Low-temperature magnetic circular dichroism spectra and magnetisation curves of 4Fe clusters in iron-sulphur proteins from Chromatium and Clostridium pasteurianum

433

Biochimica et Biophysica Acta, 667 (1981) 433--451 © Elsevier/North-Holland Biomedical Press

BBA 38623

LOW-TEMPERATURE MAGNETIC CIRCULAR DICHROISM SPECTRA AND MAGNETISATION CURVES OF 4Fe CLUSTERS IN IRON-SULPHUR PROTEINS FROM C H R O M A T I U M A N D C L O S T R I D I U M P A S T E U R I A N U M

MICHAEL K. JOHNSON a, ANDREWJ. THOMSON a, A. EDWARDROBINSON a, KRISHNA K. RAO b and DAVID O. HALL b a School o f Chemical Science, University OfEast Anglia, Norwich, Norfolk, NR4 7TJ and b Department o f Plant Sciences, King's College, 66 Half Moon Lane, London, SE24 9JF (~.g.)

(Received October 9th, 1980) Key words: Ferredoxin; High-potential iron protein; Iron cluster; Magnetic circular dichroism; Magnetization curve; (Chromatium, Cl. pasteurianum)

Summary The magnetic circular dichroism (MCD) spectra of the 4Fe clusters in the iron~ulphur proteins high-potential iron protein from C h r o m a t i u m and the 8Fe ferredoxin from C l o s t r i d i u m p a s t e u r i a n u m have been measured over the wavelength range 300--800 nm at temperatures between approx. 1.5 and 50 K and at magnetic fields up to 5 tesla. In both cases the proteins have been studied in the oxidized and reduced states. The reduced state of high-potential iron protein gives a temperature-independent MCD spectrum up to 20 K, confirming the diamagetism of this state at low temperature. The MCD spectrum of samples of oxidized ferredoxin invariably show the presence of a low concentration of a paramagnetic species, in agreement with the observation that the EPR spectrum always shows a signal at g = 2.01. The pammagnetic MCD spectrum runs across the whole of the wavelength range studied and therefore most probably originates from an iron-sulphur centre. The diamagnetic component of the MCD spectrum of oxidized ferredoxin is very similar to that of reduced highpotential iron protein. The low-temperature MCD spectra of oxidized highpotential iron protein and reduced ferredoxin reveal intense, temperature~lependent bands. The spectra are highly structured with that of high-potential iron protein showing a large number of electronic transitions across the visible region. The MCD spectra of the two different oxidation levels are quite distinctive and should provide a means of establishing the identity of these state of 4Fe clusters in more complex proteins. MCD magnetisation curves have been construcAbbreviations: H/pip, high-potent[s] iron protein; #B, Bohr magneton.

434

ted from detailed studies of the field and temperature dependence of the MCD spectra of the two paramagnetic oxidation states. These plots can be satisfactorily fitted to the theoretically computed curves for an 8 = 1/2 ground state with the g factors experimentally determined by EPR spectroscopy. The low-temperature MCD spectra of the reduced 2Fe-2S ferredoxin from Spirulina maxima are also presented and MCD magnetisation curves plotted and fitted to the experimentally determined g factors.

In~oduction

The group of iron-sulphur proteins containing 4Fe-4S clusters have been extensively studied by EPR [1~2] and Mossbauer [2] spectroscopy in order to characterise their electronic structures and also to provide spectroscopic criteria which can be used to recognise the clusters in more complex proteins. X-ray crystallography [3,4] has established that the cluster has an iron atom at each of the four alternate corners of a distorted cube, each iron being bound to a cysteine sulphur and three of the four inorganic sulphur atoms which occupy the remaining comers of the cube. This structure is present in the high-redox potential iron-sulphur protein from Chromatium [5] which has a molecular weight of 10 000 with four iron and acid-labile sulphur atoms per molecule. It is also seen in the low-potential eight iron ferredoxin from Peptococcus aerogenes [4], which has a molecular weight of 6000, with eight iron and eight sulphur atoms in two identical clusters 12 ~ apart. The same structure is assumed to be present in the 8 F e ~ S ferredoxin from Clostridium pasteurianum [2]. This assumption is based upon the similarity of the EPR and Mossbauer spectra of the clusters in these proteins to those in P. aerogenes [2]. The magnetic properties of the four iron cluster have been rationalised by the three~xidation~state hypothesis of Carter et al. [6]. This hypothesis now is supported by a wealth of evidence both on proteins themselves [5] and on inorganic models [7]. According to Carter et al. [6] the 4Fe-4S cluster can adopt one of three possible oxidation levels differing by one electron per cluster. Oxidised high-potential iron protein (Hipipox) contains a cluster isoelectronic with [Fe4S4(S~ys)4]-, where S-cys is the anion of cysteine. This state, denoted C-, is paramagnetic with spin, S = 1/2, resulting in an apparently axial EPR spectrum with g~ = 2.12 and g± = 2.04 although additional structure in the spectrum suggests a second rhombic EPR component with gl = 2.086, g2 = 2,055 and gs = 2.040 [8]. Studies of the temperature dependence of the intensities of the EPR signals indicate the presence of an excited state at 160 + 10 cm -1 above the ground state [9]. Magnetic susceptibility .data also reveal an excited state approx. 200 cm -1 above the EPR active ground state. On one electron reduction to give Hipip~d a state, denoted C 2-, is produced which is diamagnetic at low temperatures. Susceptibility evidence shows no electronic states between the ground state and at least 400 cm -1 [8]. Further one~lectron reduction of Hipip protein to produce an oxidation state C 3- is only possible if the protein is unfolded by the addition of 80% dimethylsulfoxide [10]. The clusters in the eight-iron ferredoxin from C1. pasteurianum undergo the one~lectron reduction from states C 2- to C s-. The C 2- is presumed to be dis-

435

magnetic at low temperatures. However, an EPR signal which is isotropic with g = 2.01 is always present in oxidised samples of Cl. pasteurianum ferredoxin. It has been suggested that this signal may arise from the presence of a small population of clusters in the C- state, that is, super~xidised clusters [11]. Treatment of the protein with ferricyanide increases the intensity of this signal. The C3- state is paramagnetic, each cluster having a spin S = 1/2, although the eightiron ferredoxin gives rather complex EPR signals due to weak spin coupling between the two clusters within the protein [12]. Almost no magnetic susceptibility data have been published on proteins containing clusters in the C 3state. The magnetic moment of reduced ferredoxin from Bacillus polymyxa has been determined between 276 and 295 K to be 3.4--3.2 Bohr magnetons per four Fe atoms. [13] This is much higher than the value expected from the EPR spectrum measured at liquid helium temperatures and suggests the thermal population, at room temperature, of excited electronic states. We have been investigating the magnetic circular dichroism (MCD) spectra of iron~ulphur proteins both to provide an additional set of spectroscopic criteria for identifying clusters and also to gather data potentially useful for the assignment of the electronic spectra of the clusters [14--18]. In this paper we present the MCD spectra measured at fields between 0 and 5.3 tesla over the temperature range 1.5--200 K of the C- and C 2- states of Hipip iron protein from Chromatium and the C 2- and C 3- states from Cl. pasteurianum. This study complements and adds to the low-temperature MCD spectra previously reported for the oxidised state of rubredoxin from Cl. pasteurianum [14] and for the oxidised and reduced states of the two-iron centres from Spirulina maxima, spinach ferredoxins and adrenodoxin [15]. The MCD spectra of paramagnetic chromophores are invariably temperature
Instrumentation. Absorption spectra were recorded in a Cary 14 and a Cary 17 spectrophotometer. Magnetic circular dichroism spectra were measured with a JASCO~I500D spectropolarimeter fitted with an Oxford Instruments Ltd. SM4 superconducting solenoid, capable of generating magnetic field up to 5.3 tesla. Samples were placed in specially constructed cells of measured

436

path length and frozen by immersion in liquid helium. The cells consist of two thin (1 mm thick) spectrosil silica plates spaced by a rubber gasket about 1 or 2 mm thick. The gasket is circular in shape so that the cell is sealed. This cell can then be degassed and filled by means of a narrow syringe needle which pierces the gasket. This design of cell is simple and cheap to make and is effective at holding samples anaerobic for sufficient time to permit loading of the cell into the helium atmosphere of the magnet. The optical quality of the glasses is assessed in two way. First, MCD spectra are measured at two fields, positive and negative, and at zero field. The MCD spectra measured at the two field must form mirror images of one another reflected in the zero field baseline. Secondary, the depolarisation of the light beam is assessed by measuring the natural circular dichroism of a sample of D-tris(ethylenediamine)cobalt(III) chloride placed after the magnet, with and without the sample in position, in the absence of an applied field. Throughout this work the sample depolarisations measured were never greater than 5%, and were usually zero. The magnetic fields experienced by the sample were measured with a Hall probe positioned in place of the sample. It is important to calibrate the magnet in this way rather than relying upon manufacturers quoted fields since the presence of magnetic components, such as shields, near to the magnet can markedly affect the strength of the field at the centre of the solenoid. Sample temperatures in the range 1.5--4.2 K were obtained by having the sample immersed in a bath of pumped liquid helium and measured using a carbon~lass resistor, calibrated over the range 1.5--300 K by Cryogenic Calibrations Ltd., Pitchcott, Nr. Aylesbury, U.K. Temperatures were held constant by applying a controlled reduced pressure above the liquid helium using a manustat (Oxford Instruments Ltd.). Sample temperatures in the range 4.2--200 K were achieved by having the sample in a stream of cold helium gas and were measured using both a cryogenic linear temperature sensor (C.L.T.S.) and a carbon~glass resistor. The C.L.T.S. and a heater on the sample block were connected to an Oxford Instruments DTC2 temperature controller which maintained constant temperature. EPR spectra were measured in a Varian E-3, X-band, EPR spectrometer fitted with an Oxford Instruments E.S.R.-9 continuous flow cryostat. Materials. Sp. maxima ferredoxin [20], Chromatium Hipip [21,22] and Cl. pasteurianum [23] ferredoxins were prepared as previously described. Concentrations were measured using the published extinction coefficients as follows: Sp. maxima ferredoxin e420 = 9700 (oxidised) [20]; Chromatium Hipip esTs = 20000 (oxidised) [21], and Cl. pasteurianum ferredoxin e390 -- 30 600 (oxid~ed) [24]. Hipipo~ was produced by oxidation with KsFe(CN)6 added as crystals until no further change in the absorption spectrum was observed. The excess oxidant was removed on a Sephadex G-25 column. All other proteins were reduced with addition of sodium dithionite either as a concentrated solution or a solid ground up with solid buffer. The solutions for measurement were diluted to a volume of 50% (by vol.) with ethylene glycol in order to give a solution which formed a good optical quality glass.

437

Results

Fig. 1 shows the M C D spectra of Hipipox from Chromatium at a series of temperatures between 1.48 and 50 K at 5.3 tesla.Also given for comparison is the room temperature M C D spectrum previously published [17]. The absorption spectra at room temperature and at 9 K are also shown. The broad, unstructured absorption spectrum sharpens slightly on cooling to 9 K and some slight shoulders are visible on the long-wavelength edge of the main peak. It is not possible to be certain whether the increase in the intensity of the absorption m a x i m u m is real or apparent since, inevitably, some scattering is present in the low-temperature glass which leads to a steepening of the baseline

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towards the ultraviolet. In contrast to the rather slight changes in the absorption spectrum the MCD spectrum changes dramatically on cooling. The broad envelope of positive MCD peaks are resolved into sharper bands, some with negative signs. The intensity of the spectrum is increased. The temperature and magnetic field dependence of the MCD is shown in Fig. 2. Two regions of the spectrum are plotted. The ordinate is the peak-to-trough distance at wavelengths of 393 and 437 nm and wavelengths of 513 and 554 nm, respectively, and the abscissa is ~BB/2kT, where ~B is the Bohr magneton, B is the magnetic flux density in tesla, k is Boltzmann's constant and T is the absolute temperature. Fig. 2 constitutes a magnetisation curve. There are two regions, namely, a linear portion at high temperatures and/or low fields when g~BB < < kT and an asymptotic limit which is approach as #BB/2kT goes to infinity. The latter limit is reached as g~sB >> kT and corresponds to complete magnetisation of a pammagnet and is known as the saturation limit. Fig. 3 gives the room temperature absorption and MCD spectra of reduced Hipip protein from ghromatium, and also the MCD spectrum at 4.2 and 20 K. The absorption has also been measured at low temperature (1.5 K) but there is no significant sharpening of the peak or evidence for new bands. However, the low-temperature MCD spectrum shows considerably more resolved features than the room temperature spectrum although the major peaks remain. The virtual coincidence of the MCD spectra at 4.2 and 20 K proves the dia-

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Fig. 3. R e d u c e d Chrornatium high-potentSal i r o n (Hipip~ed). Upper parLel: ~ - , a b s o r p t i o n speet~cum, 2 9 5 K, 50% (v/v) e t h a n e d i o l , a q u e o u s T r I ~ H C I b u f f e r , p H 7.4. - - - - - - , MCD, 2 9 5 K, 5 t e ~ a . L o w e r p a n e l : MCD, 1.08 m m p a t h l e n g t h , 50% (v/v) e t h a n e d i o l , a q u e o u s Tris-HCI buffex, pH 7.4, B = 5 teala: • 4 . 2 2 K, a n d - - - - , 2 0 K. P r o t e i n c o n c e n t z a t i o n ffi 4 2 2 / ~ M .

magnetism of this state of the protein and, moreover, shows the complete absence of any paramagnetic impurity absorbing in this spectral region. Excellent agreement is obtained between the Ae values obtained at low temperature and room temperature. The low-temperature MCD spectra of the oxidized and reduced forms of Cl. pasteurianurn ferredoxin have been determined and are given in Figs. 4 and 6. The EPR spectrum of the samples used have been checked and Fig. 7 gives the spectra obtained. The EPR spectrum of the dithionite-reduced form of the protein in 50% ethylene glycol, given in Fig. 7a, should be compared with the spectra reported under different solvent conditions by Cammack [25 ]. The EPR spectrum is very sensitive to solvent and salt concentrations, presumably as a result of subtle changes in the spin-spin interaction between clusters. The spectrum of Fig. 7a is intermediate between the forms of the spectra of t h e reduced protein in 10 mM Tris-HC1 buffer, pH 8.0, and of the reduced protein with 10% dimethylsulfoxide (by vol.), although more similar to the former. In the oxidised state the signal at g = 2.01 is observed (Fig. 7b) although much weaker in intensity.

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Returning to consider the MCD spectra of these oxidation states, Fig. 4 presents the data for the dithionite-reduced state. The MCD spectra at three representative temperatures, namely, 1.55, 4.22 and 18.7 K, show strong temperature dependence. Comparison of the spectra with those measured at room temperature show the wealth of spectral detail that is resolved by the low-temperature MCD spectra. The signals have increased by a factor of about twenty on cooling from 300 to 1.55 K. The room temperature absorption spectrum is also given in Fig. 4. It has also been measured at 1.55 K but is not given since no significant features emerge.

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The temperature and field dependence of the MCD spectra are displayed in the form of a magnetisation curve (Fig. 5) plotted for the prominent positive peak at 530 nm. Curves of a similar shape were obtained for other peaks in the spectrum, showing that it contains no contributions from other paramagnets. The MCD and absorption spectra of the oxidised form of the protein are given in Fig. 6. The upper panel shows the room temperature absorption and MCD spectra, replotted from Ref. 17. The absorption spectrum at 1.55 K is also given. Some new shoulders are resolved at long wavelength and a new peak at 320 nm is also resolved. It may be that the increase in intensity towards shorter wavelength at low temperature is caused by scattering from a glass of imperfect optical quality. The middle panel shows the MCD spectra at 5.1 tesla and temperatures of 1.95, 4.22, 18.0 and 49.0 K. The temperature dependence of the spectrum proves that a paramagnet is being detected. However, comparison of the Ae values with those of Fig. 4 shows that the paramagnetic component is only present in low concentration, less than 10% of the protein in all probability. Therefore at higher temperatures the MCD spectrum becomes dominated by the contribution from the major component of the protein even thought it is diamagnetic. This is borne out by the experiments. The MCD spectrum at 49.0 K is very similar to the room temperature spectrum except with considerably increased resolution. It is also similar to the low-temperature MCD spectrum of reduced Hipip (Fig. 3). By measuring the MCD difference spectrum between the 1.95 and 49.0 K MCD spectra the form of the pammagnetic MCD spectrum is obtained since the diamagnetic contribution remains unchanged in intensity with temperature. This difference spectrum is plotted in the lowest panel in Fig. 6. Hence this presumably represents the MCD spectrum of the species giving rise to the g = 2.01 signal in the EPR spectrum of the oxidized ferredoxin. We have measured the low-temperature MCD spectra of a number of different samples of the oxidised ferredoxin from Cl. pasteurianum and invariably a paramagnetic contribution to the signal is obtained. This is consistent with

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Fig. 7. (a) EPR spectrum of reduced Cl. pasteurfanum ferredoxin in 50% (v/v) ethanediol, a que ous 0.8 M NaC1/20 mM Tris-HCl buffer, pH 8.5, in the presence of e x e e u s odi um dithtonite. T e m p e r a t u r e 10 K. Protein c o n c e n t r a t i o n ffi 360/~M. S p e c t r o m e t e r settings: 2 mW microwave power, 0.63 mT m o d u l a t i o n amplitude, Gain × 1, microwave frequency 9.25 GHz. (b) EPR s pe c t rum of oxidised CI. pasteurianum ferredoxin in 50% (v/v) ethanediol, aqueous 0.8 M NaCI/20 mM Tris-HCl buffer, pH 8.5. Temperature 10 K. Protein c o n c e n t r a t i o n ffi 360 /aM. S p e c t r o m e t e r settings: 2 mW microwave power, 0.63 mT m o d u l a t i o n amplitude, Gain × 10, microwave frequency 9.25 GHz.

reports of the invariable presence of an EPR signal from the oxidised form of this protein [ 11]. Finally, the low-temperature MCD spectra of the reduced ferredoxin from Sp. maxima are shown in Fig. 8. Although this ferredoxin contains a 2Fe centre we show the spectra for comparison with the 4Fe MCD spectra. MCD spectra of the reduced form of this protein at 6.5 K have been published previously [15]. The present data extend considerably the spectra in two respects. First, the MCD spectrum has been recorded at wavelengths down to 250 nm. By using minimal quantities of dithionite the MCD signals due to the iron-sulphur centre have been detected below the dithionite absorption itself which peaks at 314 nm. We have checked that anaerobic sodium dithionite gives no temperature~lependent MCD signals. It has also been possible to follow the MCD signal underneath the absorption due to the aromatic amino acids of the protein. Second, the MCD spectra have been recorded at several magnetic fields between 0 and 5 tesla at temperatures of 1.54, 2.08 and 4.22 K. From the spectra magnetisation curves can be constructed (Fig. 8). The curves are plotted for the MCD negative peak at 330 rim and the positive peaks at 670 nm.

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F i g . 1 0 . M C D m a g n e t i s a t i o n c u r v e s , p l o t s o f E q n . 1 7 i n R e f . 2 8 ; gR ffi 2 . 0 6 , g± ffi 1 . 9 0 . L e f t - h a n d p a n e l : P l o t s i n axbitrax,y, r e l a t i v e i n t e n s i t y u n i t s f o r v a l u e s o f p o l a x i s a t i o n r a t i o m z / m + i n d i c a t e d . R i g h t - h a n d p a n e l : P l o t s n o r m a l i s e d t o a s y m p t o t i c l i m i t o f u n i t y i n a r b i t r a r y u n i t s . P o l a r l s a t i o n r a t i o s : i , + 0 . 2 ; fl, + 1 . 0 , ifl~ 0 . 6 , a n d iv, + 0 . 4 . . . . . . . . i n i t / e l s l o p e a n d a s y m p t o t i c l i m i t o f c u r v e (ii) giving a n i n t e r c e p t v a l u e (I) o f 0.51, marked by an arrow on the abscissa.

Discussion The results presented in the previous section demonstrate that the ultra-lowtemperature MCD spectra of the C- and C 3- oxidation levels of 4Fe-4S clusters in iron~ulphur proteins are relatively intense and are rich in spectra detail. Since these two oxidation levels are known from other techniques, such as EPR and Mossbauer spectroscopy, to be paramagnetic at 4.22 K it was to be expected that intense temperature
446

visible region. There is no sharp distinction possible between the two oxidation levels at room temperature. However, the M C D spectra at ultra-low temperature are well resolved, consisting of both positive and negative peaks. The broad bands observed at room temperature clearly consist of a number of electronic transitions. At low temperature the M C D spectra of the C- and C 3- oxidation levelsare very different in a distinctive way. The larger number of peaks and troughs in the M C D spectrum of Hipipox compared with the M C D spectra of ferredoxin implies a higher density of electronic states in the visible spectrum of the C- cluster compared with the C 3- cluster.'There is no detailed theoretical description of the excited states of 4Fe clusters but presumably the transitions involve charge transfer from sulphur-based orbitals to empty metal orbitals, ferric in character. A reduction in the density of excited charge-transfer states is therefore to be expected as the cluster is reduced from C- to C s- and the number of formal Fe 3÷ is changed from three to one. The diamagnetism of the C 2- cluster in reduced Hipip below 20 K is immediately evident from Fig. 3. This contrasts with the difficulty experienced in obtaining completely diamagnetic samples suitable for susceptibility studies which apparently possessed a small residual of oxidised protein [8 ]. The presence of the higher oxidation state would be immediately recognised from the form of the M C D spectrum (Fig. 1). All the samples of oxidised ferredoxin from Cl. pasteurianum examined by low-temperature M C D spectroscopy contain a pammagnetic component which has absorption bands between 300 and 800 n m and is therefore an iron-sulphur compound. W e have not measured the magnetisation curves of this component because of its relatively weak M C D spectrum. There is evidence that the spectrum obtained (Fig. 6) contains more than one pararnagnetic component which on oxidation with ferricyanide are changed in relative intensity. Experiments are underway to define more precisely the nature of these species. W e defer discussion of them until this work has been completed. MCD magnetisation curves

Variable temperature MCD spectroscopy provides an invaluable method of monitoring the ground state magnetic properties of metal centres in proteins via their optical absorption bands. In this way the magnetic properties of individual centres can be studied with no interference from other centres in the same protein. Thus the technique differs from magnetic susceptibility which responds to all the centres, including impurities. We have demonstrated recently that excellent magnetisation curves of haemoproteins can be determined using ultra-low-temperature MCD spectroscopy and, moreover, can be successfully analysed to obtai~ ground state g-factors [19]. This knowledge has been applied to a study of the magnetic properties of the EPR-undetectable haem, called a3, in oxidlsed cytochrome c oxidase [26]. However, haem MCD spectra are a particularly favourable case for analysis for two reasons. First, the MCD spectra are very intense and excellent quality magnetisation curves can be plotted from the MCD spectra. This is especially so because haem MCD signals are positively and negatively signed. Therefore, plots of peak-totrough magnitudes provide a measured of the MCD intensity which is indepen-

447 dent of any baseline artifacts due to inferior optical quality of low-temperature samples. Second, the polarisation of the electronic transitions, especially in the visible region of the spectrum, are relatively well studied and are predominantly xy polarised. This simplifies considerably the analysis of the magnetisation curves. In this paper we explore the utility of measurements of MCD magnetisation curves of iron-sulphur clusters. The difficulties are more severe than for haemoproteins both because the absolute intensities of the MCD signals are much weaker but also because there are almost no assignments of the electronic spectra of the 2- and 4Fe clusters and, indeed, no polarisation data since single crystals are required for this information to be obtained. The results presented here are the first examples of a detailed study of MCD magnetisation curves of iron,sulphur clusters and their analysis. The information contained in the curves is complementary to that from EPR, Mossbauer and magnetic susceptibility measurements. This study is a necessary prerequisite to the study of the magnetic properties of iron-sulphur clusters in more complex proteins such as hydrogenase and nitrogenase. The temperature dependence of MCD signals of paramagnetic molecules arises from a changing Boltzmann population amongst the Zeeman split sublevels of the electronic ground state. Because electronic transitions out of the Zeeman sublevels are circularly polarised a differential absorption of circularly polarised light occurs. When the temperature is high or gi~BB/kT << 1, where g is the ground state g-factor, the general expression for Ae (=eL -- eR) Can be expanded to first power in B/T and the MCD signal intensity is directly proportional to B/T. Under these conditions the MCD signal is obeying Curie's law and its magnitude is expressed by the so,ailed C term [27]. The vast majority of studies of MCD spectra of paramagnets have been carried out under this condition. However, when gtJsB/kT ~ 1, then Ae becomes a non-linear function of B/T and, eventually, the MCD signal becomes independent of B and T. At this point the paramagnet is fully magnetised and t h e MCD signal is said to be saturated. There is a Boltzmann population of the lowest Zeeman sublevel only. Schatz et al. [28] have developed theoretical expressions to analyse the tenperature and field dependence of an MCD spectrum to its saturation limit for the case of an axial 'isolated' Kramers doublet ground state. The term 'isolated' implies that no zero-field components become thermally occupied over the temperature range of the experiment. No experimental data were fitted to these expressions by Schatz et al. [28]. However, the expressions have been tested with experimental data on haemoproteins [19] and the expressions extended to cover the case of a rhombic Kramers doublet, which is of potentiai interest for metalloproteins. Throughout the present work we ignore any rhombicity in the ground state since EPR spectroscopy has shown that it is small. Indeed, the departure of the g-factors from isotropy is small for both 2and 4Fe clusters in their EPR-active states [1]. The expressions required in this work are those of Schatz et al. (Eqn. 17 in Ref. 28). This relates the intensity of the MCD signal at a given magnetic field (B) and temperature (T) to the ground state g-factors, g//and g± of an axial system, and the polarisation of the electronic transition as a ratio mz/m. where m2+ and m~ are the electric transition dipole moments in the molecular xy and z

448

directions, respectively. The expression consists of two terms, the first o f which is the contribution from the z-polarised component, and the second the contribution from the xy.polarised component. The expression is a function of the angles between the molecular principle axes and the direction o f the applied magnetic field. It requires to be averaged over these angles since a frozen glass contains all possible orientations o f an assembly o f molecules. This averaging is an integration which cannot be performed analytically but is best evaluated numerically on a computer. The expression has been evaluated for s o m e parameters of interest and the results are given in Fig. 10. The parameters chosen are g//= 2.06 and gl = 1.90, which are taken to be typical o f a C 3- cluster in a protein, assuming it to be axial by averaging gx = 1.88 and gy = 1.92 a s g . ffi [(g~ + ~ ) / 2 ] 1/2. The ratio of the z- to xy-polarised intensity is allowed to vary in steps of 0.2 from 1.00 to --1.00. Fig. 10 gives the magnetisation curves in arbitrary units of intensity for various values o f the ratio mz/rn÷. When this ratio has values of +0.4 to +1.0 the sign o f the MCD is opposite to that for the values +0.2, 0, --0.2 to --1.0. However, since we have no knowledge o f the absolute sign expected for a given MCD transition in the case o f iron,sulphur proteins all the curves are plotted as positive quantities. Because the expression is the sum o f two terms, the relative signs o f which depend u p o n the sign o f mz/m+, then the expression is zero when the two terms are equal and opposite. This occurs for the example given when the ratio mz/m+ = 0.37. For this particular polarisation ratio the MCD signal will be zero and is clearly a special case which can be readily recognised. Fig. 10 also shows the curves obtained by normalising the saturation limits to unity for selected values o f the ratio rnJrn.. The dependence o f the curve shape upon this ratio is slight except when the special value o f 0.37 is approached. For example, all the curves with values o f this ratio between 0 and --1.0 fall on the line indicated as ii in Fig. 10. When the ratio approaches 0.37 the curve first rises slightly then drops. Thus, as expected .for a system with such a small ground state g factor anisotropy there is little dependence of the form o f the magnetisation curve upon the polarisations o f the electronic transitions, except as the polarisation ratio approaches the value o f 0.37. Note that the magnetisation curves which are distinct become shallower in form, requiring higher fields and lower temperatures to achieve magnetic saturation. Fig. 10 illustrates another interesting property o f MCD magnetisation curves. The g factor o f an isotropic paramagnet is readily estimated from the initial slope and the asymptotic limit o f a magnetisation curve of the t y p e given in Fig. 10 [19~28]. The ratio of the asymptotic limit to the initial slope is the intercept (I) on the abscissa (see Fig. 10). Then g = 1/1. The value of I for a g-factor o f two is therefore 0.5. As Fig. 10 shows when rnl/m ÷ = 1.0, the intercept value obtained from the theoretical curves is given correctly. For other ratios o f mz/m+ the relationship between the g factors and the intercept value (I) can be computed readily from Eqns. 20 and 21 o f Ref. 28. These properties o f MCD magnetisation curves are valid only for paramagnets with an electronic ground state S = 1/2. However, it is possible to recognise spin states o f greater than 1/2 from an MCD magnetisation curve in several ways. First, a g factor as measured by t h e intercept value (I) will be significantly different from 2.0. Second, the magnetisation curve will only become

449

linear when kT is much greater than the energy spread of the zero-field componets. Lastly, a plot of Ae against #BB/2kT can lead to a nested set of curves rather than to a s m o o t h curve which is a simple function of B/T [19]. Armed with these concepts we n o w discuss the experimentally determined MCD magnetisation curves. Fig. 2 presents t w o curves for Hipipox plotted at t w o different wavelength regions of the MCD spectrum. The points plotted are the differences b e t w e e n t h e values at t w o fixed wavelengths, these wavelengths being chosen at points where the MCD signals are positive and negative, respectively. In this w a y we can eliminate errors due to any baseline changes as the temperature is varied. The points have been measured from MCD spectra recorded at fields of 1--5 tesla and at temperatures of 1.50--47.0 K. However, all the points can be fitted to one smooth curve regardless of the temperature o f measurement. This suggests that no excited electronic states are significantly populated over this temperature range. Futhermore, the points all lie very close to the theoretical curve c o m p u t e d with parameters of g/~ = 2.12, gi = 2.04 and mz/m+ = --1.00. There is little significance in the choice of the value of mz/m+ since any other choice, except one between +0.4 and +0.2, w o u l d give an equally good fit. Also shown in Fig. 2 are the asymptotic limit and the starting slope which give a intercept value of 0.49, the value expected for the above parameters. The magnetisation curve of reduced ferredoxin from Cl. pasteurianum given in Fig. 5 has been constructed from MCD measurements at 530 nm at eleven different magnetic fields b e t w e e n 0 and 5.1 tesla and three temperatures of 1.52, 4.22 and 18.6 K. The data for t h e t w o low temperatures fit a theoretical curve c o m p u t e d for g//= 2.06 and gi = 1.90 and mJm+ = - - 1 . 0 0 extremely well. The intercept value (I) o f 0.50 is close to the expected value for these g-values. Thus, at 4.22 K and below, the C 3- state magnetises as a simple Kramers d o u b l e t S = 1/2 system. The complex EPR spectrum arises apparently from intercluster magnetic coupling [12]. The magnitude of this coupling has not been estimated b u t it clearly must b e fractions of 1 cm -1 in order not to abolish the EPR spectrum. The MCD magnetisation curve will not be affected b y a coupling of this magnitude since the Zeeman energies available in the MCD experiment are up to 5 cm -1 (g/~sB = 4.6 cm -1 when g = 2.0 and B = 5 tesla) which will overwhelm the coupling. In an EPR experiment, by contrast, the resonant magnetic field is 0.3 tesla at X-band and 1.0 tesla at Q-band frequencies. The magnetisation curve o f Fig. 5 does show that the points measured at 18.6 K have a steeper slope than t h e initial slope of the low-temperature points. This m a y indicate the thermal population o f low-lying electronic states at this temperature. However, a more detailed study over a wide range of temperatures is required to confirm this suggestion. No magnetic susceptibility data appear to have been reported on C 3- proteins at very low temperatures. R o o m temperature measurements have been made on B. polymyxa ferredoxin in aqueous solution and a magentic m o m e n t of b e t w e e n 3.4 and 3.2 ~B reported [13]. However, there is a considerable b o d y o f magnetic data for the model compounds for t h e C 3- oxidation level o f proteins [29,30]. In solution at low temperature these model c o m p o u n d s give axial EPR spectra with g values of 2.03-2.06 and 1.93, indicative of an S = 1/2 ground state [29]. The susceptibility

450

of the solutions gives a magnetic moment of 2.03--2.05 #B at 4,? K which increases rapidly with rising temperature until at 300 K values as high as 4.54 #s are obtained. This evidence and a detailed study of the temperature~iependence of the intensity of the EPR signals of an acetonitrile solution of the models leads to the conclusion that there are electronic states lying within 30 cm -1 of the ground state [31]. The MCD spectra and magnetisation curves now provide an excellent method of comparing and contrasting the magnetic properties of Fe-S clusters within complex proteins with those of synthetic model compounds.' The magnetisation curves for the reduced 2Fe-2S ferredoxin from Sp. maxima are given in Fig. 9. Again it can be seen that the experimental points fall on or close to the theoretical curve computed for the following values: g//= 2.05, g± = 1.93 and mz/m+ = --1.0. The intercept values obtained for the plots from the two wavelength regions are in close agreement with one another and are equal to 0.51. This result is in good agreement with EPR, magnetic susceptibility and MSssbauer data on reduced 2Fe-2S centres which shows a simple S = 1/2 ground state of an anti-ferromagenticaUy coupled pair of Fe 2÷ and Fe 3÷, with no electronic excited states below 200 cm -I. Hence, over the temperature range used for the construction of the MCD magnetisation curves no influence of the excited states should be apparent. Thus the satisfactory fits of MCD magnetisation curves for Hipipox, reduced Cl. pasterianum ferredoxin and reduced Sp. maxima ferredoxin establishes the validity of our experimental procedures and theoretical analysis. Acknowledgements Thanks are due to the SRC for a Senior Research associateship (M.K.J.), a Graduate Research assistantship (A.E.R.) and equipment grants (to A.J.T. and D.O.H.). Part of this work was supported by funds from NATO. Dr. J.P. Springsll carried out preliminary studies on Hipip protein. References 1 0 r m e - J o h n s o n , W.H. and Sands, R.H. (1973) Iron-Sulfur Proteins (Lovenberg, W., ed.), Vol. II0 Academic Press, New York 2 Cammack, R , Dickson, D.P.E. and Johnson, C.E. (1977) Iron-Sulfur Proteins (Lovenberg, W., ed.), Vol. I n . Academic Press, New York 3 C--ter, C.W., Freer, S.T.. Xuang, Ng. H., Alden, A. and I~aut, J. (1971) Cold Spring Harbor Sym. Quant. Biol. 36, 381--389 4 Adman, E.T., Seiker, L.C. and Jensan, L.H. (1973) J. Biol. Chem, 248, 3987--8996 5 Carter, C.W. (1977) Iron-Sulfur Proteins (Lovenberg, W., ed.), Col. III, Academic Press, New York 6 Carter, C.W., Kraut, J., Freer, S.T., Alden, R.A., Sieker, L.C., Adam, A. and Jensen, L.H. (1972) Proc. Natl. Acad. Sci. U.S.A. 69, 8526--3529 7 Holm, R.H. and Ibers0 J.A. (1977) I r o n ~ u l f u r Proteins (Lovenberg, W.° ed.), Vol. III0 Academic Press° New York 8 Antanai~s° B.C. and Moss, TIH. (1975') Biochim. Biophys. Acta 405, 262--279 9 Blum, H., Salerno, J.C., Prince, R.C., Lelgh° J.S. and Obnlshl, T. (1977) Blophys. J. 20, 23--31 10 Cammaek, R. (1978) Bionh!m. Biophys. Res. Commun. 54, 548--554 11 Sweeney, W.V.° Beardcn, A.J. and Rabinowitz, J.C. (1974) Biochem. Biophys. Res. Commun. 59, 188--194 12 Ma~thews, R., Chazlton, S., Sands° R.H. and Palmer, G. (1974) J. Biol. Chem. 249, 4326--4828 13 PhflUps, W.D., McDonald, C.C., Stombaugh, N.A. and Orme-Johnson, W.H. (1974) Proc. Nat/. Aead. Sci. U.S.A. 71,140--143 14 Rivoal, J.C., Briar, B.0 Cammack, R., Hall, D . O , Rao, K.K., Douglas, I.N. and Thomson, A.J. (1977) Bioehim. Biophys. Acta 493, 122--131

451 15 Thomson, A.J. Cammack, R., H~I, D.O.0 Rao, K.K., Briat, B., Rivoal, J.C. and Badoz, J. (1977) Biochim. Biophys. Acta 493, 132--141 16 Stephens, P.J., Thomson, A.J., Keiderling, T.A., Rawlings, J., Rao, K.K. and Hall, D.O. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 5273--5275 17 Stephens, P,J., Thomson, A.J., Dunn, J.B.R,, Keiderling, T.A., Rawlings, J., Rao, K.K. and Hall, D.O. (1978) Biochemistry 17, 4770--4778 18 Stephens, P.J., MeKenna, C.E., Smith, B.E., Nguyen, H.T., MeKerma, M.-C., Thomson, A.J., Devlin, F. and Jones, J.B. (1979) Proe. Natl. Acad. ScL U.S.A. 76, 2585--2589 19 Thomson, A.J. and Johnson, M.K. (1980) Biochem. J. 191,411--420 20 Hall, D.O., Rao, K . K . and Cammack, It. (1972) Bioehem. Biophys. Res. Commun. 47, 798--802 21 Dus, K., Deklerk, H., Sletten, K. and Bartsch, R.G. (1967) Biochim. Biophys. Aeta 140,291--311 22 Evans, M.C.W., Hall, D.O. and Johnson, C.E. (1970) Bioehem. J. 119,289--291 23 Thompson, C.L., Johnson, C.E., Dickson, D.P.E.0 C-mm_ack, R., Hall, D.O., Weser, V. and Rao, K.K. (1974) Biochem. J. 139, 97--103 24 Hong, J.S. and Rabinowitz, J.C. (1970) J. Biol. Chem. 245, 4982--4987 25 Cammack, R. (1975) Biochem. Soc. Trans. 3,482--488 26 Thomson, A.J., Johnson, M.K., Greenwood, C. and Gooding, P.E. (1981) Biochem. J., in the press 27 Stephens, P.J. (1976) Annu. Rev. Phys. Chem. 35, 197--264 28 Schatz, P.N., Mowery, R.L. and Krausz, E.R. (1978) Mol. Phys. 35, 1537--1557 29 Laskowski, E.J., Frankel, R.B., Ginum, W.O., Papaefthymiou, G.C., Renaud, J., I b m , J.A. and Holm, R.H. (1978) J. Am. Chem. Soc. 100, 5322--5336 30 Laskowski, E.J., Reynolds, J.G., Frankel, R.B., Foner, S., Papaefthymiou, G.C. and Holm, R.H. (1979) J. Am. Chem. Soc. 101, 6562--6570 31 Papaefthymiou, G.C., Frankel, R.B., Foner, S., Laskowski, E.J. and Holm, R.H. (1980) J. Phys. 41, C1--493