Valence-Delocalized - American Chemical Society

Chapter 16 +

Valence-Delocalized [Fe S ] Clusters Downloaded via UNIV OF CALIFORNIA SANTA BARBARA on July 22, 2018 at 10:12:35 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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M . K. Johnson , E . C. Duin , B. R. Crouse , M.-P. Golinelli , and J. Meyer

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Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602 Département de Biologie Moléculaire et Structurale, CEA-Grenoble, 38054 Grenoble, France 2

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The [Fe S ] clusters in the C56S and C60S mutated forms of Clostridium pasteurianum 2Fe ferredoxin are shown to reversibly interconvert between valence-delocalized S = 9/2 and valence-localized S = 1/2 forms as a function of pH. This provides a unique opportunity to investigate the ground and excited state electronic properties and the vibrational modes of a valence-delocalized [Fe S ] cluster using the combination of EPR, variable-temperature magnetic circular dichroism (VTMCD) and resonance Raman spectroscopies. Near-IR electronic transitions arising from Fe-Fe interactions have been identified and assigned by VTMCD and resonance Raman studies. Moreover, the same set of transitions are also observed in the VTMCD spectra of [Fe S ] , [ZnFe S ] and [Fe S ] clusters, indicating the presence of valence-delocalized [Fe S ] fragments in these systems. Assignment of the z-polarizedσ-->σ* transition provides the first direct measurement of the magnitude of spin dependent resonance derealization in F e - S clusters. The double exchange parameter, B, is shown to be in the range 790-930 c m for valence-delocalized [Fe S ] units in [Fe S ] , [Fe S ] , [ZnFe S ] and [Fe S ] clusters indicating resonance delocalization energies in the range 3950-4650 c m . 2

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Detailed understanding of intracluster valence delocalization in F e - S centers is crucial for understanding ground and excited state properties as a well as rationalizing the thermodynamics and kinetics of intercluster electron transfer. Structural studies have shown that F e ^ - S ^ units are the building block for more complex clusters and the electronic properties of these units hold the key to understanding electron delocalization in F e - S clusters. Although synthetic and biological [Fe S ] clusters are valence localized with 5=1/2 ground states (i), Môssbauer and NMR data for higher nuclearity homometallic and heterometallic Fe-S clusters have been interpreted in terms of valence-delocalized S = 9/2 [Fe S ] fragments in at least one oxidation state (2-7). Moreover, theoretical studies have shown that the ground and excited +

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©1998 American Chemical Society Solomon and Hodgson; Spectroscopic Methods in Bioinorganic Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

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properties of [Fe^SJ* clusters are critically dependent on the interplay of Heisenberg exchange, spin-dependent resonance delocalization (double exchange) and vibronic interactions (8-14). This has lead to rationalization of the ground state properties of a range of trinuclear and tetranuclear clusters in terms of antiferromagnetic exchange interactions involving valence-delocalized S = 9/2 [Fe S ] fragments: [Fe S ]~ (in [ZnFe S ] ) (2); [Fe S ]° (2,72); [Fe S ] (13); [Fe S ] (14). However, recent analyses of the hyperfine coupling constants for [Fe S ] and [Fe S ] clusters suggest that S = 7/2 or intermediate between S = 1/2 and 5/2 are more appropriate formal spin states for the valence-delocalized [Fe S ] fragments in these cases (15,16). The dependence of the ground state properties of a [Fe S ] cluster fragment on the relative magnitude of Heisenberg exchange and spin-dependent resonance delocalization is illustrated in Figure 1. The energy level scheme is based on a Hamiltonian with both Heisenberg (J) and double exchange (B) terms that results in Ε = -JS(S + 1) ± B(S + Vi) (17). This simple model neglects vibronic interactions and assumes that the valence-localized species with the extra electron on Fe and Fe are isoenergetic. As the extent of resonance delocalization (B/J) increases, the ground state changes from S = 1/2 to 9/2 in integer steps, becoming S = 9/2 for \B/J\ > 9. Inclusion of the factors responsible for valence localization, i.e. vibronic coupling and inequivalence in the energies of the valence trapped species, decreases the B/J range in which the ground state has 3/2 < S < 7/2 (18,19). This diminishes the likelihood of observing these intermediate-spin ground states and leads towards a situation in which the ground state changes directly from valence-localized S = 1/2 to valence-delocalized S = 9/2 with increasing B/J (18,19). Hence the value of B, relative to J and the energetic factors responsible for valence localization, determines both the ground state spin and the extent of valence delocalization. However, there is as yet no reliable experimental estimate of B, and the theoretical estimates span a wide range, 30-970 cm" , as gauged by the recent set of JBIC commentaries on exchange versus double exchange in polymetallic systems (19-22). Figure 1 illustrates a potential experimental method for measuring B. For a valence-delocalized S = 9/2 [Fe S ] cluster, the model predicts a spin-allowed, electric-dipole-allowed, z-polarized σ -» σ* transition in the near-IR region at energy 105 (= 20, where β is the resonance energy). This type of direct measurement of Β requires detailed optical studies of a valence-delocalized S = 9/2 [Fe S ] cluster and the discovery and characterization of such clusters in C56S and C60S mutated forms of Clostridium pasteurianum (Cp) 2Fe ferredoxin (Fd) are the subject of this chapter. +

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C56S and C60S Mutated Forms of Cp 2Fe Fd Cp 2Fe Fd is a low molecular weight protein (102 amino acids) containing one [Fe S ] cluster with complete cysteinyl-S ligation. Although structural characterization by x-ray crystallography or NMR is not available, the combination of mutagenesis and spectroscopic studies has revealed a unique arrangement of coordinating cysteine residues (in positions 11, 24, 56 and 60) (23-25) compared to other [Fe S ]-containing proteins (26). In the wild-type protein, the [Fe S ] cluster is valence localized with an S = 1/2 ground state and the non-reducible Fe site is coordinated by cysteines 11 and 24 which occur in a flexible loop region (25). The 2 + +

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Solomon and Hodgson; Spectroscopic Methods in Bioinorganic Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

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initial discovery of 5 = 9/2 [Fe S ] clusters in the C56S and C60S mutated forms camefromVTMCD and EPR studies (27). Moreover, the close similarity in the nearIR VTMCD spectra to those of clusters known to contain valence-delocalized 5 = 9/2 [Fe^J" " clusters lead to the suggestion that these unique ground state properties were the consequence of valence delocalization. This was subsequently confirmed by Mossbauer studies, which provided the definitive proof that these 5 = 9/2 [Fe S ] clusters were valence delocalized on the time scale of ~10~ s (18). However, detailed characterization of the properties of this unique [Fe S ] cluster were hindered by the observation that the reduced samples were mixtures of valencelocalized (5 = 1/2) and valence-delocalized (5 = 9/2) species (18,27). The subsequent observation that the C56S and C60S mutated forms, but not the wild-type Fd, had pH dependent midpoint potentials, led to the discovery of reversible interconversion between valence-delocalized 5 = 9/2 and valence-localized 5 = 1 / 2 forms as a function of pH (28). This has provided a unique opportunity to compare the ground state, excited state and vibrational properties of valence-delocalized S = 9/2 [Fe S ] and valence-localized 5 = 1 / 2 [Fe S ] clusters in the same protein (28) and a summary of these recent results is presented below 2

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Redox Properties. At pH 7.0, the C56S and C60S mutations result in a 102-108-mV decrease in midpoint potential compared to wild type (E = -260 mV vs NHE and independent of pH over the range 5-10). Negative shifts in midpoint potential have previously been reported for [Fe S ] ' couples on replacing a cysteine ligating the reducible Fe site by a serine: AE = -100 and -240 mV at pH 7 in Escherichia coli fumarate reductase (29) and AE = -18 mV (pH not specified) in Anabaena Fd (30). However, the absolute magnitudes of these serine-induced changes in midpoint potential should not be compared, since the pH dependence was not investigated for E. coli fumarate reductase and Anabaena Fd. The midpoint potentials of the C56S and C60S mutated forms are both strongly pH dependent; approximately constant at -460 mV above pH 10 and increasing with a slope of -50 mV/pH unit below pH 8. Such behavior is indicative of protonation of the reduced clusters with a pK « 9, with the serine oxygen as the logical site for protonation. These redox results are, therefore, interpreted in terms of serinate ligation at pH > 10, with the nature of the fourth ligand at pH < 8 unclear at present, i.e. serine OH, water or some other amino acid side chain. Precedent for serinate ligation of a [Fe S ] cluster comes form the crystallographic studies of the C49S mutated form of Anabaena Fd (31) and redoxdependent serinate coordination of an Fe-S cluster has recently been observed for the nitrogenase P-clusters (32). As shown below, the pH-induced changes in the nature of the fourth cluster ligand in the C56S and C60S mutated forms have dramatic consequences for the properties of the [Fe S ] clusters. m

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Ground State Properties. The ground state properties of [Fe^]"*" clusters in the high pH (pH 10-11) and low pH (pH 6-8) forms of the C56S and C60S variants have been investigated by EPR and MCD saturation magnetization studies (28). Both approaches indicate that the low-pH forms are exclusively 5 = 1/2 and that the high-pH forms are predominantly (>80%) 5 = 9/2. The low pH forms exhibit identical 5 = 1/2 EPR signals (g = 2.01, 1.92, 1.88) with increased anisotropy compared to wild-type (g = 2.00, 1.95, 1.92), indicative of a change of ligation at the F e site of a localized 2+


289 valence [Fe^J* cluster (29,33,34). MCD magnetization data at selected wavelengths are well fit at all temperatures by theoretical data constructed using the EPRdetermined g-values, indicating that all transitions originate from the S = 1/2 ground state responsible for the EPR signal. Both EPR spin quantitations and the incomplete reversibility of absorption changes following redox cycling show that the reduced cluster is stable at pH 8, but readily degraded at pH 6. For both the C56S and C60S variants at pH 11, weak S = 1/2 resonances were observed, accounting for 0.18 and 0.10 spins/molecule, respectively. The resonances are distinct from those associated with low pH forms in terms of both g-value anisotropy (g = 2.01, 1.91, 1.87 for C56S and g = 2.02, 1.93, 1.86 for C60S) and substantially increased linewidths. Hence they are interpreted as minor components of localized-valence species with serinate as the fourth cluster ligand (as opposed to protonated serine, water, or some other protein ligand at low pH). However, the majority (> 80%) of the serinate-coordinated clusters in the high pH forms of both variants are present as valence-delocalized S = 9/2 [Fe S ] clusters as evidenced by EPR and MCD studies. Low field resonances that are uniquely indicative of S = 9/2 ground states (E/D = 0.12, D = -2.7 c m for C56S, and E/D = 0.17, D = —1.4 c m for C60S) were observed at temperatures between 4 and 60 Κ (28). For the C56S variant, Fig. 2 shows the temperature-dependence of the low-field EPR signals and the analysis in terms of a conventional S = 9/2 spin Hamiltonian with g = 2: +

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Observed g-values are given in parenthesis and the low transition probability of the resonances within the two lowest doublets results in the sample being almost devoid of low field resonances below 4.5 Κ except for the derivative feature centered at g = 4.3 from adventitious Fe . The value of D was determined by estimating the energy difference between the M = ±3/2 and ±5/2 doublets from the slope of a plot of the log of the ratio of the intensities at g = 9.59 and 8.53 versus 1/T. MCD magnetization studies for both variants are consistent with the dominant bands being xy-polarized transitions originating from the highly anisotropic M = ± 9/2 doublet, #11 « 18 and g « 0, of an S = 9/2 ground state with D < 0 (see below). 3+

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Excited State Properties. The UV/visible/near-IR absorption spectra of both variants in the oxidized state (23) are independent of pH over the range 6-11. In contrast, the reduced absorption and VTMCD spectra are strongly pH dependent (28) with analogous changes for both variants. This is illustrated by the high and low pH VTMCD spectra for reduced C60S shown in Figure 3. At or below pH 8, both the absorption and VTMCD spectra are characteristic of valence-localized 5 = 1 / 2 [Fe S ] clusters (35,36). The variants and wild type all exhibit a pronounced absorption and positive MCD band at - 540 nm that has been tentatively attributed to the Fe * -> F e intervalence transition (35,36). We have noted previously that the temperature-dependent MCD intensity of this band argues against this assignment, since it is formally a uniaxial transition and MCD C-terms require two non-zero perpendicular transition dipole moments. It is of course possible that the anomalously high MCD intensity arises from out-of-state spin orbit coupling involving an +

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S = 9/2--3K

60 -Γ σ-*σ* z-polarized, electric-dipole allowed transition

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Figure 1. Energy level diagram for an exchange coupled [Fe S ] cluster as a function of increasing spin dependent resonance delocalization. 2

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Figure 2. Temperature dependence of the low-field EPR spectrum of dithionitereduced C56S Cp 2Fe Fd at pH 11. The tabulated data is the analysis in terms of a S = 9/2 spin Hamiltonian with the g-values in parenthesis corresponding to observed values. Adaptedfromref. 28.


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Figure 3. UV/visible/near-IR VTMCD spectra (6.0 T) of the dithionite-reduced C60S mutated form of Cp 2Fe Fd at pH 6.0 and 11.0. All transitions increase in intensity with decreasing temperature. Adapted from ref. 28.


292 energetically similar and orbitally degenerate excited state (37). However, the observation that the energy of this transition is not significantly perturbed by changes in ligation at the F e site (i.e. replacing a cysteinate by an oxygenic ligand as in this work or replacing both cysteinates with histidines in Rieske protein (unpublished MCD results from this laboratory)) is more difficult to reconcile with this assignment. In light of the resonance Raman excitation profiles (35), we therefore currently favor assignment of this band to a μ ~$ ~ -* F e charge transfer transition, with Cys-S ~ F e charge transfer transitions occurring to higher energies (350-525 nm). The most significant differences in the low-pH VTMCD spectra of the variants compared to wild-type, occur in the 250-350 nm region, which is primarily associated with charge transfer transitions involving the F e site (35). Hence, the VTMCD data for the variants are also consistent with a change in ligation at the F e site of a valencelocalized [Fe S ] cluster. The absorption and VTMCD spectra of both variants at pH 11 are unique compared to any known type of [Fe S ] cluster. In the visible region, the absorption spectrum comprises a broad shoulder with inflections at 420 and 470 nm and weaker lower energy band centered at 670 nm (28). These features correlate with intense positive MCD bands at 420 and 480 nm and a derivative-shaped feature centered at 660 nm (positive and negative components at 705 and 610 nm, respectively), see Figure 3. MCD magnetization studies for the positive bands centered near 480 nm and 705 nm indicate that both arise almost exclusively from xy-polarized transitions from an S = 9/2 ground state with D < 0. For example, the lowest temperature data for the C60S variant collected at 705 nm (Figure 4) are well fit by theoretical data (computed according to the equations in ref. 38 and indicated by the solid line) corresponding to 93% of the MCD intensity at this wavelength arising from an 5 = 9/2 ground state (transition originating from the lowest doublet with g = 18 and g = 0) and 7% from an 5 = 1/2 ground state (g = 2.02, 1.93, 1.86). The minor components of serinate-ligated localized-valence 5=1/2 [Fe S ] clusters that were apparent in the EPR studies of both variants at pH 11, are also evident in the VTMCD spectra by the presence of bands with 5 = 1/2 temperature dependence in the 500-580 nm region (Figure 3). The 480-nm MCD band of valence-delocalized 5 = 9/2 [Fe^J* clusters is the logical counterpart of the 540-nm band of the valence-localized 5 = 1/2 [Fe S ] clusters, and is, therefore, assigned to a M -S ~ -* F e charge transfer transition. Likewise, the higher energy bands of the 5 = 9/2 clusters are assigned to Cys-S ~ -» Fe charge transfer transitions with each shifted to higher energy relative to the Cys-S" -> F e counterparts in 5 = 1/2 clusters. However, the near-IR MCD bands, i.e. intense positive bands at 710 nm and weaker positive bands at - 850 nm (shoulder in C60S) and 1070 nm (Figure 3), are unique to the 5 = 9/2 species and hence appear to be a direct consequence of the Fe-Fe interactions that lead to the spin dependent valence delocalization (37). Since MCD magnetization studies indicate that the 710 nm is xy-polarized, it is a not a candidate for the z-polarized σ -> σ* transition that would resultfromdirect σ overlap of the Fe d 2 orbitals. Rather this transition is assigned to the weak MCD band at 1070-nm which is shown to be predominantly zpolarized by MCD magnetization studies (Figure 4). The anomalous magnetization behavior at this wavelength, i.e. increasing to a maximum and then decreasing as a function of βΒ/lkT, is predicted for predominantly z-polarized C-terms arising from 2+

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Figure 4. MCD magnetization data collected at 705 and 1070 nm for the dithionite-reduced C60S mutated form of Cp 2Fe Fd at pH 11.0. Adapted from ref. 28.


294 highly anisotropic doublet ground states, when both the z-polarized and xy-polarized transition dipole moments (m and wz , respectively) have the same sign (38). The theoretical data shown by the solid line in Figure 4 was computed using the effective g-values for the M = ± 9/2 doublet of the C60S variant derived from the spin Hamiltonian analysis of the EPR resonances (g = 17.96 and g = 0.003) and m /m = 300. These polarization assignments facilitate rationalization of the near-IR MCD bands in terms of a schematic molecular orbital diagram for the Fe-Fe interactions in the valence-delocalized S = 9/2 [Fe S ] cluster (Figure 5). The estimate of the tetrahedral crystal field flitting at each Fe site (10Dq ~ 5000 cm" ) is based on the detailed electronic studies of tetrahedral F e and F e complexes with thiolate ligands (39AO). The dominant interaction (responsible for the S = 9/2 ground state) is the σ overlap between the pair of d 2 orbitals, with progressively smaller π interactions, between pairs of d^ and dy orbitals, and δ interactions, between pairs of d and d 2. 2 orbitals. The σ -* σ* transition (1070 nm, 9300 cm" ) is electric dipole allowed, but z-polarized resulting in weak VTMCD intensity. The a -> τ and σ -* π* transitions will be effectively xy-polarized if the d and dy orbitals are close in energy and both should therefore exhibit broad derivative shaped temperaturedependent MCD bands (pseudo 4-terms). The former is electric-dipole forbidden and hence weak in the MCD. Only the positive feature is observed as a shoulder at -850 nm in C60S (it is more clearly observed in the C56S variant (28)) with the negative component buried under the intense band 710 nm. The latter is electric-dipole allowed and xy-polarized and hence gives rise to the intense pseudo ^-term centered at 660 nm (15000 cm" ). The energy of the σ -> σ* transition corresponds to 102? (2β) and hence provides the first direct measurement of the double exchange parameter and resonance energy for a valence-delocalized [Fe S ] cluster; Β = 930 cm" and β = 4650 cm" . These values are in good agreement with those estimated for a [Fe S ] cluster (B = 965 c m and β = 4825 cm ) based on extrapolation from the Fe-Fe electronic coupling observed in [Fe (OH) (tmtacn) ] (37). This synthetic complex is by far the best characterized valence-delocalized S = 9/2 diiron system investigated thus far (37,41). z

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Vibrational Properties. The high pH forms of dithionite-reduced C56S and C60S Cp 2Fe Fd offer the first opportunity to investigate the vibrational consequences of valence delocalization in a [Fe S ] unit. Moreover, since vibrations involving displacement along the Fe-Fe coordinate should be enhanced in the Raman spectrum by excitation into the low energy σ -> σ* and σ -* π* transitions, resonance Raman spectroscopy provides a method for testing the above assignments. Thus far resonance Raman studies have been confined to the C56S variant using excitation wavelengths in the range 406-676 nm, and a detailed discussion of spectra and the rationale for the preliminary assignments presented in Table I can be found in ref. 28. As indicated by the frequencies listed in Table I, the valence-delocalized and valence-localized [Fe S ] clusters in the C56S variant have remarkably different resonance Raman spectra, showing that the former is valence delocalized on the vibrational time scale (10" -10" s) ^Fe- and S-isotope shifts are not yet available for Cp 2Fe Fd, since cluster +

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reconstitution by the standard protocols produces a protein different from wild-type (42). Nevertheless, Fe-S stretching modes have been assigned for both the [Fe S ] and valence-localized [Fe S ] clusters in wild type Cp 2Fe Fd (35,43,44), under effective D and C symmetry, respectively, by analogy to other Fds and model complexes (35,43-45), see Table I. The resonance Raman spectrum of the oxidized C56S variant is very similar to that of the wild-type except for significant upshifts (7-9 cm" ) in the predominantly terminal A * and B F e - S stretching modes. This is attributed to the mass effect resultingfromreplacement of a coordinating S by Ο (24). The same general trend is observed in comparing the frequencies of the valencelocalized reduced cluster in the low pH C56S variant and wild-type, i.e. upshifts of 0-14 cm" . However, the significant upshifts that are observed for the B mode, which is almost exclusively F e - S stretching at the F e site, and for the modes at 370 and 390 cm" in the wild-type, which primarily involve F e - S sketching, are unexpected and probably reflect a change in the overall cluster environment resulting from replacing the serinate ligand with water or another protein ligand in the C56S variant at low pH. The resonance Raman spectra of the valence-delocalized cluster in the reduced C56S variant at pH 11 are assigned under D symmetry by analogy to the oxidized C56S sample. Each band is shifted down in frequency by 12-35 cm" , which is consistent with weakening of the Fe-S bonds on goingfromF e to Fe . 2+

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Table I. F e - S stretching frequencies (cm" ) for wild-type and C56S Cp 2Fe Fd Mode" WT C56S Mode C56S WT C56S (red) (red, VL) (red, VD) (ox) (ox) (D h) (C ) 404 406 385 404 390 Bi 387 387 368 375 370 Ai Α.* 366 366 341 310 320 Ai S1 ι Λ 2 g 353 353 341 B 341 328 335 344 317 320 310 Ai 313 314 299 280 280 Β, 290 297 267 268 280 B Aj A (Fe-Fe) 208 211 176 *The t and b superscripts refer to F e - S stretching modes that predominantly involve terminal (cysteinyl) and bridging (inorganic) S, respectively. VD, valance delocalized; VL, valence localized. Adapted from ref. 28 2

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A particularly striking difference between the resonance Raman spectra of the valencelocalized and valence-delocalized [Fe S ] clusters in the C56S is seen with 676-nm excitation (Figure 6). In accord with the assignment of the absorption and MCD bands near 670 nm to the Fe-Fe σ ττ* transition in the high-pH form of C56S, excitation at this wavelength results in enhancement of the totally symmetric Fe-Fe stretching mode at 176 cm" and the totally symmetric breathing mode of the F e ^ core at 368 cm" . Both modes involve displacement along the Fe-Fe coordinate. Hence their enhancement with near-IR excitation provides strong evidence that the near-IR electronic transitions of the valence delocalized cluster arise from Fe-Fe interactions. Confirmation of these assignments via Fe-isotope shifts and attempts to use excitation into the σ -> σ* transition at 1050 nm are in progress. +

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Fe-Fe interaction Fe

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Figure 5. Schematic MO diagram for the Fe-Fe interactions in the valencedelocalized S = 9/2 [Fe S ] cluster based on the observed electronic transitions. The z-axis is along the Fe-Fe coordinate. Adapted from ref. 28. +

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Figure 6. Comparison of the resonance Raman spectra (676-nm excitation) of dithionite-reduced C56S Cp 2Fe Fd at pH 8 and 11. The asterisks indicate lattice modes of ice.


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Origin of Valence Delocalization. The discovery of valence-delocalized 5 = 9/2 [Fe S ] clusters in the C56S and C60S mutated forms of Cp 2Fe Fd begs for the question of why such clusters are observed in this case, whereas all other biological and synthetic [Fe^J* clusters investigated thus far are valence localized with 5 = 1/2 ground states. A detailed discussion of the extensive literature on "static" and "vibronic" mechanisms for electron trapping in exchange-coupled systems is beyond the scope this chapter. The interested reader is referred to reviews by Blondin and Girerd (9) and the recent JBIC commentary articles (19-22). However, we have tried below to summarize our initial thoughts on this interesting question. Valence delocalization resulting in an 5 = 9/2 ground state requires \BIJ\ > 9 in an idealized system in which the two valence-localized configurations have the same energy (Figure 1). This can be accomplished by increasing B, decreasing / , or a combination of both. Asymmetric Fe coordination as a result of serinate coordination might be expected to decrease / (46), and this is probably an important contributing factor to the observed valence delocalization. Whether or not Β increases in the serinate-coordinated valence-delocalized forms remains to be determined. Since Β is expected to correlate with the Fe-Fe distance (37), this question can be directly addressed by EXAFS studies of low-pH (valence-localized) and high-pH (valencedelocalized) forms of these two variants and such studies are in progress. The degree of valence delocalization for a given spin state is determined by the ratio of Β to the localization energy, AE, which contains the energetic terms reflecting both the vibronic and static preference for valence localization. On the basis of the simple resonance Hamiltonian model, valence trapping will generally occur unless 215(5 +1/2)| > AE. Although it is difficult to separate the vibronic and static contributions to AE, the static component, which corresponds to the energy difference when the extra electron is on Fe or Fe , is likely to be minimized in the C56S and C60S variants. By decreasing the potential at the "reducible" Fe site by approximately 200 mV, we hypothesize that serinate coordination accidentally makes the two Fe sites equipotential and that this is a major determinant for valence delocalization in the C56S and C60S variants. On the basis of this discussion, it seems probable that further examples of valence delocalized 5 = 9/2 [Fe S ] clusters will come through mutagenesis studies of proteins rather than from chemical synthesis. In order to decrease the Heisenberg exchange coupling, asymmetric Fe coordination is probably required, but it is difficult to see how this can be accomplished in a synthetic cluster while maintaining isopotential Fe sites. In contrast, the nature of the Fe ligands in a protein-bound cluster is only one of the factors that determines the redox potentials at the Fe sites, making it possible to obtain isopotential Fe sites with asymmetric Fe coordination via site-directed mutagenesis. +

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Implications for Higher Nuclearity F e - S Clusters Now that the unique electronic and vibrational properties of a valence delocalized [F^SJ* clusters have been established, can this information be used to identify and characterize similar units in higher nuclearity clusters? At least in the case of VTMCD spectroscopy, the answer appears to be a resounding yes. Figure 7 shows a comparison of the low temperature MCD spectra of


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Figure 7. Comparison of the low temperature MCD spectra of different types of paramagnetic F e - S clusters. Adapted from ref. 28.


299

representative examples of each type of paramagnetic Fe-S cluster that has been proposed to contain a valence delocalized [Fe S ] fragment, i.e. S = 5/2 [Fe S ]~ fragment in [ZnFe S ] Fd (2), S = 2 [Fe S ]° Fd, S = 1/2 [Fe S ] Fd, S = 3 or 4 [Fe S ] nitrogenase P-cluster, and S = 1/2 [Fe S ] HiPIP. The origin of the intense positive band between 700-800 nm that dominates the near-IR MCD of many paramagnetic Fe-S has long been a puzzling feature of the spectra of F e - S proteins (47). It is now evident that this is the hallmark of a valence delocalized [Fe S ] fragment. Indeed inspection of Figure 7, suggests that the electronic transitions from thisfragmentdominate the UV/visible/near-IR MCD spectra of [ZnFe S ] , [Fe S ]°, [Fe S ] and [Fe S ] clusters. This is not surprising since, with the exception of [Fe^J cluster, the remaining Fe is all F e which is expected to contribute charge transfer bands only below 400 nm. What is particularly striking is that [ZnFe S ] , [Fe S ]°, [Fe S ] clusters all exhibit the same pattern of near-IR MCD bands that were assigned to Fe-Fe σ -» σ*, σ -* τ and σ π* transitions for the valencedelocalized [Fe S ] clusters. Hence we conclude that all three clusters contain a valence-delocalized [Fe S ] cluster fragment and the energy of the σ -* σ* transition provides a direct measure of the double exchange parameter, Β = 790 c m in [ZnFe S ]\ 870 cm" in [Fe S ]°, and 890 cm" in [Fe S ] . In the case of the [ZnFe^SJ"*" and [Fe S ]° clusters, the Môssbauer data leave little doubt that the valence-delocalized [Fe S ] fragment is S = 9/2 (2,72). Based on the similarity in the VTMCD data, it is tempting to draw the same conclusion for the spin state of this fragment in [Fe S ] clusters. However, a similar set of MCD bands might also be expected for valence-delocalized [Fe S ] clusters with S = 7/2 or 5/2 ground states, based on the schematic energy level scheme shown in Figure 5. It may prove possible to discriminate between these alternatives by obtaining MCD data to lower energies, since only the lower spin states would be expected to have observable transitions at wavelengths > 1400 nm. Such measurements are in progress for a wide range of Fe-S clusters. At this stage, it remains to be determined if nearIR VTMCD can provide a means of assessing the spin state of the valence-delocalized [Fe S ] fragments in higher nuclearity clusters. The presence of an intense MCD band at 800 nm in oxidized P-clusters clearly suggests the presence of valence-delocalized [Fe S ] fragments. Although near-IR VTMCD data clearly needs to be extended to lower energy in this case, analysis will be more difficult, since there are likely to be two different valence delocalized [Fe S ] fragments in the oxidized form of this double cubane cluster (32). A surprising aspect of the MCD data shown in Figure 7, is that the VTMCD spectrum of the [Fe^J * cluster in oxidized HiPIPs does not conform to the pattern expected for cluster containing a valence delocalized [Fe S ] fragment. Whether this is a consequence of overlapping electronic transitions from the F e pair and/or a spin state < 9/2 for the valence-delocalized [Fe S ] fragment is unclear at present. This brief discussion demonstrates that the discovery and characterization of 5 = 9/2 valence-delocalized [Fe S ] clusters in mutated forms of Cp 2Fe Fd represents a major step forward in understanding the electronic structure of F e - S clusters. Moreover, the ability of VTMCD spectroscopy to identify and determine the resonance energy of these fragments in higher nuclearity clusters indicates that this technique is destined to play an increasingly important role in investigations of valence delocalization in Fe-S clusters. +

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Valence-Delocalized - American Chemical Society

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