Ligand Conformational Effects on the Resonance Raman Signature of

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Ligand Conformational Effects on the Resonance Raman Signature of [Fe4S4(SAryl)4]2Clusters† Estelle M. Maes,‡ Michael J. Knapp,§,| Roman S. Czernuszewicz,*,‡ and David N. Hendrickson§ Departments of Chemistry, UniVersity of Houston, Houston, Texas 77204, and UniVersity of California at San Diego, La Jolla, California 92093 ReceiVed: January 26, 2000; In Final Form: June 29, 2000

Low-temperature (77 K) resonance Raman (RR) spectra are reported for a series of analogues of [4Fe-4S] iron-sulfur proteins, (nBu4N)2[Fe4S4(SR)4], where SR- ) tp (1), 3,5-dmtp (2), 2,4-dmtp (3), and 2,6-dmtp (4) (tp ) thiophenol and dmtp ) dimethylthiophenol). Structural influences of these different terminal thiolates on the RR signature of the [4Fe-4S]2+ core are presented. Both the cluster and terminal ligand Fe-S vibrational modes are identified and assigned through 34S isotope substitution of analogues 1 and 4 at the cluster sulfur position. The characteristic RR frequencies of the four compounds provide new insights into the vibrational and electronic properties of the iron-sulfur cluster due to the ligand conformation changes. The RR spectra demonstrate a significant D2d distortion of each cluster, and an energy crossing is observed for the T2 terminal (t) and bridging (b) Fe-S stretching modes above 350 cm-1 of 1 and 4 in response to changes in the SbFe-St bond angles imposed by different geometries about the Fe-St bonds. The results affirm the absolute necessity of isotope substitution for an exact assignment of the Fe-St and Fe-Sb vibrational modes in [4Fe4S] clusters when the [4Fe-4S] proteins are analyzed and compared. The blue-shifted to 404 nm electronic absorption band of 4 is identified as a predominantly S(Aryl) f Fe charge-transfer transition by excitation profiles for two prominent Fe-S RR bands, the A1 bridging stretch at 336 cm-1 and the T2 terminal stretch at 351 cm-1.

Introduction Proteins containing [4Fe-4S] chromophores belong to a large family of iron-sulfur proteins that are known for their strong light absorptions in the UV-vis region, diverse structural motifs, variable redox and spin-state properties, and facile electrontransfer dynamics.1,2 The standard [4Fe-4S] chromophores, involved in fundamental biological redox processes of photosynthesis,3,4 nitrogen fixation,5 and respiration,6 share a common cubane-like structure with interpenetrating, concentric Fe4 and S4 tetrahedra and one terminal cysteine ligand on each Fe.7 Proteins containing these structures include the bacterial ferredoxins (Fds),8-12 high-potential iron proteins (HiPIPs),11,13,14 and other electron-transfer proteins such as the Fe-protein of nitrogenase.15-17 A given [4Fe-4S] protein in vitro operates between two of the three oxidation levels, [4Fe-4S]2+/1+ (Fdox/ Fdred) with redox potential Em ) ∼-0.4 V or [4Fe-4S]3+/2+ (HiPIPox/HiPIPred) with Em ) ∼+0.4 V, the former couple being more common. However, the redox potential18,19 and electronic properties of the couple20,21 are species dependent, the observed variations making evident the environmental differences at the active sites of these proteins. These characteristics and the role that the protein matrix plays in modulating the properties of the [4Fe-4S] cluster continue to engender biophysical and theoretical studies.22-28 †

Part of the special issue “Thomas Spiro Festschrift”. * To whom correspondence should be addressed. Phone: (713) 7433235. Fax: (713) 743-2709. E-mail: [email protected]. ‡ University of Houston. § University of California at San Diego. | Present address: Department of Chemistry, University of California, Berkeley, CA 94720.

The cores of the synthetic compounds of the [Fe4S4(SR)4]2-/3(R ) alkyl or aryl) type constitute good analogues of [4Fe4S]2+/1+ redox states, providing convenient model systems for Fe-S electronic and structural elucidation,21,29-32 including the effects of subtle environmental differences. Recently, spectrochemical measurements have been carried out by Knapp et al. on a series of (nBu4N)2[Fe4S4(SAryl)4] compounds that contain different arylthiolate terminal ligands such as thiophenol (tp) (1), 3,5-dimethylthiophenol (3,5-dmtp) (2), 2,4-dmtp (3), 2,6dmtp (4), or 2,6-dichlorothiophenol (2,6-dctp) (5).33 Techniques such as X-ray crystallography, magnetic susceptibility, cyclic voltammetry, and NMR, optical, and EPR spectroscopies were used to investigate the terminal ligand conformational effects on the electronic properties of the [4Fe-4S]2+/1+ cores. In the present work, we report the results of the vibrational study on the series of [Fe4S4(SAryl)4]2- dianions (where SAryl ) tp, 3,5dmtp, 2,4-dmtp, or 2,6-dmtp) using cryogenic resonance Raman (RR) spectroscopy and isotope substitution. RR spectroscopy, which provides a record of molecular vibrations that are coupled to allowed excited electronic transitions, has been a sensitive structural probe for directly assessing the coordination environment of chromophoric metal centers in complex biological systems,34,35 including the Fe-S protein sites.36,37 The [Fe4S4(SAryl)4]2- clusters of this work are used to establish conformational influences on the [4Fe-4S]2+ RR signature of the different terminal arylthiolate ligands as well as to provide useful information on the electronic and bonding properties of these biologically significant iron-sulfur clusters. Furthermore, the experimental data through the control of ligand conformation on vibrational frequencies of the cluster provide new reference points for analyzing the [4Fe-4S] protein spectra.

10.1021/jp0003104 CCC: $19.00 © 2000 American Chemical Society Published on Web 09/14/2000

RR Signature of [Fe4S4(SAryl)4]2- Clusters

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Materials and Methods All (nBu4N)2[Fe4S4(SAryl)4] complexes were prepared using standard Schlenk-line techniques by the method of Christou et al.,38 and were recrystallized from a CH3CN/MeOH solution. The analogues enriched with 34S at the cluster positions were prepared by the same procedure as for the natural abundance sulfur using the elemental 34S (99.8% isotopic purity) purchased from Cambridge Isotope Laboratories. Room-temperature UV-vis absorption spectra of the complexes in solution (CH3CN) were collected using a HewlettPackard 8452 diode-array spectrophotometer. The RR spectra of the solid analogues were obtained at 77 K using excitation lines provided by Coherent K-2 Kr+ (406.7568.2 nm) and 90-6 Ar+ (457.9-514.5 nm) ion lasers and collecting backscattered photons directly from the surface of a pellet held in a vacuum and attached to a coldfinger of a liquid N2 cryostat. Under these conditions no sample damage was observed, even during prolonged (12 h) spectral data acquisition at laser powers of 200-300 mW. Preparation of the pellets by grinding up crystalline complexes with KCl, pressing with a hand press, and mounting of the pellet onto the copper finger were performed in an O2-free glovebox. Details of this sampling technique have been presented elsewhere.39 To ensure accurate determination of isotope shifts, natural abundance and 34Slabeled samples were affixed in pairs on the coldfinger so that RR spectra were recorded under identical conditions. The Raman spectrometer consisted of a Spex 1403 double monochromator equipped with a pair of 1800 grooves/mm gratings and a cooled Hamamatsu 928 photomultiplier detector, and the spectra were recorded under the control of a Spex DM3000 microcomputer system.40 The spectral slit widths were set to 6 cm-1. All spectra were collected with 0.5 cm-1 increments at 1 s/data point. Multiple scans (8-10) were averaged to improve the signalto-noise ratio. Band positions were calibrated using the excitation frequency. Polarization measurements were carried out on the solid sample41 (KCl pellet) by analyzing the scattered light in front of the monochromator slit. Excitation profiles for solid 4 were obtained by recording Raman spectra with K2SO4 as an internal standard. The relative intensity of the desired bands to the standard band (984 cm-1) was then corrected for the ν4 dependence of the scattered radiation and the monochromator sensitivity, and normalized at 568.2 nm excitation. Raman data manipulation (signal averaging) was performed using LabCalc software (Galactic Industries Corp.) mounted on a 486/66 PC computer. IGOR Pro (version 3.12) software (WaveMetrics, Inc.) installed on a Power Macintosh G3 was used to prepare the spectral figures. Results and Discussion Core Structures and Resonance Raman Signatures. Raman excitation within the S f Fe charge-transfer (CT) electronic absorption in the visible region of iron-sulfur proteins and model complexes42-44 selectively enhances vibrational bands associated with bridging (b) and terminal (t) Fe-S stretching vibrations, ν(FeSb) and ν(FeSt).36,37 The enhanced ν(FeSb) and ν(FeSt) bands are distinctive for different types of iron-sulfur clusters (1-Fe, 2-Fe, 3-Fe, and 4-Fe), their vibrational signatures providing a sensitive probe of structure. In tetranuclear [4Fe4S]2+ clusters, the ν(FeSb) modes of the 12 bridging Fe-Sb bonds occur over the wavenumber range ∼250-400 cm-1, while the predominantly ν(FeSt) modes of the four terminal FeSt bonds are confined to a much narrower range, ∼350-400 cm-1. The most diagnostic vibrational marker of the [4Fe4S]2+ core structure is a bridging breathing mode (A1 species

Figure 1. UV-vis absorption spectra of solutions in CH3CN of (s) 4, (‚‚‚) 3, and (- - -) 2. The absorption spectrum for [Fe4S4(tp)4]2- (1) is not shown, as it virtually overlaps that of 2. Excitation profiles for the (]) 336 and (2) 351 cm-1 Fe-S stretching bands of 4 are superimposed on its electronic spectrum. Raman intensities were determined relative to the SO42- band at 984 cm-1.

in the limiting Td symmetry) that gives rise to the most intense band centered near 338 cm-1 in the RR spectra of proteins and analogues. The remaining ν(FeSb) modes produce weaker but characteristic RR bands in the 250 (T2), 275 (T1), 300 (E), and 385 (T2) cm-1 regions. Stretching of the [4Fe-4S]2+ cluster four terminal Fe-St bonds results in RR bands in the 360 (T2) and 390 (A1) cm-1 regions, the latter stretch often coinciding with the higher wavenumber T2 ν(FeSb) stretch (∼385 cm-1). This tetrahedral vibrational signature is only approximate; the symmetry lowering (approximately D2d) due to a slight compression of the [4Fe-4S]2+ cube along an S4 symmetry axis splits the degenerate vibrational modes (E f A1 + B2, T2 f B2 + E, T1 f A2 + E), leading to a characteristic 9-10 RR bands for the [4Fe-4S]2+ clusters.36,37,45 Arylthiolato complexes 1-4 studied in this work are no exception and exhibit the RR spectral signatures that are uniformly nontetrahedral. This is demonstrated in Figure 2, which shows the low-temperature (∼77 K) RR spectra in the 225-475 cm-1 ν(FeS) region taken from crystalline tetrabutylammonium salts of [Fe4S4(tp)4]2- (1), [Fe4S4(3,5-dmtp)4]2- (2), [Fe4S4(2,4-dmtp)4]2- (3), and [Fe4S4(2,6-dmtp)4]2- (4) with excitation at 514.5 nm. The 514.5 nm excitation wavelength, which falls on the lower energy side of the electronic absorption band centered about 460 nm in dianions 1-3 (Figure 1), was found to be effective in enhancing and resolving nearly all the arylthiolato cluster ν(FeS) vibrational modes. This resonance enhancement behavior is consistent with excitation profiles previously measured for the ν(FeS) RR bands of [Fe4S4(SCH2Ph)4]2(6) (Ph ) phenyl) that maximize at the red edge (∼460 nm) of the dianion 6 absorption band and fall off at the absorption maximum (∼425 nm).45 Figure 3, however, displays the spectra of 3 using excitation wavelengths at 457.9 and 514.5 nm. For dianion 3, the visible absorption of which is broadened and slightly blue shifted (Figure 1), the 457.9 nm excitation line enabled detection of an additional band at 354 cm-1. Because analogue 4 absorbs light at drastically lower wavelength (404

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Figure 3. Low-temperature (77 K) RR spectra of [Fe4S4(2,4-dmtp)4]2(3) obtained with (a) 457.9 and (b) 514.5 nm excitation wavelengths.

Figure 2. Low-temperature (77 K) RR spectra of (a-d) 1-4 obtained in the 225-475 cm-1 region with a 514.5 nm excitation wavelength, 200 mW laser power, and 6 cm-1 slit widths.

nm), its RR spectra were recorded using excitation lines at 406.7 and 413.1 nm, in addition to the 514.5 and 459.7 nm lines (Figure 4). Similarly to other Fe-S clusters containing a [4Fe4S]2+ core, the [Fe4S4(SAryl)4]2- dianions 1-4 also have RR spectra that are characterized by a 9-10 D2d-symmetry band pattern in the Fe-S stretching region (250-400 cm-1). In particular, the appearance of at least four bands in the 225325 cm-1 region of 1-4 where only two Fe-S bridging modes are Raman active under Td symmetry (E and T1) makes the lowered symmetry of a [4Fe-4S]2+ core in these complexes a certainty. This observation is consistent with the X-ray structures of 1 and 2, which exhibit eight long and four short Fe-Sb bonds across and along the S4 compression axis, respectively.33,46 Careful examination of the 413.1 nm excitation spectrum of 4 (Figure 4) reveals a larger number of Fe-S bands in the 230400 cm-1 region than in the same spectral region of 1 and 2. Again, this result agrees with the crystallographic data, which indicate the [4Fe-4S]2+ core structure of 4 to be more distorted than those of 1 and 2.33 Raman Spectral Assignments. Previously, Czernuszewicz et al.45 provided the detailed vibrational assignments for [Fe4S4(SR)4]2- species on the basis of 34Sb, 34St, and 54Fe isotope shifts and normal coordinate analysis (NCA) calculations of the benzyl thiolate complex salt (Et4N)2[Fe4S4(SCH2Ph)4] (6). Figures 5 and 6 display the RR spectra of the arylthiolate analogues 1 (514.5 nm excitation) and 4 (413.1 nm excitation),

respectively, together with their 34S-substituted derivatives at the bridging sulfides positions (34Sb). Table 1 gives the observed band frequencies, with assignments to bridging and terminal Fe-S stretching modes, for all four arylthiolate complexes (14) and the frequency shifts on 34S substitution of the bridging atoms for 1 and 4. Also given in Table 1 are the Fe-S mode frequencies and 34Sb isotope shifts of crystalline benzyl cluster 6 for comparison. In general, the arylthiolate complexes exhibit similar sets of at least eight Fe-S stretching modes associated with a D2d-distorted [Fe4S4(SAryl)4]2- anion. The validity of the vibrational assignments for all four complexes is supported by 34S labeling of the bridging sulfurs in 1 and 4, and by comparison with the aforementioned vibrational analysis of 6. Thus, the assignment for [Fe4S4(SPh)4]2- (1) is essentially identical to that of 6 except that the 359 cm-1 band of 1 is attributed to ν(FeSb) rather than ν(FeSt) due to a larger 34Sb downshift (∼5 cm-1) relative to that displayed by the benzyl thiolate complex (2 cm-1).45 Likewise, the observed 34Sb shifts for the 240-340 cm-1 bands of [Fe4S4(2,6-dmtp)4]2- (4) are very similar to those obtained for 6, identifying these bands with the ν(FeSb) stretches (Table 1). The 34Sb dependence of bands above 340 cm-1 is also similar to that for 6 but different from that for 1, and the bands at 351 and 368 cm-1, which have relatively smaller 34Sb downshifts (2.0 and 3.5 cm-1, respectively), are assigned as predominantly ν(FeSt) modes, whereas the bands at 382 and 388 cm-1 are assigned to mainly ν(FeSb) modes (6.5 cm-1 34Sb downshifts), in analogy to those of 6. The assignment of vibrational modes for analogues 2 and 3 is limited due to the absence of isotopically dependent RR data. However, the very high similarity in the metrical details from the X-ray structures of 146 and 233 allows the assignment of 2 to be made to a first approximation by analogy with that of 1. In the case of analogue 3, the 400 cm-1 band is certainly partially due to a thiolate ligand internal vibrational mode. As shown in Figure 2, this band is much broader than all the other bands and must therefore contain more than one component.

RR Signature of [Fe4S4(SAryl)4]2- Clusters

Figure 4. Low-temperature (77 K) RR spectra of [Fe4S4(2,6-dmtp)4]2(4) obtained with (a) 514.5, (b) 457.9, (c) 413.1, and (d) 406.7 nm excitation wavelengths. The inset shows the parallel (|) and perpendicular (⊥) scattering excited from 4 at 514.5 nm.

Since the 457.9 nm excitation line, which is closer to the CT transition of the compound (Figure 1), shifts down this band by 4 cm-1 (Figure 3), the unresolved component most likely comes from a vibrational mode of the [4Fe-4S] cluster itself. Although the absence of crystal structure and isotopic shifts for 3 impeded its spectral interpretation, a tentative assignment is made by analogy with that of 4. Unlike [Fe4S4(SCH2Ph)4]2-,45 arylthiolate analogues 1-4 show an intense RR band at 435, 431, 400, and 426 cm-1, respectively (Figure 2). Previously, Moulis et al. proposed the 435 cm-1 band in the RR spectrum of [Fe4S4(SPh)4]2- (1) to arise from the Fe-St stretching mode because it slightly downshifted upon replacement of the core sulfur atoms with selenium.47 The observation of such a high frequency for FeSt stretching modes was assumed to be the consequence of a partial delocalization of the phenyl ring π electrons toward the electrophilic [4Fe-4S]2+ core, leading to a strengthening of the Fe-S terminal bonds. We find that the 435 and 426 cm-1 bands (Figures 5 and 6) in the RR spectra of 1 and 4, respectively, are not sensitive to 34Sb isotope substitution. In addition, the reported average Fe-S(Aryl) bond lengths for 1 (2.264 Å), 2 (2.269 Å), and 4 (2.263 Å)33 are larger than that of FeS(Benzyl) in 6 (2.251 Å),48 for which no intense RR band around 430 cm-1 is detected.45 The band occurring at ∼400440 cm-1 for analogues 1-4 is rather ascribed to aryl ring

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Figure 5. Low-temperature (77 K) RR spectra of (a) [Fe4S4(tp)4]2(1) in natural abundance and (b) its 34Sb-labeled derivative obtained with a 514.5 nm excitation wavelength. The magnitude of isotope sensitivity (32S minus 34S) is indicated above each band of the RR spectrum for the 34Sb-labeled derivative.

vibrational motions since a similar band is reported at 412 cm-1 in the Raman spectra of thiophenol itself,49,50 and its frequency is sensitive to the presence of methyl substituents (Figure 2). Blue-Shifted Absorption Spectrum of [Fe4S4(2,6-dmtp)4]2(4). The 406.7 and 413.1 nm excitation RR spectra of 4 are anomalous in that they show a dramatic intensity increase for the T2 terminal band at 351 cm-1 as compared to the other bands in the ν(FeS) region (Figure 4). Figure 1 shows excitation profiles for the T2 terminal band at 351 cm-1 and the A1 bridging band at 336 cm-1. The mode symmetry and terminal versus bridging character of these bands have been confirmed by measuring their 34Sb isotope shifts (Figure 6) and depolarization ratios (inset in Figure 4). The profiles maximize at the red edge (∼500 nm) of the electronic absorption band and increase near the absorption maximum (404 nm), but to different extents. While the intensity of the A1 bridging stretch at 336 cm-1 seems to increase only slightly, the corresponding resonance enhancement factors for the T2 terminal stretch at 351 cm-1 are strikingly much greater and appear to track the absorption band into the near-UV region. Certainly, there are multiple electronic transitions in the absorption envelope of 4, but the near-UV band at 404 nm is largely of terminal S f Fe CT character. Arylthiolate ligand conformations were suggested to be responsible for the spectral position of the predominant absorption band of 1-5 in the visible region.33 The crystal structures of 1, 2, and 433,46 reveal that the aryl rings of tp (1) and 3,5dmtp (2) ligands form dihedral angles of ∼45° with the Fe-St

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TABLE 1: Resonance Raman Frequency (cm-1) and 34Sb Shift Comparison for [Fe4S4(SAryl)4]2- (1-4) with [Fe4S4(SCH2Ph)4]2- (6) [Fe4S4(SCH2Ph)4]2- (6)

D2d (Td) assignment

obsda

calcdb

A1 B2 (T2)

391 (1)d 367 (1)

391.3 (2.1) 366.0 (0.8)

[Fe4S4(SAryl)4]21, obsdc Mainly Terminal ν(Fe-S) 390 (1.0) 379 (3.0)e

E (T2)

359 (2)

3, obsdc

4, obsdc

385

390 368

368 (3.5)

354f

351 (2.0)

371e

360.0 (2.5) Mainly Bridging ν(Fe-S) 383.0 (6.8) 359 (5.0)

B2 (T2)

2, obsdc

348

386 (6)

382 (6.5) 377

E (T2) A1 A1 (E) B1 (E) E (T1)

335 (8) 298 (5) 283 (4)e 283 (4)e

386.3 (5.2) 335.1 (7.2) 291.0 (4.3) 280.3 (3.8) 285.4 (4.1)

A2 (T1) B2 (T2) E (T2)

270 (3) 249 (6) 243 (5)

274.0 (3.9) 247.9 (5.3) 242.4 (5.1)

379 (9.5)e 345 (8.0)

371e 334

291 (4.0)

295 280

271 (2.0) 258 (4.0) 240 (4.5)

273 252

339 313 295

388 (6.5) 336 (7.0) 307 (3.5) 295 (4.5)

274

279 (3.5)

254 238

264 (2.5) 244 (4.5)

a Tetraethylammonium salt in KCl pellet at low temperature (77 K); data from Czernuszewicz et al.45 b Calculated for a D cube via the force 2d field given in Czernuszewicz et al.45 c Tetra-n-butylammonium salts in a KCl pellet at low temperature (77 K); this work. d Numbers in parentheses are observed or calculated downshifts upon 34S substitution for the bridging S atoms. e Overlapping bands. f Seen with 457.9 nm excitation.

Figure 6. Low-temperature (77 K) RR spectra of (a) [Fe4S4(2,6dmtp)4]2- (4) in natural abundance and (b) its 34Sb-labeled derivative obtained with a 413.1 nm excitation wavelength.

bonds, whereas the sterically bulkier 2,6-dmtp ligands of 4 favor a dihedral angle of nearly 90° in the solid state. Proton NMR spectra of 4 in CD3CN indicate that this ∼90° geometry is maintained in solution, and has a significant blue-shift effect upon the electronic absorption spectrum of 4 through interactions between the aryl-ring π system and the terminal sulfur p

orbitals.33 The excitation profile for the T2 Fe-St stretch at 351 cm-1 identifies this blue-shifted absorption band of 4 as having a predominantly S(Aryl) f Fe CT transition. It is interesting to notice that the intensity of the A1t band at 354 cm-1 in 3 also slightly increases when the excitation wavelength varies from 514.5 to 457.9 nm (Figure 3). This behavior and the fact that the analogue 3 is characterized by a broadened and slightly blue shifted CT transition with respect to those of 1 and 2 (Figure 1) suggest a thiolate ligand conformation for 3 that is intermediate between those of 1 and 4. Influence of Thiolate Ligands on Fe-S Vibrations. Although they are similar in appearance, the bridging and terminal Fe-S stretching RR bands show some sensitivity to the presence of methyl substituents in the [Fe4S4(SAryl)4]2- structures. The frequency of the dominant bridging breathing mode (A1) is different for each cluster and varies from 345 cm-1 in 1 to 334, 339, and 336 cm-1 in 2, 3, and 4, respectively, when the phenyl ring on each tp ligand of 1 is substituted with two methyl groups at different positions (Figure 2, Table 1). The decrease in frequency of the cluster breathing mode suggests a small expansion of the Fe4S4 cubane structure, consistent with the crystallographic data that the mean Fe-Sb distances in 3,5-dmtp (2) and 2,6-dmtp (4) clusters appear larger than the unsubstituted tp (1) cluster, in particular along the long axes of the compressed Fe4S4 cube (Table 2).33,46 Careful inspection of the Fe-S RR frequencies and 34Sb isotope shifts for 1 and 4 in Table 1 reveals additional striking changes with the arylthiolate ligands. The observed frequencies for 1 and 4 are diagrammatically correlated in Figure 7. Besides the A1 cluster breathing frequency, which is raised by 9 cm-1 in 1 (345 cm-1) compared to 4 (336 cm-1), the most surprising difference lies in the bands near 360 and 385 cm-1. RR bands in these regions have been assigned by 34Sb and 34St isotopic dependencies and NCA calculations to predominantly terminal and bridging T2 Fe-S stretches, respectively, in [4Fe-4S] clusters.45 This vibrational behavior is also observed for 4 but not for 1. 34Sb isotope shifts indicate that the terminal T2 Fe-S stretch occurs at 351/368 cm-1 in 4 and shifts up to 379 cm-1 in 1, while the bridging T2 Fe-S stretch is lowered from 382/ 388 cm-1 in 4 to 359/379 cm-1 in 1 (Figure 7). A structural

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TABLE 2: Selected Fe4Sb4St4 Averaged Bond Distances (Å) and Angles (deg) for [Fe4S4(SAryl)4]2- Dianionsa

[Fe4S4(SAryl)4]2structural parameter t

t, d(Fe-S ) (4) l, d(Fe-Sb) (8) s, d(Fe-Sb) (4) Rts, ∠(St-Fe-Sb) (4) βtl, ∠(St-Fe-Sb) (4) δtl, ∠(St-Fe-Sb) (4)

1b

2c

4c

2.264 2.287 2.263 119.2 103.9 119.3

2.269 2.302 2.254 122.2 101.5 118.1

2.263 2.297d 2.270 117.2 110.2 115.7

a The number of values averaged is given in parentheses; esd values are not shown. Sb and St refer to bridging and terminal sulfur, respectively. b Tetraethylammonium salt; data from Gloux et al.46 c Tetra-n-butylammonium salts; data from Knapp et al.33 d The mean of seven distances is given; the remaining Fe-Sb bond is unusually short (2.254 Å).

This kinematic coupling between ν(FeSb) and ν(FeSt) modes and, hence, the predominantly bridging or terminal Fe-S stretching character of the observed RR bands is expected to be dependent on the arylthiolate ligand conformation. The sterically bulky 2,6-dimethylthiophenolate ligands of 4 impose a geometry about the Fe-St bonds in which the aryl ring and Fe-St bond are arranged almost perpendicular to each other, constraining both of the Sb-Fe-St angles near 120° (110° and 116°); in contrast, the thiophenol ligands of 1 have a nearly parallel conformation, allowing one of the Sb-Fe-St angles to approach 90° (104° and 119°). The mean values of the SbFe-St angles of 4 are close to those found in the crystal structure of benzyl thiolate cluster 6, and as mentioned above, the Fe-S vibrational mode patterns in the RR spectra of 4 and 6 are the same (Table 1). Extension of the benzyl thiolate cluster NCA calculations45 without changing the force field (data not shown) showed that compressing the Sb-Fe-St angle along or across the S4 symmetry axis (seen in 1) increases coupling between terminal and bridging T2 stretching modes, scrambling in consequence their 34Sb isotope shifts. Furthermore, if the FeSt stretching force constant is increased by ∼10% from that found for 6,45 the frequencies of the T2 modes above 350 cm-1 will crossover as indicated by the calculated 34Sb isotope shifts. These results emphasize the importance of 34S isotope incorporation (at both bridging and terminal S positions) into the protein [4Fe-4S] clusters because, while protein cluster structures are indistinguishable within the experimental uncertainty of the X-ray diffraction analysis, considerable variability is seen in the RR bands of [4Fe-4S] proteins, especially in those assigned to terminal Fe-S stretches.45,51,52 Much of this variability could reflect kinematic coupling between bridging and terminal modes that are due to conformational changes in the cysteinyl ligands. As vibrational assignments have only been confirmed for [Fe4S4(SCys)4]2- clusters of Clostridium pasteurianum Fdox by RR spectra of 54Fe and 34S2- reconstituted protein samples,45 further work in this area could uncover some interesting information about how protein structure relates to the electronic structure of the Fe-S cofactors. Acknowledgment. This research was supported by grants from the Robert A. Welch Foundation (E-1184 to R.S.C.), the National Institute of General Medical Sciences (GM48370 to R.S.C.), and the National Institutes of Health (2 T32-DK07233 to D.N.H.). References and Notes

Figure 7. Correlation diagram of RR frequencies for [Fe4S4(tp)4]2(1) and [Fe4S4(2,6-dmtp)4]2- (4). b refers to bridging Fe-S modes; t refers to terminal Fe-S(Aryl) modes.

connection for this crossover of the ν(FeSt) and ν(FeSb) T2 frequencies is not immediately apparent from crystallographic data; although there are noticeable differences in the mean bridging Fe-S bond lengths between the two structures, their mean terminal Fe-S bond lengths are indistinguishable (Table 2). However, the calculated eigenvectors of the Fe4S4bS4t cluster stretching vibrations45 are complex and consist of kinematically coupled ν(FeSb) and ν(FeSt) coordinates. Clearly, the T2 Fe-S stretching modes of 1 and 4 above 350 cm-1 have substantial contributions from both coordinates, as demonstrated by mixed 34Sb isotope shifts of their corresponding RR bands (Figures 5 and 6).

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