Synthetic analogues of ½Fe4S4ðCysÞ3ðHisÞ� inhydrogenases and ½Fe4S4ðCysÞ4� in HiPIP derivedfrom all-ferric ½Fe4S4fNðSiMe3Þ2g4�Yasuhiro Ohki, Kazuki Tanifuji, Norihiro Yamada, Motosuke Imada, Tomoyuki Tajima, and Kazuyuki Tatsumi1

Department of Chemistry, Graduate School of Science and Research Center for Materials Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan

Edited by Douglas C. Rees, Caltech/HHMI, Pasadena, CA, and approved June 13, 2011 (received for review April 27, 2011)

The all-ferric ½Fe4S4�4þ cluster ½Fe4S4fNðSiMe3Þ2g4� 1 and its one-electron reduced form ½1�− serve as convenient precursors forthe synthesis of 3∶1-site differentiated ½Fe4S4� clusters and high-potential iron-sulfur protein (HiPIP) model clusters. The reactionof 1 with four equivalents (equiv) of the bulky thiol HSDmp (Dmp¼2,6-ðmesitylÞ2C6H3, mesityl¼2,4,6-Me3C6H2) followed by treatmentwith tetrahydrofuran (THF) resulted in the isolation of ½Fe4S4ðSDmpÞ3ðTHFÞ3� 2. Cluster 2 contains an octahedral iron atomwith three THF ligands, and its FeðSÞ3ðOÞ3 coordination environ-ment is relevant to that in the active site of substrate-bound aco-nitase. An analogous reaction of ½1�− with four equiv of HSDmpgave ½Fe4S4ðSDmpÞ4�− 3, which models the oxidized form of HiPIP.The THF ligands in 2 can be replaced by tetramethyl-imidazole(Me4Im) to give ½Fe4S4ðSDmpÞ3ðMe4ImÞ� 4 modeling the ½Fe4S4ðCysÞ3ðHisÞ� cluster in hydrogenases, and its one-electron reducedform ½4�− was synthesized from the reaction of 3 with Me4Im. Thereversible redox couple between 3 and ½3�− was observed atE1∕2¼−820 mV vs. Ag∕Agþ, and the corresponding reversiblecouple for 4 and ½4�− is positively shifted by þ440 mV. The cyclicvoltammogram of 3 also exhibited a reversible oxidation couple,which indicates generation of the all-ferric ½Fe4S4�4þ cluster,½Fe4S4ðSDmpÞ4�.

Fe4S4 cluster ∣ thiolates

Cuboidal ½Fe4S4� clusters are ubiquitous metal-centers inproteins, expediting electron transfer and enzymatic reac-

tions. These ½Fe4S4� cores are usually bound to four cysteinyl thio-lates (Cys) as found in the high-potential iron-sulfur proteins(HiPIP) and widely distributed ferredoxins (Fd). Some ½Fe4S4�clusters carrying an N- or O-donor ligand and three Cys ligandsare also known, for example the ½Fe4S4ðCysÞ3ðHisÞ� cluster(His ¼ histidinyl imidazole) in [NiFe] and [FeFe] hydrogenases(Fig. 1) (1–6), and the ½Fe4S4ðCysÞ3ðO-donorÞ� cluster in aconi-tase (7–9) and protochlorophyllide reductase (10). All of these½Fe4S4� clusters are present in three oxidation states, ½Fe4S4�3þ(HiPIPox), ½Fe4S4�2þ (HiPIPred∕Fdox), and ½Fe4S4�þ (Fdred), whilethe ½Fe4S4�0 state has been suggested for the cluster in the Fe-pro-tein of nitrogenase (11, 12). To date, no ½Fe4S4�4þ cluster has beenfound in proteins.

The influence of the ligands around the ½Fe4S4� core on thecluster properties is an important issue. For Fd and HiPIP theformation of hydrogen bonds with water has been suggested toaccount for most of the difference between the redox potentialsof HiPIP and Fd (13), indicating the importance of hydrophobicshielding of the ½Fe4S4� clusters in the more oxidized form. Syn-thetic analogues of HiPIPox with thiolates derived from bulkyhydrocarbon groups are valuable for the investigation of thedependency of the redox potentials on hydrogen bonding. In thecase of the hydrogenase ½Fe4S4ðCysÞ3ðHisÞ� cluster, a possiblerole of the His ligand is to modulate the redox potential, ashas been observed for the Rieske proteins featuring His-bound½Fe2S2� centers (14). However, there is only limited data onthe effect on the redox potentials of ½Fe4S4� clusters (4, 15).

Thus synthetic analogues of ½Fe4S4ðCysÞ3ðHisÞ� clusters, with aðthiolateÞ3ðimidazoleÞ ligand set, are required to elucidate theinfluence of histidine coordination. Although a number of syn-thetic ½Fe4S4� clusters have been reported, most of these are ofthe type ½Fe4S4ðSRÞ4�2−, which has the ½Fe4S4�2þ (HiPIPred∕Fdox)oxidation state (16). However synthesis of other family memberssuch as analogues of HiPIPox and ½Fe4S4ðCysÞ3ðHisÞ� clustersremains difficult. There is only one isolated compound forthe ½Fe4S4�3þ cluster modeling HiPIPox, ½Fe4S4ðSTipÞ4�− (Tip ¼2;4;6-i Pr3 C6H2) (17, 18), which was prepared by chemical oxida-tion of ½Fe4S4ðSTipÞ4�2−. As for ½Fe4S4ðCysÞ3ðHisÞ� analogues,generation of ½Fe4S4ðLS3ÞðimidazolesÞ�− [LS3 ¼ 1,3,5-tris((4,6-dimethyl-3-mercaptophenyl)thio)-2,4,6-tris(p- tolylthio)benze-nate)] was inferred from an 1H NMR spectrum of a mixturecontaining ½Fe4S4ðLS3ÞCl�2−, excess imidazoles, and NaBF4 (19),

Fig. 1. The iron-sulfur clusters in [NiFe] hydrogenases. The coordinates havebeen taken from the crystal data for the protein ofD. v. Miyazaki F, where thePDB code is 1WUL (5).

Author contributions: Y.O. and K.T. designed research; Y.O., K.T., N.Y., M.I., and T.T.performed research; Y.O. and K. T. analyzed data; and Y.O. and K.T. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The atomic coordinates and structure factors have been depositedwith the Cambridge Crystallographic Data Centre, Cambridge CB2 1EK, United Kingdom(CSD reference nos.: CCDC823086–823090).1To whom correspondence should be addressed. E-mail: i45100a@nucc.cc.nagoya-u.ac.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1106472108/-/DCSupplemental.

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however isolation and detailed characterization of the imidazole-bound ½Fe4S4� cluster have not been accomplished.

We (20) and Lee and coworkers (21) have reported the synth-esis of an all-ferric cluster in the ½Fe4S4�4þ oxidation state carryingfour amide ligands, ½Fe4S4fNðSiMe3Þ2g4� 1. This ½Fe4S4�4þ oxida-tion state is not yet known in proteins. This all-ferric cluster hastwo less electrons than the ½Fe4S4�2þ state in the common½Fe4S4ðSRÞ4�2− clusters, and its high oxidation state begs reduc-tion. In fact, the cyclic voltammetry of 1 in THF shows thatthe first reduction process occurs at E1∕2 ¼ 0.10 V vs. Ag∕Agþ,which opens a unique possibility for preparation of syntheticanalogues of HiPIPox and ½Fe4S4ðCysÞ3ðHisÞ� clusters, via reduc-tion. Here we report the successful synthesis of an ½Fe4S4�3þcluster ½Fe4S4ðSDmpÞ4�− 3 modeling HiPIPox as well as 3∶1-sitedifferentiated ½Fe4S4� clusters having a tetramethyl-imidazole(Me4Im) ligand, ½Fe4S4ðSDmpÞ3ðMe4ImÞ�n− (n ¼ 0 4, n ¼ 1

½4�−); Dmp ¼ 2;6-ðmesitylÞ2C6H3), starting from the all-ferriccluster 1 or from its one-electron reduced form ½1�−. We have alsoprepared another 3∶1-site differentiated ½Fe4S4� cluster ½Fe4S4ðSDmpÞ3ðTHFÞ3� 2 containing an octahedral iron atom withthree THF ligands, which is structurally relevant to the activesite of aconitase. The electrochemical properties of the unique½Fe4S4� clusters and the influence of the bulky -SDmp ligandon the redox potential are also discussed.

Results and DiscussionSynthesis of ½Fe4S4fNðSiMe3Þ2g4�−½1�−. Chemical reduction of 1 wasattained by treatment of 1 with one equiv of sodium naphthale-nide (NaC10H8) in THFat 0 °C (Scheme 1, top). Formation of theanionic cluster ½1�− is manifested in the electro-spray ionizationmass spectrum (ESI-MS), showing an anionic signal at m∕z ¼992.4. After removal of naphthalene by sublimation, NaðTHFÞ2½Fe4S4fNðSiMe3Þ2g4� was isolated in 71% yield as black crystals.This anionic cluster has been also prepared recently using NaSHor Na2S as reductants (21). Two redox couples at E1∕2 ¼ 0.11 Vand −0.98 V vs. Ag∕Agþ were observed in the cyclic voltammo-gram (CV) of ½1�−, as in the case of 1.

The crystal structure of NaðTHFÞ2½Fe4S4fNðSiMe3Þ2g4� wasdetermined by X-ray analysis, using single crystals obtainedfrom a hexane solution. As the structure of the same cluster hasbeen reported (21), here we describe only its salient features. Anamide nitrogen atom (N1) and a sulfur atom of ½1�− interactweakly with a NaðTHFÞ2 unit, and the Na-N1 and Na-S distancesare 2.528(2) Å and 3.1992(11) Å, respectively. The Na-N1 inter-action results in a slight pyramidalization at N1, displacing theN atom 0.3721(20) Å from the plane defined by the Fe and Sineighbors, while displacement of the other nitrogen atoms is lessthan 0.0948(22) Å. The planarity of the amide nitrogen suggests astrong N → Fe π-interaction, which shortens the Fe-N distancesand efficiently stabilizes the oxidized ½Fe4S4�3þ core of ½1�−. In-deed the iron-amide (N1) distance of 1.9466(19) Å is notablylonger than the other iron-amide distances (Fe-N2, N3, N4)[1.892(2)–1.897(2) Å]. Interestingly, the Fe-Fe distances of ½1�−,ranging from 2.8044(4) Å to 2.9151(5) Å, are shorter than thoseof 1 [2.8667(7)–3.0014(5) Å], while they are longer than those of½Fe4S4ðSRÞ4�2−ð2.71–2.82 ÅÞ (16).

Reactions of 1 and ½1�− with HSDmp (Dmp¼2,6-ðmesitylÞ2C6H3). Theamide nitrogen of NðSiMe3Þ2 bound to iron is a Brϕnsteadbase, and we have utilized this property to synthesize variousiron-sulfide clusters (22–25). Here, we describe the reactionsof clusters 1 and ½1�− with the bulky thiol HSDmp ðDmp ¼2;6-ðmesitylÞ2C6H3Þ (26).

Reaction of 1. Addition of four equiv of HSDmp to a toluenesolution of 1 led to a color change from reddish black to pur-plish black, and the removal of volatile materials afforded ablack solid. Although characterization of this black solid hasbeen unsuccessful, treatment with THF resulted in the isolationof ½Fe4S4ðSDmpÞ3ðTHFÞ3� 2 in 67% yield as black crystals[Scheme 1, second-row (left)]. On the other hand, the reactionof 1 with HSDmp in THF did not afford cluster 2, and the pro-duct(s) of this reaction remains uncharacterized. Cluster 2consists of one ferrous and three ferric iron atoms, and thus theall-ferric ½Fe4S4�4þ core in 1 is reduced by one electron duringthe reaction with HSDmp. We presume that one of the SDmp−ligands introduced onto the ½Fe4S4� core has been oxidized gen-erating the disulfide (1∕2 DmpS-SDmp), while the resultingvacant coordination site on iron is occupied by three THF mo-lecules.

The generation of one unique iron site in a ½Fe4S4�3þ cubanecluster by reduction of 1 is intriguing, and this reaction offers anew synthetic route to 3∶1-site differentiated ½Fe4S4� clusterswithout invoking the use of tridentate thiolate auxiliaries (19,27, 28). This route complements the recently reported oxidationreactions of the all-ferrous ½Fe4S4�0 clusters ½Fe4S4ðPi Pr3Þ3�2 or½Fe4S4ðPi Pr3Þ2�4 with disulfides, diselenides, or I2, yielding 3∶1-site differentiated ½Fe4S4�þ clusters (29).

Cluster 2 was structurally identified by X-ray crystallographicanalysis. The molecule displays threefold crystallographic sym-metry, and there are two unique molecules in the asymmetricunit, and one of these is shown in Fig. 2, along with selected bonddistances and angles. The threefold axis lies along the Fe1-S2Scheme 1.

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vector, and thus three of the four iron atoms (Fe2, Fe2*, Fe2′) arecrystallographically equivalent. These iron atoms have a distortedtetrahedral geometry surrounded by four sulfur atoms, threefrom the ½Fe4S4� core and fourth from a thiolate ligand. Onthe other hand, Fe1 is nearly octahedral, bound to three oxygenatoms of THF molecules and three sulfur atoms from the core,with the S1-Fe1-S1* and S1-Fe1-O1 angles being 95.53(4)° and87.72(11)°, respectively. Due to the octahedral coordination geo-metry at Fe1, the Fe1-Fe2 distance [2.9600(9) Å] is significantlyelongated compared with the Fe2-Fe2* distance [2.7328(11) Å],indicating no Fe1-Fe2 bonding interaction. The Fe1-S1 bond[2.3938(12) Å] is also longer than the Fe2-S1 and Fe2-S2 bonds[2.2412(10) and 2.2732(13) Å] by 0.1–0.15 Å. It is noteworthy thatthe octahedral geometry of Fe1 coordinated by three O-donorsand three S-donors resembles that of the active site of aconitase(7–9), which catalyzes the reversible isomerization of citrate andisocitrate. In fact, the Fe1-S and Fe1-O distances of 2 [Fe-S;2.3939(12) Å, Fe-O; 2.234(3) Å] are similar to those for theisocitrate complex of aconitase [Fe-S; 2.33–2.36 Å, Fe-O; 2.20(water), 2.28 and 2.37 Å (isocitrate)] (7–9). This similarity opensthe possibility that the unique iron atom of molecule 2 may havecatalytic properties. The THF ligands bound to Fe1 may beweakly held, so that 2 may serve as a synthetic precursor to3∶1-site differentiated ½Fe4S4� clusters.

Reaction of ½1�−. Treatment of ½1�− with four equiv of HSDmp intoluene resulted in the isolation of ½Fe4S4ðSDmpÞ4�− 3 [Scheme 1,second-row (right)] in 69% yield as crystals. In this reaction, allfour amide ligands of ½1�− were all replaced by thiolates, withoutchanging the ½Fe4S4�3þ oxidation state. The ESI-MS spectrum ofa THF solution of 3 gave an anionic signal at m∕z ¼ 1;732.4, andthe isotope pattern matches that calculated. The oxidation stateof 3, ½Fe4S4�3þ, is the same as that of HiPIPox, for which onlyone isolated model cluster, ½Fe4S4ðSTipÞ4�−, has been reported(17, 18). The EPR spectrum of cluster 3 gave a rhombic signalat g ¼ 2.076, 2.035, and 2.018 in frozen toluene at 8 K, whoseg values are similar to those of ½Fe4S4ðSTipÞ4�− with g ¼ 2.10,2.05, and 2.03 (18). The averaged g value of 3, gav ¼ 2.043, is alsoclose to those for HiPIPox with gav ¼ 2.056 (Rubrivivax gelatinosa)(30), 2.065 (Thiobacillus ferrooxidans) (31), and 2.048 (Ectothior-hodospira halophila) (32), although these HiPIPox signalsare axial.

The structure of 3 was determined by X-ray analysis, usingsingle crystals obtained from a toluene-hexamethyldisiloxane

(HMDSO) solution (Fig. 3). The mean values of the Fe-S(core),Fe-S(thiolate), and Fe-Fe distances of 3, are compared with thoseof ½Fe4S4ðSTipÞ4�− (17, 18), ½Fe4S4ðSPhÞ4�2− (33), and ½Fe4S4ðSPhÞ4�3− (34), in Table 1. The Na cation of 3, which interactswith THF, one of the SDmp sulfur atoms, one of the core sulfuratoms, and one of the mesityl groups of SDmp, is disordered overtwo positions with 75∶25 occupancy factors, and only one of theseis shown for clarity. The Na-C(mesityl) distances [2.885(4)–2.979(5) Å] are in the range of the Na-C(Tip) distances determinedfor NaSð2;6-Tip2C6H3Þ [2.839(5)–3.249(5) Å] (35). The higheroxidation state of 3 compared to the ½Fe4S4ðSPhÞ4�2−∕3− clustersresults in a smaller ionic radius for iron, and therefore the Fe-S(core) and Fe-S(thiolate) bond lengths of 3 are slightly butevidently shorter than those of the ½Fe4S4ðSPhÞ4�2−∕3− clusters.The Fe-S(core) and Fe-S(thiolate) bond lengths of 3 and ½Fe4S4ðSTipÞ4�− are similar, while the mean Fe-S(thiolate) length islonger for 3 by ca. 0.02 Å probably because of the Na-SDmpinteraction. Whereas the charge on the ½Fe4S4� core affects theFe-S bond lengths, the Fe-Fe distances of 3 are similar to the½Fe4S4ðSRÞ4�1−∕2−∕3− clusters.

Synthesis and Structures of 3∶1-Site Differentiated ½Fe4S4� Clusterswith a Tetramethyl-Imidazole Ligand. In toluene the THF ligandsof 2 were replaced by one tetramethyl-imidazole (Me4Im), and½Fe4S4ðSDmpÞ3ðMe4ImÞ� 4 was isolated in 46% yield as crystals[Scheme 1, bottom (left)]. Alternatively, cluster 4 was synthesizeddirectly from 1 in 54% yield, via successive treatment with fourequiv of HSDmp and with one equiv of Me4Im. An analogous

Fig. 2. Molecular structure of ½Fe4S4ðSDmpÞ3ðTHFÞ3� 2 with thermal ellip-soids at the 50% probability level. The THF ligand on Fe1 is disordered overtwo positions, and one of these is shown for clarity. Selected distances (Å) andangles (°): Fe1-Fe2 2.9600(9), Fe2-Fe2* 2.7328(11), Fe1-S1 2.3938(12), Fe2-S12.2412(10), Fe2-S2 2.2732(13), Fe2-S3 2.2234(14), Fe1-O1 2.234(3), S1-Fe1-S1*95.53(4), and S1-Fe1-O1 87.72(11).

Fig. 3. Structure of ½NaðTHFÞ�½Fe4S4ðSDmpÞ4� 3with thermal ellipsoids at the50% probability level. The Na(THF) group is disordered over two positions,and one of these is shown for clarity. Selected distances (Å): Fe1-Fe2 2.7282(8), Fe1-Fe3 2.8127(10), Fe1-Fe4 2.7894(8), Fe2-Fe3 2.7561(6), Fe2-Fe4 2.7306(6), Fe3-Fe4 2.7889(7), Fe1-S1 2.2810(12), Fe1-S2 2.2885(11), Fe1-S3 2.2316(11), Fe2-S1 2.3020(12), Fe2-S2 2.2625(13), Fe2-S4 2.2716(13), Fe3-S1 2.2471(12), Fe3-S3 2.3057(13), Fe3-S4 2.2791(13), Fe4-S2 2.2177(11), Fe4-S3 2.2716(12), Fe4-S4 2.2674(14), Fe1-S5 2.2445(15), Fe2-S6 2.2615(9), Fe3-S7 2.2408(11), Fe4-S8 2.2128(10), and S6-Na1 2.894(3).

Table 1. Selected distances (Å) for clusters 3, ½Fe4S4ðSTipÞ4�−,½Fe4S4ðSPhÞ4�2−, and ½Fe4S4ðSPhÞ4�3−

3 ½Fe4S4ðSTipÞ4�−* ½Fe4S4ðSPhÞ4�2−† ½Fe4S4ðSPhÞ4�3−‡

Oxidation state ½Fe4S4�3þ ½Fe4S4�3þ ½Fe4S4�2þ ½Fe4S4�1þav. Fe-Fe 2.7677(10) 2.74(1) 2.736(3) 2.744(17)av. Fe-S(core) 2.2688(14) 2.266(8) 2.286(5) 2.288(12)av. Fe-S(thiolate) 2.2268(11) 2.206(7) 2.263(3) 2.295(10)

*Reference 17.†Reference 33.‡Reference 34.

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½Fe4S4�3þ cluster with histidine ligation has been generated asthe oxidized form of the site-directed Cys → His mutant of theDNA repair enzyme MutY, and this cluster has been reportedto degrade readily into an ½Fe3S4�þ cluster by release of an ironatom (36).

The one-electron reduced form of 4, ½Fe4S4ðSDmpÞ3ðMe4ImÞ�− ½4�−, was obtained in 72% yield as black crystals fromthe reaction of ½Fe4S4ðSDmpÞ4�− 3 with one equiv of Me4Imin toluene [Scheme 1, bottom (right)], and the ESI-MS spectrumof a THF solution of ½4�− exhibits an anionic signal at m∕z ¼1;511.6 (M−). Interestingly, liberation of a SDmp ligand in 3induces one-electron reduction of the ½Fe4S4�3þ core to ½Fe4S4�2þof ½4�−, presumably via formation of DmpS-SDmp. Thus single-site ligand replacement via reduction is also applicable to theHiPIPox-type ½Fe4S4�3þ cluster 3. In a similar manner to the directsynthesis of 4 from 1, the one-pot reaction of ½1�− with four equivof HSDmp and one equiv of Me4Im in toluene gave crystalsof ½4�− in 86% yield. As demonstrated here by the formationof the Me4Im-adducts 4 and ½4�−, this ligand exchange reactionaccompanied by one-electron reduction is a promising tool forthe incorporation of one external biologically relevant ligandonto ½Fe4S4ðSRÞ4�n− clusters. We have also examined chemicaloxidation/reduction of 4∕½4�−. However, oxidation of ½4�− with½Cp2Fe�½PF6� and reduction of 4 with NaðC10H8Þ yielded unchar-acterizable black solids.

The coordination of three thiolates and a tetramethyl-imida-zole to the ½Fe4S4� core of 4 and ½4�− models the structure ofthe ½Fe4S4ðCysÞ3ðHisÞ� cluster found in hydrogenases. The overallsimilarity among the core structures of 4, ½4�−, and the ½Fe4S4ðCysÞ3ðHisÞ� cluster from Desulfovibrio gigas can be seen in Fig. 4.One feature common to all three clusters is a slight bending ofthe imidazole coordinaton, as is manifested by the deviation ofthe S*-Fe1-N angles from 180°, namely, 167.70(15)° (4), 165.67(8)° (½4�−), and 160.9° (D. gigas). As a consequence, the tetrahe-dral geometry at Fe1 is distorted. The distances in D. gigas, Fe-Fe(2.70–2.75 Å), Fe-N(imidazole) (1.99 Å), Fe-S(thiolate) (2.25–2.42 Å), and Fe-S(core) (2.23–2.36 Å), are comparable to those

listed in Table 2 for 4 and ½4�−, taking the lower accuracy of dis-tances in protein structures into consideration. The oxidationstate of the ½Fe4S4ðCysÞ3ðHisÞ� cluster in the crystal structure ofD. gigas has not been clarified yet, although the ½Fe4S4�2þ∕1þ

states have been suggested to be involved in the enzymaticprocess. Successful isolation of 4 and ½4�− suggests that the½Fe4S4�3þ∕2þ states might be another possibility for the ½Fe4S4ðCysÞ3ðHisÞ� clusters. It should be also noted that 4 and ½4�−are structurally analogous to the His-bound ½Fe4S4ðCysÞ3ðHisÞ�clusters serving as one of the electron-transfer sites of the mem-brane-bound nitrate reductase from Escherichia coli (37), and asthe active site of 4-hydroxybutyryl-CoA dehydratase (38).

Fig. 4. The core structures of 4, ½4�−, and the ½Fe4S4ðCysÞ3ðHisÞ� cluster from D. gigas (PDB code 2FRV) (2). For clarity, only the central aromatic rings of -SDmpligands are shown for 4 and ½4�−.

Table 2. Selected distances (Å) and angle (°) for clusters 4 and ½4�−

4 ½4�−Oxidation state ½Fe4S4�3þ ½Fe4S4�2þav. Fe-Fe 2.7450(11) 2.7116(8)Fe1-Fe 2.7272(11) 2.7102(7)other Fe-Fe 2.7628(10) 2.7130(8)av. Fe-S(core) 2.2681(17) 2.2846(12)Fe1-S(core) 2.2782(17) 2.2796(12)other Fe-S(core) 2.2647(16) 2.2862(12)av. Fe-S(thiolate) 2.2179(17) 2.2627(10)Fe-N(imidazole) 1.992(5) 2.017(2)S4-Fe1-N 167.70(15) 165.67(8) Fig. 5. Structures of ½Fe4S4ðSDmpÞ3ðMe4ImÞ� 4 and ½Fe4S4ðSDmpÞ3ðMe4ImÞ�−

½4�− with thermal ellipsoids at the 50% probability level.

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While the structures of 4 and ½4�− shown in Fig. 5 are very muchalike, there are some differences in distances due to the differentoxidation states, as listed in Table 2. The higher oxidation statefor 4 leads to slightly shorter Fe-N(imidazole), Fe-S(core), andFe-S(thiolate) distances than those for ½4�−. The most notabledifference among these can be found in the Fe-S(thiolate) dis-tances, 2.2090(17)–2.2351(15) Å [average (av.) 2.218 Å] for 4and 2.2361(10)–2.2866(10) Å (av. 2.263 Å) for ½4�−, indicatingmore flexibility for the thiolate ligands than the core sulfur atomsin response to the change of oxidation state. Similarly, the Fe-S(thiolate) distances in the ½Fe4S4�3þ cluster 3, 2.2128(10)–2.2615(9) Å (av. 2.227 Å), are shorter than those for ½4�−, although theNa-S(thiolate) interaction elongates one of the Fe-S(thiolate)bonds of 3 to 2.2615(9) Å. In contrast to the Fe-N(imidazole),Fe-S(core), and Fe-S(thiolate) distances, the Fe-Fe distances for4, 2.6946(10)–2.7978(10) Å (av. 2.745 Å), and 3, 2.7282(8)–2.8127(10) Å (av. 2.768 Å), are slightly longer than those for ½4�−,2.6834(6)–2.7335(7) Å (av. 2.712 Å).

Electronic Properties of 3, 4, and ½4�− as Models of HiPIP and½Fe4S4ðCysÞ3ðHisÞ�. The CV for the HiPIP model 3 and the ½Fe4S4ðCysÞ3ðHisÞ� models 4 and ½4�− have been measured and com-pared to assess the influence of the imidazole ligand on the redoxpotentials. Fig. 6 shows the CV data for 3 and ½4�− in the region of0 ∼ −1.2 V, where the potentials are referenced to Ag∕Agþ.

The ½Fe4S4�3þ∕2þ redox couple between 4 and ½4�− was foundto be reversible at E1∕2 ¼ −380 mV, and the corresponding rever-sible redox couple for 3 and ½3�− appeared at E1∕2 ¼ −820 mV.Thus the redox potential is shifted by þ440 mV, when one ofthe -SDmp ligands in 3 is replaced by Me4Im. The positive shiftis reasonable, as the electron-donating abilities of anionic thiolateligands should be greater than those of neutral imidazoles. Simi-larly, the in situ generated ½Fe4S4� cluster with a 4-methyl-imida-zole (4-MeIm) ligand, ½Fe4S4ðLS3Þð4-MeImÞ�−, shows a redoxcouple more positive by 320 mV than the cluster having anethane-thiolate instead of 4-MeIm, ½Fe4S4ðLS3ÞðSEtÞ�2− (19).

The significantly higher redox potential for 4 relative to 3implies that facile electron transfer could occur from the½Fe4S4ðCysÞ4� cluster, which is proximal to the Ni-Fe active siteof [NiFe] hydrogenases, to the distal-½Fe4S4ðCysÞ3ðHisÞ� cluster.This direction is the electron flow that promotes the oxidationof H2 at the active site. On the other hand, hydrogenases alsopromote the reduction of Hþ that requires the reverse electrontransfer from the distal- to proximal-½Fe4S4� clusters. Therefore,there must be a mechanism, which promotes the reverse electrontransfer process, by modulating the redox potentials of the

½Fe4S4� clusters. The unique positioning of the distal-½Fe4S4ðCysÞ3ðHisÞ� cluster in the protein may give a clue. The distalcluster is located at the protein surface with the imidazole N-Hgroup oriented toward outside of the protein (Fig. 1). We hypo-thesize here that the imidazole N-H group is exposed to water,and that coordination of the imidazole to iron enhances the N-Hacidity facilitating its deprotonation. This deprotonation at imi-dazole would cause a substantial negative shift of the potential ofthe ½Fe4S4ðCysÞ3ðHisÞ� cluster, due to the addition of an extranegative charge with retention of the oxidation state of the ironatoms. Therefore, protonation/deprotonation at the imidazolemay act as a changeover switch reversing the electron transferbetween the distal- and proximal-½Fe4S4� clusters.

Interestingly, upon scanning a CV of 3 toward positive poten-tial, a reversible redox couple at E1∕2 ¼ þ80 mV was observed(Fig. 7), which indicates the generation of an all-ferric ½Fe4S4�4þcluster, ½Fe4S4ðSDmpÞ4�0. Cluster 1 is the sole example of anall-ferric ½Fe4S4�4þ cluster, and a thiolate-bound ½Fe4S4�4þ clusteris unprecedented. Stabilization of the all-ferric state of the½Fe4S4�4þ core requires a strongly basic, electron donating setof ligands to counter the large positive charge of the iron atoms.Even the removal of a small amount of negative charge due to theformation of hydrogen bonds appears to destabilize the all-ferricoxidation state. As for the HiPIPox cluster, the ½Fe4S4�3þ core isburied in a hydrophobic pocket of the protein, while the½Fe4S4�2þ∕1þ core of Fd is exposed to hydrogen bonding by water(13). In this context, the unique ability of higher oxidation statesof 3 may arise from the environment provided by the bulkyhydrocarbon groups of the -SDmp ligands, which mimics a hydro-phobic pocket of the protein.

Materials and MethodsAll reactions and the manipulations were carried out using standard Schlenktechniques and a glove box with a nitrogen atmosphere. Toluene, THF,hexane, and HMDSO were purified by columns of activated alumina and asupported copper catalyst supplied by Hansen and Co. Ltd.

All experimental conditions and procedures, spectroscopic data, anddetails of characterization of complexes are given in SI Text, which ispublished on the PNAS web site.

ACKNOWLEDGMENTS. We thank Roger E. Cramer for his help in X-raycrystallographic analysis and for careful reading of the manuscript. Thisresearch was financially supported by Grant-in-Aids for Scientific Research(Nos. 18GS0207 and 18064009) from the Ministry of Education, Culture,Sports, Science and Technology, Japan.

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