite-reduced cluster. The iron associated with the lower occupancy site is high-spin ferric in character, but the other two iron atoms are indistinguishable from one another and presumably share the single electron added on reduction, as both are approximately at the oxidation level of Fe 2"s+. The list of proteins in which this novel cluster has been identified by M6ssbauer spectroscopy is growing rapidly. The centre has now been identified in ferredoxin I from D. gigas, in glutamate synthase from Azotobacter vinelandii and in aconitase from bovine heart mitochondria [1,3]. The last report is of particular interest in view of an earlier account, in which it is shown that a binuclear cluster is extruded from aconitase using the standard techniques [6]. This therefore leaves M6ssbauer spectroscopy as the only technique currently which can positively identify threeqron centres in proteins. The EPR signal at g = 2.01 has in the past been mistaken for the signals of Fe4S4 clusters in the same oxidation level as Hipipox [1]. Therefore other techniques which can unambiguously identify this cluster, especially in the presence of other centres, are needed. We have been developing the use of low-temperature MCD spectroscopy as a probe of the optical and magnetic properties of metal centres in metalloproteins [7]. The particular feature of MCD spectroscopy which makes it invaluable for this purpose is that the MCD spectra of diamagnets are independent of temperature, whereas the spectra of paramagnetic centres

increase as the temperature is lowered. The magnitudes of the MCD signals of paramagnets at 4.2 K and below are invariably at least an order of magnitude greater than those of diamagnetic species. At these temperatures only the MCD spectrum of the paramagnetic species appears. Therefore, in contrast to M6ssbauer spectroscopy, which observes all the iron atoms present in a protein, MCD spectroscopy, at low temperatures, only detects the iron atoms or other chromophores which have a paramagnetic ground state. We have shown that the technique can probe the electronic ground states of haems in proteins, even when the haem is not detectable by EPR spectroscopy, as in the case of the high-spin ferrous haem (S = 2) [7] and the haem a3 in oxidized cytochrome c oxidase [8]. We have also reported low-temperature MCD studies on the single iron centre in rubredoxin [9], the two-iron centres [10] in the proteins from spinach, Spirulina maxima and adrenodoxin and, more recently, the four-iron clusters in Chromatium Hipip and in C. pasteurianum [11 ]. These studies have shown that the MCD spectra at low temperature of the paramagnetic forms of all these proteins are quite distinctive and readily recognised. The most recent account shows that the C- and C 3- oxidation levels of Fe4S4 give readily identifiable spectra which are distinctive from one another [11]. This contrasts with room temperature MCD spectra of the Fe4S4 clusters, which are broadly similar [12]. It has also been demonstrated that the form of the field and temperature dependence of the MCD spectra can be analysed satisfactorily to provide the spin of the ground state [ 11 ]. It is of great interest, therefore, to extend these studies of low-temperature MCD spectra of ironsulphur centres to the threeqron clusters of ferredoxin II from D. gigas. These results we now report. There are two aspects. One is the recording of the form of the spectra of the two oxidation states. The second is the investigation of the electronic ground states of these oxidation levels by means of MCD magnetization curves. The reduced form of ferredoxin II has no EPR signal, but is paramagnetic, although the spin of the ground state has not been determined [3]. Therefore this state is especially interesting to examine.

95

Materials and Methods

10

The growth conditions for D. gigas and the isolation of the tetrameric form of ferredoxin, ferredoxin II, have been described previously [4]. The measurement o f low temperature MCD spectra has been described previously [ 11 ]. Spectra are expressed as Ae = e L - eR, where e L and eR are the molar extinction coefficients in left and right circularly polarised light, respectively. All extinction values are based on the minimum molecular weight of 6456 [4]. The reduced form was produced by the action of solid dithionite on an anaerobic solution for 35 rain. Solutions of proteins in 100 ~vl Tris-HC1 buffer, pH 7.6, were diluted with ethylene glycol (50 : 50, v/v) to ensure the formation o f good optical quality glasses at low temperature.

Results Fig. 1 shows the absorption spectrum of oxidized ferredoxin II at 4.22 K and a set of MCD spectra at temperatures of 1.52, 1.945 and 22.5 K at a magnetic field of 5.1 Tesla. The absorption spectrum at 4.22 K reveals no extra features compared with the room

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Fig. 1. Upper panel, absorption spectrum at 4.2 K of oxidized ferredoxin II. Lower panel, MCD spectra of oxidized ferredoxin II. - - , 1.53 K; - - - , 4.22 K; . . . . . . . 20.0 K. Magnetic fields, 5.1 Tesla, path length = 1.053 ram. The sample was dissolved in 100/2M Tris-HCl, pH 7.6, buffer diluted to 50% (v/v) with ethylene glycol

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Fig. 2. Upper panel, absorption spectrum at 4.2 K of reduced ferredoxin II. Lower panel, MCD spectra of reduced ferredoxin II. - - , 1.50 K; - - - , 4.22 K; . . . . . . . 20.0 K. Magnetic field, 5.1 Tesla, path length = 1.103 mm. Sample was dissolved in 100/2M Tris-HC1, pH 7.6, buffer, made anaerobic and reduced for 35 min with solid sodium dithionite. The sample was diluted to 50% by volume with ethylene glycol.

temperature spectrum [4], although the shoulder at 570 nm and the weak peak at 700 nm are better resolved. However, the MCD spectra consist of a number of positive and negative peaks running across the whole of the wavelength range 2 6 0 - 7 5 0 nm. The absorption spectrum at 4.22 K appears to have a base-line increasing in slope towards the ultraviolet, due to the low temperature glass being o f tess than perfect optical quality. The corresponding spectra of the dithionitereduced ferredoxin II are given in Fig. 2. The absorption spectrum at 4.22 K compares well with the room temperature spectrum [4]. The shoulder at 425 nm is not significantly better resolved, although a weak peak at 660 nm is more obvious at low temperature. The set of MCD spectra shown has been recorded at .1.53, 4.22 and 21.9 K at a magnetic field of 5.1 Tesla. Well.resolved peaks and troughs are seen, although the spectrum contrasts sharply with that of the oxidized protein. The forms of the field and temperature dependences of the MCD spectra of the oxidized and reduced ferredoxin II are best displayed in the form

96

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Fig. 3. a. MCD magnetization curve of oxidised ferredoxin II. ZXepis the Ae value measured from peak at 448 to trough 466 nm, expressed in arbitrary units, o, 1.5 K; z~, 1.945 K; o, 4.22 K; O, 11.1 K and +, 22.5 K. See text for details of magnetic field values at each of these temperatures~ Solid line is the curve computed for isotropic g = 2.01 according to Ref. 13. The dotted lines are drawn to the initial slope and the asymptotic limits of the curve. Their intersection occurs at a value marked on the abscissa with an arrow. This is the intercept value, I. b, MCD magnetization curves of reduced ferredoxin II. AXepis the 6e value measured from the peak at 380 to the trough at 351 nm, expressed in arbitrary units, n, 1.53 K; A, 1.995 K; o, 4.215 K; o, 9.5 K and +, 21.9 K. See text for details of magnetic field values at each of these temperatures. Solid line is the curve computed for gu = 8.00,g± = 0.20, rnz/m + = 0.2 according to Ref. 13. The dotted lines are drawn to the initial slope and the asymptotic limits of the curve. Their intersection occurs at a value marked on the abscissa with an arrow. This is the intercept value I. The inset shows the zero-field splitting of a state with spin S = 2 in the presence of an axial distortion and zero rhombic distortion. The energy levels are given by a spin Hamiltonian/t, of the following form: /:] =DSz2 +g0 " #" (Bz' Sz" + B x " Sx + B y " Sy). D is taken to be negative in sign to place the M s = -+2 levels lowest, and go is given the free spin value of 2.00. For applied fields such that goB1? ~ 3D and in t h e absence of a rhombic distortion it is possible to describe the magnetic properties o f the two doublets s e p ~ a t e l y using a spin Hamiltonian, with an effective spin S = ~, ,

//e = ~S .g .B. The resulting effective g values, ~', are given in the diagram.

o f magnetization curves [7]. The intensity o f the MCD signal at a given wavelength is plotted against {3B/2kT, where/3 is the Bohr m a g n e t o n , B is the field in Tesla, k is Boltzman's constant and T the absolute temperature. Fig. 3 gives examples o f such curves for b o t h oxidation states o f the protein. Fig. 3a, for the oxidized ferredoxin II, is a plot o f the MCD intensity difference b e t w e e n the positive peak at 448 n m and the negative trough at 466 nm.

There is an advantage in plotting peak to trough distances wherever possible, since this eliminates possible errors due to base-line shifts during the course o f an e x p e r i m e n t . Data points have been recorded at fields o f 0.445, 1.019, 2.157, 3.089, 4.095 and 5.1 Tesla for each o f the temperatures 1.52, 1.945, 4.22, 11.1 and 22.5 K. All 30 data points are shown in Fig. 3a, in addition to the theoretical curve calculated on the assumption o f an isotropic ground-state g

97

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0

All are plotted in Fig. 3b. In addition, a theoretical curve is plotted which has been calculated on the assumption o f a low-lying state with g factors ofg//= 8.0 and gl = 0.20. The justification for this is given in the Discussion. A qualitatively similar magnetization curve has also been obtained by plotting the MCD peak intensity at 710 nm. Further data points at wavelengths of 351 and 380 nm have been measured at a single field of 5.1 Tesla and temoeratures of 34, 48.5, 68, 93 and 121 K. These points are plotted in Fig. 4 against 1IT. .05

.10

.~ (K -1) Fig. 4. MCD intensities at constant field plotted against lIT for the oxidized (o) and reduced (a) ferredoxin II. Aep, in arbitrary units, are the peak to trough distances at wavelengths of 448 and 466 nm and 380 and 351 nm, for the oxidized and reduced forms, respectively. The magnetic field was 5.1 Tesla. Temperatures are as follows: Oxidized: 11.1, 22.5, 36, 51, 69, 91, 117.5, 138, 162.5 K. Reduced: 9.5, 21.9, 34, 48.5,68,93,121 K.

factor of 2.01 [5]. The expressions o f Schatz et al. [13] have been used in the manner described earlier by us [7,11 ]. In addition, MCD data points have been collected at a single field of 5.1 Tesla and temperatures of 36, 51, 69, 91, 117.5, 138 and 162.5 K. These are plotted in Fig. 4 against 1/T in order to determine whether any low-lying, excited electronic states are populated over this temperature range. Fig. 3b shows the magnetization plots for reduced ferredoxin II, using the MCD intensity difference between the positive peak at 380 nm and the negative trough at 351 nm. Data points have been recorded as follows:

T (K)

Magnetic field, B (T)

1.53

0.146, 0.29, 0.445, 0.59, 0.88, 1.16, 1.415, 2.02, 3.089, 4.095, 5.1

1.995

0.29, 0.59, 0.88, 1.16, 1.415, 2.06, 3.089, 4.095, 5.1

4.215 9.5 21.9

0.445, 1.019, 2.157, 3.089, 4.095, 5.1 1.019, 2.157, 3.089, 4.095,5.1

Discussion The low temperature MCD spectra o f both the oxidized and reduced forms o f ferredoxin II from D. gigas are intense and dependent upon temperature. Hence the paramagnetism o f both oxidation states, first observed with M6ssbauer spectroscopy [3], is readily confirmed with MCD. The reduced state is EPR silent but gives a paramagnetic MCD signal. The analysis of the MCD magnetization curves yields the nature of ground-state spins. We discuss the curves of each oxidation state in turn. Oxidized ferredoxin 11. Fig. 3a shows that the MCD signal intensity at all fields and temperatures between 1.52 and 22.5 K falls on a smooth curve which approaches an asymptotic limit as BIT increases. The behaviour is characteristic of an electronic ground state which is a doublet with no lowlying zero-field components' thermally accessible over the temperature range 0 - 2 2 . 5 K. Using the expressions derived by Schatz et al. [13] it is possible to fit a curve to the experimental points of Fig. 3a using an isotropic g factor o f 2.0. When the ground state g factor is isotropic then there is no dependence o f the MCD magnetization curves upon the polarisation of the electronic transition and hence upon the wavelength. The EPR signal is not perfectly isotropic but can be simulated by choosing gl = 2.02, g2 = 2.00 and g3 = 1.97 using gaussian widths of 15, 35 and 80 G. [3] However, these values are tentative. The MCD magnetization curves for the two cases, namely, an isotropic g = 2.00 and the rhombic set with an average g value of 2.00 and a very small anisotropy, will not be distinguishable within the experimental accuracy. As anisotropy of the ground state increases

98 the MCD magnetization curves become increasingly dependent upon the polarization of the electronic transitions. Fig. 4 shows for the oxidized form o f ferredoxin II a plot o f the MCD intensity against lIT where T ranges from 11.1 to 162.5 K. The curve is sigmoidal with only a short linear section between 36 and approx. 7 0 K . At higher temperatures the curve begins to flatten off. This is evidence for the thermal population o f at least one excited electronic state at about 80 c m ' [ However, a much more detailed study o f the MCD in the range 1 0 - 3 0 0 K will be required before firm estimates can be made o f the magnitude of the exchange coupling. Reduced ferredoxin 11. The magnetization curves o f reduced ferredoxin II are of particular interest since no EPR signals are obtained from this state and the only data so far concerning the nature of this state are the M6ssbauer spectra [3]. Fig. 3b shows that the magnetization curves are o f quite a different character compared with those of the oxidized form. The points form a set of nested curves such that it is not possible to fit all the MCD intensity points at fields between 0 and 5.1 Tesla and temperatures between 1.53 and 21.9 K on a single, smooth curve. The curves are nested in such a manner that, at a given field, as the temperature is raised the MCD signal intensity falls more steeply than a smooth curve would predict. This implies that, as the temperature is raised, low-lying excited states are becoming thermally populated, but these states give weaker MCD signals than the lowest state. However, the magnetization curves measured at 1.53 and 1.995 K are superimposable with excellent precision. Therefore we conclude that below 2 K only the lowest level is populated. Moreover, because the curves at 1.53 and 1.995 K approach an asymptotic limit, that is, the MCD signal saturates, the ground state must be an electronic doublet. Comparison of the curves, Fig. 3b, with those in Fig. 3a, shows that the former magnetize much more steeply. There is a simple procedure which enables the ground state g factor to be estimated from a magnetization curve [7]. The ratio of the initial slope of the curve to the asymptotic limit gives the intercept, I, on the x-axis, see Fig. 3b. If the ground state is isotropic then g = lIT On the other hand if the ground state is completely axial, such that gu = 4S and g± = 0, then gu = 3 / 2 / [ 7 ] . From

Fig. 3b I = 0.185, giving an isotropic g value of 5.4 or anisotropic g values g//= 8.1, g± = 0. We reject the former value since an isotropic g value is usually close to 2.0. The latter estimate gives a value o r S = 2. This is the value used to simulate the magnetic M6ssbauer spectrum of the reduced form of ferredoxin II [3]. A theoretical magnetization curve can be computed on the assumption of an isolated electronic doublet with M s = -+2 as the ground state and g factors of gu = 8.0 and gi = 0.00 using the expression of Schatz et al. [13]. The fit to the experimental curve below 2 K is not perfect. However, an improved fit is obtained by allowing a small, non-zero g± component. The best fit is obtained with parameters gu = 8.0, g±= 0.20 and mz/m+ = 0.20 where mz/m+ is the polarization ratio of the z- to the x,y-polarized electronic transitions at the wavelength at which the MCD is being monitored. Fig. 3b shows the excellence of the fit. A state with S = 2 and an axial distortion which is much larger than any rhombic component will give a set of zero-field levels shown inset in Fig. 3b, with the effective g values indicated. Thus, as the temperature is raised above 0 K thermal population of M s = -+1, and 0 levels will take place. At temperatures such that kT is greater than the spread of the zero-field levels of the S = 2 state, the MCD signal intensity will obey the Curie law. Fig. 4 shows that the Curie law is obeyed between temperatures of 21.9 and 121 K. Hence all the zero-field levels of the S = 2 state are within approx. 15 cm -1 and no other electronic states lie closer than 85 cm -1. These two numbers are upper and lower limits, respectively, for those energy gaps and hence only place limits on the zero-field splitting and the exchange coupling parameters. Therefore we conclude that the electronic ground state of reduced ferredoxin II has S = 2, with a predominantly axial zero-field distortion leaving the lowest levels as M s = -+2. All five zero-field levels lie below approx. 15 cm -1, and no other electronic levels have been detected below approx. 85 cm -1. It is appropriate to add one or two comments about the possible limitations of our analysis. First, for complete verification of these proposals it is necessary to simulate a complete set of nested MCD magnetization curves. However, this task has not yet been accomplished even for a well-defined magnetic

99 state. The number of parameters that would be needed are high, and cumbersome expressions require to be computed. Thus, the approach to such a simulation will involve a preliminary analysis of the type carried out here to suggest values of S, the likely g values, and the likely range of the zero-field splitting parameters. Secondly, we have assumed that any rhombic splitting of the ground state is zero or negligible compared with the axial distortion. If the rhombic distortion, which leads to a zero-field splitting of the M s = -+2 levels, since we are dealing with a non-Kramers ion, is much smaller than Zeeman energies (g~B) then the MCD magnetization curves below 2 K will be insensitive to it. When the rhombic distortion is zero then gu = 8.0 and gi = 0. As the distortion increases then g± is replaced by gx :/=gy=/=0. Computation of the MCD magnetization curves for a rhombic paramagnet involves an increased number of parameters, including the polarisations of the electronic transitions. Therefore we have fitted the curve maintaining gx =gy =gi but allowing g± to depart from zero. Finally, the expressions which relate the g values to the initial slopes and asymptotic limits of a magnetization are strictly valid only for an isolated electronic doublet [13]. Specifically, field-induced mixing of the zero-field components of the ground state has been ignored. This assumption is only valid provided that go[3B ,~ D, where go is the g factor in the Zeeman term of the spin Hamiltonian andD is the axial zero-field splitting parameter. It is only under this condition that the effective g factor remains valid over all magnitudes of B, the applied field. The fact that the magnetization curves at 1.53 and 1.995 K saturate completely, giving field independent MCD signals above 4 Tesla, strongly supports the validity of the assumption. Effects arising from the second-order Zeeman effect will become most pronounced at high fields. We next consider briefly the form of the MCD spectra of the two oxidation states of ferredoxin II. There is a remarkable contrast between the spectra in Figs. 1 and 2. Both sets of MCD spectra reveal much more structure than the corresponding absorption spectra do even at 4.2 K. The MCD spectrum of the oxidized ferredoxin II reveals a considerable number of excited electronic states between 250 and 750 nm. Quantities of protein have, as yet, been insufficient to enable the spectra to be followed

down to 2 000 nm but there is no reason to assume that all the transitions have so far been detected. The spectrum appears to consist of two distinct regions. The first from 550-750 nm consists only of a weak tail in absorption but over seven positive and negative MCD peaks are recorded. It is natural to assign this region to intra-d shell transitions. The second region from 250-550 nm shows nine peaks or troughs below the broad absorption peaks at 305 and 430 nm. The intensity of these bands suggests they are associated with charge-transfer transitions from sulphur ligands to the ferric core. The MCD spectrum of oxidized Hipip at low temperature [ 11] also shows a large number of peaks across the region 300-800 nm. However, there is no correspondence between the peaks in the spectra from the different centres. Furthermore, the MCD intensity in the spectrum of oxidized ferredoxin II is fairly evenly distributed between negative and positive bands. This contrasts with the spectrum of oxidized Hipip in which most of the intensity is due to positive MCD peaks. When the exact structure of the three-ion centre has been defined a thorough assignment of the excited electronic states may well be possible with the aid of this distinctive MCD spectrum. Certainly the spectrum provides an excellent fingerprint to distinguish between two centres with very similar EPR characteristics that appear to have been confused in the past, [14,5] namely, the oxidized three-ion centre and the oxidized Hipip cluster. In contrast to the MCD spectrum of the oxidized ferredoxin II the spectrum of the reduced state shows much less structure. Many fewer peaks are found, pointing to a much lower density of excited electronic states in the reduced form. The spectrum has been measured beyond 750 to 1000 nm but only weak, positive peaks are found. There are some marked simi•a•ties between the MCD spectra of Fig. 2 and the MCD spectra of the reduced 4Fe-4S centre in the ferredoxin from Clostridium pasteuriahum [11]. Both spectra are dominated by positive MCD peaks, with a pronounced peak at 710 nm in ferredoxin II and at 730 nm in Cl. pasteurianum ferredoxin. Similarly the region between 400 and 500 nm contains a number of positive peaks. However, the critical distinction between the two reduced forms of the cluster is made because of the distinctive magnetization properties of the MCD signals from

100 ferredoxin II. As shown this saturates as an S = 2 system, whereas the reduced ferredoxin from Cl. pasteurianum magnetizes as an S = ½ Kramers doublet. L. In conclusion the low temperature MCD spectra of the three-iron centre in ferredoxin II can be unambiguously identified and distinguished from the four iron clusters in the C- and C 3- states in oxidized Hipip and reduced Cl. pasteurianum. It will be o f great interest to collect the MCD spectra o f other examples o f three-iron and four-iron clusters to discover how dependent upon origin they are.

Acknowledgements This work was supported by grants from the Science Research Council, the Royal Society and NATO (to A.J.T.), by the National Institutes of Health through Grant GM 25879, by the Instituto National de Investigaqao Cientifica, Portugal, and by the Calouste Gulbenkian Foundation, Portugal (to A.V.X.).

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