The low temperature magnetic circular dichroism spectra of iron-sulphur proteins II. Two-iron ferredoxins

Biochimica et Biophysica Acta, 493 (1977) 132-141 © Elsevier/North-Holland Biomedical Press BBA 37705

T H E LOW T E M P E R A T U R E M A G N E T I C C I R C U L A R D I C H R O I S M SPECTRA OF I R O N - S U L P H U R P R O T E I N S II. T W O - I R O N F E R R E D O X I N S

A. J. T H O M S O N a, R. C A M M A C K b, D. O. H A L L b, K. K. R A O b, B. B R I A T c, J. C. R I V O A L c and J. B A D O Z c a School of Chemical Sciences, University of East Anglia, Norwich, NPA 7TJ, b School of Biological Sciences, University of London, King's College, 68 Half Moon Lane, London SE24 9JF (U.K.) and Laboratoire d'Optique Physique, EPC1, 10, rue Vauquelin, 75231 Paris Cedex 05 (France) (Received October 7th, 1976) (Revised manuscript received M a r c h 21st, 1977)

SUMMARY

Variable temperature magnetic circular dichroism (MCD) spectra of a number of two-iron ferredoxins have been measured. The spectra of fully oxidised spinach and Spirulina maxima ferredoxin are independent of temperature between room temperature and 18 K, showing that no contribution to the room temperature M C D spectrum arises from the small population of low-lying excited states originating from the exchange coupling. However, the low temperature M C D spectra of the half-reduced proteins spinach and Spirulina maxima ferredoxin and adrenodoxin are all reasonably intense and temperature dependent. An interpretation of the spectrum of the charge-transfer region is suggested by starting with the assignments previously obtained from rubredoxin.

INTRODUCTION

Although no crystal structure determination of a two-iron two sulphur protein has yet been carried out, the structure of a synthetic model compound is known [1]. The model has spectroscopic properties closely resembling those of the proteins. The fully oxidised form contains two ferric ions whereas the half-reduced state formed by the addition of one electron contains, formally, one ferric and one ferrous ion [2]. To account for the diamagnetism at low temperature of the oxidised protein it has been proposed that there are two high spin ferric ions anti-ferromagnetically coupled [3]. This results in a ground state of total spin, S, equal to zero. The first excited state, S = 1, is populated above 77 K in oxidised spinach ferredoxin. The susceptibility measurements enabled a value of 365 cm -1 to be estimated for the separation between the S = 0 and S = 1 states [3]. The magnetic properties of the reduced form of the proteins can be accounted for in term of a pair of high-spin ferrous and ferric ions antiferromagnetically coupled to give a ground state with S = 1/2 and an excited state with S = 3/2. The separation between the two is estimated to be 200 cm -1 in

133 the case of spinach ferredoxin [3]. Adrenodoxin contrasts with these results [4]. The oxidised form remains diamagnetic up to 250 K so the paramagnetic excited state must be at least 700 cm -1 above the S -- 0 state, while the reduced protein exhibits a simple Curie law behaviour over the whole range up to 250 K. This again requires a sufficiently large separation between the S ~ 1/2 and S ~ 3/2 states in order that the S = 3/2 state remains virtually unpopulated at high temperature. MSssbauer spectroscopy confirms that the oxidised form of two-iron sulphur proteins contains two ferric ions whereas the reduced form possesses a ferric and ferrous ion with valences distinct on the M6ssbauer time-scale [5, 6]. In the case of oxidised spinach ferredoxin two pairs of quadrupole split doublets are observed, suggesting that the two ferric ions have inequivalent sites [5]. The spectra of oxidised adrenodoxin do not reveal clearly two types of ferric ion but the breadth of the lines suggests that this is so [6]. Previous attempts to understand the optical spectra of the two-iron sulphur proteins have concentrated upon the near infrared region of the spectrum below approx. 17 000 cm -1 [7]. Several bands are observable which have been interpreted as the ferric d-d bands in the oxidised protein and as a mixture of ferric and ferrous d-d transition in the reduced form. Positive assignment of these bands is of paramount importance as it provides confirmation of the dimer being a valence-trapped compound with iron atoms antiferromagnetically coupled. The absence of analogous bands in the four-iron sulphur clusters indicates that the compounds are valence delocalised clusters [8]. R o o m temperature M C D spectra of oxidised and reduced spinach ferredoxin have been reported, although no attempt at assignment was undertaken [9]. RESULTS Details of the technique and references to the method of preparation of the protein samples are given in the previous paper [10]. The low temperature M C D spectra have been recorded from three different two-iron sulphur proteins, namely, spinach ferredoxin, Spirulina maxima ferredoxin and adrenodoxin. The room temperature MCD, C D and absorption spectra of oxidised S. maxima ferredoxin are shown in Fig. 1. The magnetic field used was 5.5 T. It is important to employ as high a field as possible as the M C D and CD signals are comparable in magnitude even at this field strength. Since the magnetic susceptibility measurements show that in the oxidised form of some two-iron sulphur proteins the first excited state is populated at room temperature it is necessary to show that optical transitions out of this state do not contribute to the room temperature M C D spectrum. This is especially important to establish because the ground state is 1A 1 whereas the first excited state is aA 2. Thus transition out of the 3A 2 state could give rise to relatively intense M C D co terms whereas the transition originating from the 1m 1 state can be only al and b0 terms. The M C D spectrum of oxidised spinach ferredoxin at 18 K with a field of 4 tesla is shown in Fig. 2. Since the M C D signal is weak compared with the natural circular dichroism, three spectra were recorded, in positive, negative and zero fields. The M C D spectrum is shown at the bottom of the figure as well as the low temperature

134

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5.0 Fig. 1. T h e a b s o r p t i o n (upper curves) a n d r o o m t e m p e r a t u r e M C D a n d C D spectra o f oxidised S. maxima ferredoxin. B = 5.5 tesla; concentration = 1 . 2 8 m M ; p a t h length = 1 . 0 r a m . Tris buffer, 0.8 M NaC1, p H 8.0. Left h a n d scale on lower curve refers to C D a n d right h a n d scale to MCD.

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Fig. 2. T h e absorption ( - - ' - - ) s p e c t r u m of oxidised spinach ferredoxin. T h e three u p p e r curves are the circular dichroism spectra with B = =k 4 tesla ( . . . . . . . . . ) a n d zero ( . . . . . . ). T h e M C D s p e c t r u m derived f r o m these is s h o w n below ( x • × • x ). All m e a s u r e m e n t s were m a d e on the s a m e sample at 18 K. C o n c e n t r a t i o n -- 1 m M ; p a t h length = 1.0 m m . Tris buffer, 0.8 M NaCI, p H 8.0, saturated with sucrose.

135

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Fig. 3. The absorption ( . . . . . . ), CD (-- --) and MCD (-) spectra of half-reduced spinach ferredoxin. Conditions as for Fig. 2 with the addition of sodium dithionite.

a b s o r p t i o n spectrum. T h e M C D spectrum is u n ch an g ed as c o m p a r e d with the r o o m t e m p e r a t u r e spectrum previously published [9]. A similar result was o b t a i n e d for oxidised S. m a x i m a ferredoxin. T h e r e f o r e we conclude that the small p o p u l a t i o n o f the 3A 2 state at r o o m t e m p e r a t u r e does n o t c o n t r i b u t e to the M C D spectrum in the ~

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136 case of spinach and spirulina maxima ferredoxin. The magnetic susceptibility studies show no population of the 3A2 state in oxidised adrenodoxin [4]. The M C D spectra and natural circular dichroism spectra of the reduced forms at 6-7 K are shown in Figs. 3 and 4. The M C D spectra are temperature dependent, as expected for a 2A1 ground state; they are also more intense and structured than our natural circular dichroism spectra. It is important to note that the natural CD spectra at low temperature agree well with those reported for room temperature [11]. The CD spectrum provides an excellent internal calibration of the optical quality of the low temperature sucrose glasses. Nevertheless it is necessary to establish with certainty the position of the base-line. This can be achieved by carrying out reverse field and zero field experiments as shown in Fig. 2. However, an alternative and valuable technique is to study the signal as a function of magnetic field. In order to illustrate the point we show Fig. 5 which is a photograph of the actual recorder traces obtained from reduced spinach ferredoxin. The spectrum was recorded three times at 0, 2 and 4 tesla. The quality of the glass and the strength of the M C D signal at 6.2 K led to the excellent results shown.

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~.j a'l I03CM"~ Fig. 5. Direct tracings of the recorder chart for the spectra shown in Fig. 3. The three spectra were run with B -- 0.2 and 4 tesla respectively. DISCUSSION The spectra obtained are clearly complex and contain many overlapping bands so that any assignment, even with the aid of M C D spectra, must be only tentative. However, we offer here one plausible assignment which takes as its starting point our interpretation of the rubredoxin charge-transfer spectrum which has been considerably clarified by the M C D spectra [10]. Since the two iron-sulphur centres contain exchange-coupled pairs of valencelocalised iron atoms, we regard them as two distinct FeS4 chromophores. The spectra are then the sum of two ferric units in the case of the oxidised protein and the sum of

137 one ferric and one ferrous unit in the reduced protein. This approach can be adopted provided that the exchange forces between the units are small compared with interelectronic repulsion and crystal field splittings within a single centre. Although the exchange field strengths in the excited states are not known the ground state values are small compared with the two perturbations. Therefore our model implies two FeS4 chromophores with effective spin determined by the exchange coupling. Furthermore, it is clear from the spectra of the oxidised and reduced rubredoxin that, below 27 000 cm -1, the only contribution to the spectrum comes from the ferric centre involving two single electron chargetransfer transitions from sulphur [10]. Indeed, consistent with this interpretation is the observation that, on reduction of one ferric ion, the intensity of the absorption spectrum between approx. 27 000 and 17 000 cm -1 drops by one half. In the two iron proteins there are two types of sulphur ligand, the acid labile S2- and the -CH2-S- group of the cysteinyl residues. Charge transfer transitions from Sz- to tetrahedral ferric ion have been identified in the Fe(III) doped into GazSa [12]. They occur at energies reasonably close to their counterparts in rubredoxin. Therefore, although transitions from both Sz- and -CH2-S- ligands to ferric ion will be present in the visible spectrum of the dimer they appear to lie at similar energies and may not be resolved. With these assumptions it is possible to draw up the energy level schemes shown in Fig. 6. The symmetry of the Fe(II1)S4 chromophore is assumed to be D2d. The strict crystallographic point group is presumably Czv and this will lift the degeneracy of the E state but will not necessarily alter the form of the MCD spectrum if the departure from Dzd symmetry is small. Interpretation of the near infrared d-d spectrum of spinach ferredoxin has led to the suggestion of an effective Dzd symmetry for the FeS4 unit [7]. Our interpretation of the rubredoxin spectrum [10] implies that the lowestenergy sulphur to ferric ion charge-transfer band will involve the orbital transition from the non-bonding sulphur molecular orbital tl to the e d-orbitals of the ferric ion. This gives rise to a T2 state of tetrahedral parentage which splits into E and Bz components under a Dza distortion. Squashing the tetrahedra will bring the Bz component lower in energy. The exchange coupling gives an effective spin to the state. In the case of the oxidised form of the protein a set of energy states are produced by coupling with the neighbouring ground ferric ion as shown in Fig. 6a. However, as we have shown that only the transitions from the 1A1 state contribute to the spectrum, even at room temperature, we need consider only spin-allowed transitions within the spin singlet manifold. Therefore the lowest energy band would consist of a b0 term followed at higher energy by an a~ term of negative sign. The analysis of the lowest energy charge-transfer bands of the thioanion MS4 z- can be applied [13]. This is apparently observed (Fig. 2) in the MCD spectrum of the oxidised protein, S. maxima, between approx. 23 000 and 17 000 c m - L Comparison of the absorption spectrum of the two-iron cluster with that of rubredoxin shows that the peak at 24 000 cm -a in the former has no obvious counterpart in the latter. One possibility is that these two ferric centres in the two-iron protein are sufficiently different to give two sets of overlapping spectra separated in energy by the gap between the peaks at 24 000 cmand 22 000 cm-1. The MCD should then be the result of two overlapping al and b0

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Fig. 6. Schematic energy levels for a one-electron transition, sulphur-to-ferric, in (a) fully oxidised and (b) half-reduced two-iron protein. The states of the uncoupled ions are shown in parentheses. In (b) assumption is made of couplings between the excited states of a ferric centre (6E and 6Bz under D2d) and a ground state ferrous ion (SA~under D2d). (See ref. 15). On the right-hand side of (b) the results of applying a spin-orbit perturbation, weaker than exchange coupling, is shown for the S = 1/2 manifold only.

terms o f the same signs which w o u l d generate the shape observed between approx. 27 000 a n d 15 000 cm -1. In s u p p o r t o f this view we note that the spectrum o f the t w o - i r o n analogue c o m p o u n d , [FeS(SCH2)zC6H4] 2-, contains only one p e a k at approx. 24 000 cm -~ and a b r o a d shoulder to low energy [1]. However, all the p r o teins show a distinct splitting o f this peak. Clearly the M C D spectrum o f a m o d e l c o m p o u n d would be o f great value. The high energy region a b o v e 27 000 cm -1 is then p r e s u m e d to contain the second charge-transfer t r a n s i t i o n o f two Fe(III)S4 units overlapping. W e merely note

139 the similarity of the sign of the al term centred at 29 500 cm -1 with the sign of the second charge-transfer transition of many thioanions, such as VS a-, WS]- and MoS]- [131. In the half-reduced protein there is one Fe(III)S4 unit. The energy level scheme of Fig. 6b was built up in the following manner. The assumed geometry is D2d. The ferric unit is exchange coupled with a ground state Fe(II)Sa unit leading to a set of levels characterised by different spin states. The ground state of the ferrous ion may be either 5A1 o r SB 1 depending on whether the sixth electron occupies the dz~ or dx2_y2 orbital. It turns out to be immaterial to the resulting form of the electronic transitions whether we assume a SA~ or 5B~ ground state for the ferrous unit. The resulting effective spins, after exchange coupling, are S = 1/2, 3/2, 5/2, 7/2 and 9/2. Only the lowest 2A 1 ground state is populated at the temperature at which MCD spectra were measured. The fact that the resulting MCD spectra are temperature dependent even though the ground state is an orbital singlet implies that spin-orbit coupling in the excited states is significant and must be taken into account. Inclusion of both spinorbit coupling and exchange interaction can result in a highly complex level scheme. For example, as spin-orbit coupling mixes states which differ in their spinangular momentum, manifolds of different total S are scrambled. In the limit of a large spin-orbit perturbation compared with exchange interaction all states, regardless of their S values, become spectroscopically accessible from the 2A~ ground state. Such a situation would be virtually impossible to interpret. However, if the exchange coupling is much larger than spin-orbit coupling then it is permissible to derive the resulting spin-orbit components merely by coupling the spin and orbital moment of each exchange-coupled component. An example of this approach is shown in Fig. 6b. The unknown factors are the magnitudes of the exchange forces operating in the excited states and those of spin-orbit coupling. Our rubredoxin analysis suggests that at least in the first allowed excited configuration, the spin-orbit coupling constant is about 300 cm -1. However, we have no means of estimating the magnitude of exchange forces in the excited state. If we assume that the exchange coupling is larger than spin-orbit coupling then the energy level scheme of Fig. 6b may be appropriate. In this case the MCD spectrum arising from the one-electron transition h ~ e will consist of three c0 terms, two of one sign, (E'---~ E") and one of the opposite sign (E' -+ E'). It is therefore worthwhile to see if the observed spectra can be interpreted in terms of a model with J >> ~" in the excited state, corresponding to the limit shown in Fig. 6. Figs. 3 and 4 show how remarkably similar are the spectra of spinach and spirulina maxima ferredoxin both species having similar properties and redox potentials [5]. However, adrenodoxin is markedly different, in accord with the known differences in the magnitude of the exchange coupling in this protein [3]. Clearly the spectra are sensitive to the magnitude of the exchange coupling which presumably is related to the iron-iron distance. The portion of the spectra below approx. 18 000 cm -a arises from the d-d transitions and no attempt to interpret this region is made here. Potentially this region is simpler to understand than the charge-transfer spectrum. The sharp positive peak just above 18 500 cm -~ is a feature of the MCD spectra of all three proteins and we

140 leave its assignment on one side at this point. The peak at approx. 21 500 cm -~ in the low temperature absorption spectrum of spinach ferredoxin presumably corresponds to the 2A 1 --> 2E transition with the 2B 2 shoulder to low energy. Then the three M C D peaks, two positive on either side of the small negative peak at approx. 20 500 c m -1, would correspond to the three spin-orbit components resulting from the 2E and 2B 2 states. Three further M C D peaks, two positive and the large negative one, can be found under the second absorption maximum at 25 000 cm -1. The main features of this analysis are still discernible in the spectrum of adrenodoxin. We return to the question of the assignment of the prominent positive peak at approx. 19 000 cm -1. This corresponds to the absorption maximum at this same energy and has no clearly resolved counterpart in the spectrum of the oxidised proteins. It could be that this peak results from a mixed valence transition Fe(II) -+ Fe(III). I f so, it is remarkably narrow for such a transition and its energy invariance from protein to protein would be of great interest. However, such an assignment is admittedly conjectural. CONCLUSION We have attempted to show how the charge-transfer spectra of oxidised and reduced two-iron sulphur proteins are related to the analogous spectrum of the single iron protein rubredoxin which we have been able to assign with some certainty. The type of energy level scheme likely to be appropriate for a full and rigorous assignment has been discussed. It is clear that there are so many parameters of unknown magnitude and a large number of overlapping transition anticipated that an unambiguous assignment cannot be made with certainty. However, we have shown that, at low temperature, the paramagnetic, halfreduced two-iron cluster has a reasonably intense M C D spectrum. A large range of well-characterised proteins need to be examined now before it is possible to conclude that the M C D spectrum is diagnostic of this type of centre. Nevertheless, it would be of great interest to attempt to detect, at low temperature, the M C D spectrum of a two-iron sulphur centre in proteins suspected of containing them but which also contain other chromophores. Xanthine oxidase, containing molybdenum and flavin as well as two iron-sulphur centres, is one example [14]. ACKNOWLEDGEMENTS The Science Research Council has provided funds for preparative work (King's College), apparatus and travel (Norwich). One of us (A.J.T.) was the recipient of a Ciba-Geigy Senior Fellowship. REFERENCES 1 Mayerle, J. J., Frankel, R. B., Holm, R. H., Ibers, J. A., Phillips, W. D. and Weiker, J. F. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2429 2 Tsibris, J. C. M, and Woody, R. W. (1970) Coord. Chem. Rev. 5, 417 3 Palme, G., Dunham, W. R., Fee, J. A., Sands, R. H., Iizuka, T. and Yonetain, T. (1971) Biochim. Biophys. Acta 245,201

141 4 5 6 7 8 9 10 11 12 13 14 15

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