METTL17 is an Fe-S cluster checkpoint for mitochondrial translation.

Friedreich's ataxia (FA) is a debilitating, multisystemic disease caused by the depletion of frataxin (FXN), a mitochondrial iron-sulfur (Fe-S) cluster biogenesis factor. To understand the cellular pathogenesis of FA, we performed quantitative proteomics in FXN-deficient human cells. Nearly every annotated Fe-S cluster-containing protein was depleted, indicating that as a rule, cluster binding confers stability to Fe-S proteins. We also observed depletion of a small mitoribosomal assembly factor METTL17 and evidence of impaired mitochondrial translation. Using comparative sequence analysis, mutagenesis, biochemistry, and cryoelectron microscopy, we show that METTL17 binds to the mitoribosomal small subunit during late assembly and harbors a previously unrecognized [Fe4S4]2+ cluster required for its stability. METTL17 overexpression rescued the mitochondrial translation and bioenergetic defects, but not the cellular growth, of FXN-depleted cells. These findings suggest that METTL17 acts as an Fe-S cluster checkpoint, promoting translation of Fe-S cluster-rich oxidative phosphorylation (OXPHOS) proteins only when Fe-S cofactors are replete.

An Iron-Sulfur Cluster with a Highly Pyramidalized Three-Coordinate Iron Center and a Negligible Affinity for Dinitrogen.

Attempts to generate open coordination sites for N2 binding at synthetic Fe-S clusters often instead result in cluster oligomerization. Recently, it was shown for Mo-Fe-S clusters that such oligomerization reactions can be prevented through the use of sterically protective supporting ligands, thereby enabling N2 complex formation. Here, this strategy is extended to Fe-only Fe-S clusters. One-electron reduction of (IMes)3Fe4S4Cl (IMes = 1,3-dimesitylimidazol-2-ylidene) forms the transiently stable edge-bridged double cubane (IMes)6Fe8S8, which loses two IMes ligands to form the face-bridged double-cubane, (IMes)4Fe8S8. The finding that the three supporting IMes ligands do not confer sufficient protection to curtail cluster oligomerization prompted the design of a new N-heterocyclic carbene, SIArMe,iPr (1,3-bis(3,5-diisopropyl-2,6-dimethylphenyl)-2-imidazolidinylidene; abbreviated as SIAr), that features bulky groups strategically placed in remote positions. When the reduction of (SIAr)3Fe4S4Cl or [(SIAr)3Fe4S4(THF)]+ is conducted in the presence of SIAr, the formation of (SIAr)4Fe8S8 is indeed suppressed, permitting characterization of the reduced [Fe4S4]0 product. Surprisingly, rather than being an N2 complex, the product is simply (SIAr)3Fe4S4: a cluster with a three-coordinate Fe site that adopts an unusually pyramidalized geometry. Although (SIAr)3Fe4S4 does not coordinate N2 to any appreciable extent under the surveyed conditions, it does bind CO to form (SIAr)3Fe4S4(CO). This finding demonstates that the binding pocket at the unique Fe is not too small for N2; instead, the exceptionally weak affinity for N2 can be attributed to weak Fe-N2 bonding. The differences in the N2 coordination chemistry between sterically protected Mo-Fe-S clusters and Fe-only Fe-S clusters are discussed.

Cluster-selective 57Fe labeling of a Twitch-domain-containing radical SAM enzyme.

57Fe-specific techniques such as Mössbauer spectroscopy are invaluable tools in mechanistic studies of Fe-S proteins. However, they remain underutilized for proteins that bind multiple Fe-S clusters because such proteins are typically uniformly enriched with 57Fe. As a result, it can be unclear which spectroscopic responses derive from which cluster, and this in turn obscures the chemistry that takes place at each cluster. Herein, we report a facile method for cluster-selective 57Fe enrichment based on exchange between the protein's Fe-S clusters and exogenous Fe ions. Through a combination of inductively coupled plasma mass spectrometric and 57Fe Mössbauer spectroscopic analysis, we show that, of the two [Fe4S4] clusters in BtrN (a Twitch-domain-containing radical S-adenosyl-l-methionine (SAM) enzyme), the Fe ions in the SAM-binding cluster undergo faster exchange with exogenous Fe2+; the auxiliary cluster is essentially inert under the reaction conditions. Exploiting this rate difference allows for either of the two [Fe4S4] clusters to be selectively labeled: the SAM-binding cluster can be labeled by exchanging unlabeled BtrN with 57Fe2+, or the auxiliary cluster can be labeled by exchanging fully labeled BtrN with natural abundance Fe2+. The labeling selectivity likely originates primarily from differences in the clusters' accessibility to small molecules, with secondary contributions from the different redox properties of the clusters. This method for cluster-selective isotopic labeling could in principle be applied to any protein that binds multiple Fe-S clusters so long as the clusters undergo exchange with exogenous Fe ions at sufficiently different rates.

Exploiting Molecular Symmetry to Quantitatively Map the Excited-State Landscape of Iron-Sulfur Clusters.

Cuboidal [Fe4S4] clusters are ubiquitous cofactors in biological redox chemistry. In the [Fe4S4]1+ state, pairwise spin coupling gives rise to six arrangements of the Fe valences ("valence isomers") among the four Fe centers. Because of the magnetic complexity of these systems, it has been challenging to understand how a protein's active site dictates both the arrangement of the valences in the ground state as well as the population of excited-state valence isomers. Here, we show that the ground-state valence isomer landscape can be simplified from a six-level system in an asymmetric protein environment to a two-level system by studying the problem in synthetic [Fe4S4]1+ clusters with solution C3v symmetry. This simplification allows for the energy differences between valence isomers to be quantified (in some cases with a resolution of <0.1 kcal/mol) by simultaneously fitting the VT NMR and solution magnetic moment data. Using this fitting protocol, we map the excited-state landscape for a range of clusters of the form [(SIMes)3Fe4S4-X/L]n, (SIMes = 1,3-dimesityl-imidazol-4,5-dihydro-2-ylidene; n = 0 for anionic, X-type ligands and n = +1 for neutral, L-type ligands) and find that a single ligand substitution can alter the relative ground-state energies of valence isomers by at least 103 cm-1. On this basis, we suggest that one result of "non-canonical" amino acid ligation in Fe-S proteins is the redistribution of the valence electrons in the manifold of thermally populated excited states.

Connecting the geometric and electronic structures of the nitrogenase iron-molybdenum cofactor through site-selective 57Fe labelling.

Understanding the chemical bonding in the catalytic cofactor of the Mo nitrogenase (FeMo-co) is foundational for building a mechanistic picture of biological nitrogen fixation. A persistent obstacle towards this goal has been that the 57Fe-based spectroscopic data-although rich with information-combines responses from all seven Fe sites, and it has therefore not been possible to map individual spectroscopic responses to specific sites in the three-dimensional structure. Here we have addressed this challenge by incorporating 57Fe into a single site of FeMo-co. Spectroscopic analysis of the resting state informed on the local electronic structure of the terminal Fe1 site, including its oxidation state and spin orientation, and, in turn, on the spin-coupling scheme for the entire cluster. The oxidized resting state and the first intermediate in nitrogen fixation were also characterized, and comparisons with the resting state provided molecular-level insights into the redox chemistry of FeMo-co.

An Open-Cuboidal [Fe3S4] Cluster Characterized in Both Biologically Relevant Redox States.

Synthetic analogues of the three common types of Fe-S clusters found in biology─diamond-core [Fe2S2] clusters, open-cuboidal [Fe3S4] clusters, and cuboidal [Fe4S4] clusters─have been reported in each biologically relevant redox state with one exception: the open-cuboidal [Fe3S4]+ cluster. Here, we describe the synthesis and characterization of an open-cuboidal [Fe3S4] cluster in both biologically relevant redox states: [Fe3S4]+ and [Fe3S4]0. Like their biological counterparts, the oxidized cluster has a spin-canted, S = 1/2 ground state, and the reduced cluster has an S = 2 ground state. Structural analysis reveals that the [Fe3S4] core undergoes substantial contraction upon oxidation, in contrast to the minimal structural changes observed for the only [Fe3S4] protein for which high-resolution structures are available in both redox states (Azotobacter vinelandii ferredoxin I; Av FdI). This difference between the synthetic models and Av FdI is discussed in the context of electron transfer by [Fe3S4] proteins.

Facile and dynamic cleavage of every iron-sulfide bond in cuboidal iron-sulfur clusters.

Nature employs weak-field metalloclusters to support a wide range of biological processes. The most ubiquitous metalloclusters are the cuboidal Fe-S clusters, which are comprised of Fe sites with locally high-spin electronic configurations. Such configurations enhance rates of ligand exchange and imbue the clusters with a degree of structural plasticity that is increasingly thought to be functionally relevant. Here, we examine this phenomenon using isotope tracing experiments. Specifically, we demonstrate that synthetic [Fe4S4] and [MoFe3S4] clusters exchange their Fe atoms with Fe2+ ions dissolved in solution, a process that involves the reversible cleavage and reformation of every Fe-S bond in the cluster core. This exchange is facile-in most cases occurring at room temperature on the timescale of minutes-and documented over a range of cluster core oxidation states and terminal ligation patterns. In addition to suggesting a highly dynamic picture of cluster structure, these results provide a method for isotopically labeling pre-formed clusters with spin-active nuclei, such as 57Fe. Such a protocol is demonstrated for the radical S-adenosyl-l-methionine enzyme, RlmN.

Valence Localization in Alkyne and Alkene Adducts of Synthetic [Fe4S4]+ Clusters.

Reported herein are alkyne and alkene adducts of synthetic [Fe4S4]+ clusters that model intermediates and inhibitor-bound states in enzymes involved in isoprenoid biosynthesis. Treatment of the N-heterocyclic carbene-ligated cluster [(IMes)3Fe4S4(OEt2)][BArF4] (IMes = 1,3-dimesitylimidazol-2-ylidene; [BArF4]- = tetrakis(3,5-bis(trifluoromethyl)phenyl)borate) with phenylacetylene (PhCCH) or cis-cyclooctene (COE) results in displacement of the Et2O ligand to yield the corresponding π complexes, [(IMes)3Fe4S4(PhCCH)][BArF4] and [(IMes)3Fe4S4(COE)][BArF4]. EPR spectroscopic analysis demonstrates that both clusters are doublets with giso > 2 and thus are spectroscopically faithful models of the analogous species characterized in the isoprenoid biosynthetic enzymes IspG and IspH. Structural and Mössbauer spectroscopic analysis reveals that both complexes are best described as [Fe4S4]+ clusters in which the unique Fe site engages in modest back-bonding to the π-acidic ligand. Paramagnetic NMR studies show that, even at room temperature, the alkyne/alkene-bound Fe centers harbor minority spin and therefore adopt an Fe2+ valence. We propose that such valence localization could likewise occur in Fe-S enzymes that interact with π-acidic molecules.

The Elusive Mononitrosylated [Fe4 S4 ] Cluster in Three Redox States.

Iron-sulfur clusters are well-established targets in biological nitric oxide (NO) chemistry, but the key intermediate in these processes-a mononitrosylated [Fe4 S4 ] cluster-has not been fully characterized in a protein or a synthetic model thereof. Here, we report the synthesis of a three-member redox series of isostructural mononitrosylated [Fe4 S4 ] clusters. Mononitrosylation was achieved by binding NO to a 3 : 1 site-differentiated [Fe4 S4 ]+ cluster; subsequent oxidation and reduction afforded the other members of the series. All three clusters feature a local high-spin Fe3+ center antiferromagnetically coupled to 3 [NO]- . The observation of an anionic NO ligand suggests that NO binding is accompanied by formal electron transfer from the cluster to NO. Preliminary reactivity studies with the monocationic cluster demonstrate that exposure to excess NO degrades the cluster, supporting the intermediacy of mononitrosylated intermediates in NO sensing/signaling.

Characterization by ENDOR Spectroscopy of the Iron-Alkyl Bond in a Synthetic Counterpart of Organometallic Intermediates in Radical SAM Enzymes.

Members of the radical S-adenosyl-l-methionine (SAM) enzyme superfamily initiate a broad spectrum of radical transformations through reductive cleavage of SAM by a [4Fe-4S]1+ cluster it coordinates to generate the reactive 5'-deoxyadenosyl radical (5'-dAdo•). However, 5'-dAdo• is not directly liberated for reaction and instead binds to the unique Fe of the cluster to create the catalytically competent S = 1/2 organometallic intermediate Ω. An alternative mode of reductive SAM cleavage, especially seen photochemically, instead liberates CH3•, which forms the analogous S = 1/2 organometallic intermediate with an Fe-CH3 bond, ΩM. The presence of a covalent Fe-C bond in both structures was established by the ENDOR observation of 13C and 1H hyperfine couplings to the alkyl groups that show isotropic components indicative of Fe-C bond covalency. The synthetic [Fe4S4]3+-CH3 cluster, M-CH3, is a crystallographically characterized analogue to ΩM that exhibits the same [Fe4S4]3+ cluster state as Ω and ΩM, and thus an analysis of its spectroscopic properties─and comparison with those of Ω and ΩM─can be grounded in its crystal structure. We report cryogenic (2 K) EPR and 13C/1/2H ENDOR measurements on isotopically labeled M-CH3. At low temperatures, the complex exhibits EPR spectra from two distinct conformers/subpopulations. ENDOR shows that at 2 K, one contains a static methyl, but in the other, the methyl undergoes rapid tunneling/hopping rotation about the Fe-CH3 bond. This generates an averaged hyperfine coupling tensor whose analysis requires an extended treatment of rotational averaging. The methyl group 13C/1/2H hyperfine couplings are compared with the corresponding values for Ω and ΩM.

Reversible Alkyl-Group Migration between Iron and Sulfur in [Fe4S4] Clusters.

Synthetic [Fe4S4] clusters with Fe-R groups (R = alkyl/benzyl) are shown to release organic radicals on an [Fe4S4]3+-R/[Fe4S4]2+ redox couple, the same that has been proposed for a radical-generating intermediate in the superfamily of radical S-adenosyl-l-methionine (SAM) enzymes. In attempts to trap the immediate precursor to radical generation, a species in which the alkyl group has migrated from Fe to S is instead isolated. This S-alkylated cluster is a structurally faithful model of intermediates proposed in a variety of functionally diverse S transferase enzymes and features an "[Fe4S4]+-like" core that exists as a physical mixture of S = 1/2 and 7/2 states. The latter corresponds to an unusual, valence-localized electronic structure as indicated by distortions in its geometric structure and supported by computational analysis. Fe-to-S alkyl group migration is (electro)chemically reversible, and the preference for Fe vs S alkylation is dictated by the redox state of the cluster. These findings link the organoiron and organosulfur chemistry of Fe-S clusters and are discussed in the context of metalloenzymes that are proposed to make and break Fe-S and/or C-S bonds during catalysis.

Evidence for Low-Valent Electronic Configurations in Iron-Sulfur Clusters.

Although biological iron-sulfur (Fe-S) clusters perform some of the most difficult redox reactions in nature, they are thought to be composed exclusively of Fe2+ and Fe3+ ions, as well as mixed-valent pairs with average oxidation states of Fe2.5+. We herein show that Fe-S clusters formally composed of these valences can access a wider range of electronic configurations─in particular, those featuring low-valent Fe1+ centers. We demonstrate that CO binding to a synthetic [Fe4S4]0 cluster supported by N-heterocyclic carbene ligands induces the generation of Fe1+ centers via intracluster electron transfer, wherein a neighboring pair of Fe2+ sites reduces the CO-bound site to a low-valent Fe1+ state. Similarly, CO binding to an [Fe4S4]+ cluster induces electron delocalization with a neighboring Fe site to form a mixed-valent Fe1.5+Fe2.5+ pair in which the CO-bound site adopts partial low-valent character. These low-valent configurations engender remarkable C-O bond activation without having to traverse highly negative and physiologically inaccessible [Fe4S4]0/[Fe4S4]- redox couples.

Dinitrogen binding and activation at a molybdenum-iron-sulfur cluster.

The Fe-S clusters of nitrogenases carry out the life-sustaining conversion of N2 to NH3. Although progress continues to be made in modelling the structural features of nitrogenase cofactors, no synthetic Fe-S cluster has been shown to form a well-defined coordination complex with N2. Here we report that embedding an [MoFe3S4] cluster in a protective ligand environment enables N2 binding at Fe. The bridging [MoFe3S4]2(µ-η1:η1-N2) complex thus prepared features a substantially weakened N-N bond despite the relatively high formal oxidation states of the metal centres. Substitution of one of the [MoFe3S4] cubanes with an electropositive Ti metalloradical induces additional charge transfer to the N2 ligand with generation of Fe-N multiple-bond character. Structural and spectroscopic analyses demonstrate that N2 activation is accompanied by shortened Fe-S distances and charge transfer from each Fe site, including those not directly bound to N2. These findings indicate that covalent interactions within the cluster play a critical role in N2 binding and activation.

Postbiosynthetic modification of a precursor to the nitrogenase iron-molybdenum cofactor.

Nitrogenases utilize Fe-S clusters to reduce N2 to NH3 The large number of Fe sites in their catalytic cofactors has hampered spectroscopic investigations into their electronic structures, mechanisms, and biosyntheses. To facilitate their spectroscopic analysis, we are developing methods for incorporating 57Fe into specific sites of nitrogenase cofactors, and we report herein site-selective 57Fe labeling of the L-cluster-a carbide-containing, [Fe8S9C] precursor to the Mo nitrogenase catalytic cofactor. Treatment of the isolated L-cluster with the chelator ethylenediaminetetraacetate followed by reconstitution with 57Fe2+ results in 57Fe labeling of the terminal Fe sites in high yield and with high selectivity. This protocol enables the generation of L-cluster samples in which either the two terminal or the six belt Fe sites are selectively labeled with 57Fe. Mössbauer spectroscopic analysis of these samples bound to the nitrogenase maturase Azotobacter vinelandii NifX reveals differences in the primary coordination sphere of the terminal Fe sites and that one of the terminal sites of the L-cluster binds to H35 of Av NifX. This work provides molecular-level insights into the electronic structure and biosynthesis of the L-cluster and introduces postbiosynthetic modification as a promising strategy for studies of nitrogenase cofactors.

A Terminal Imido Complex of an Iron-Sulfur Cluster.

We report the synthesis and characterization of the first terminal imido complex of an Fe-S cluster, (IMes)3 Fe4 S4 =NDipp (2; IMes=1,3-dimesitylimidazol-2-ylidene, Dipp=2,6-diisopropylphenyl), which is generated by oxidative group transfer from DippN3 to the all-ferrous cluster (IMes)3 Fe4 S4 (PPh3 ). This two-electron process is achieved by formal one-electron oxidation of the imido-bound Fe site and one-electron oxidation of two IMes-bound Fe sites. Structural, spectroscopic, and computational studies establish that the Fe-imido site is best described as a high-spin Fe3+ center, which is manifested in its long Fe-N(imido) distance of 1.763(2) Å. Cluster 2 abstracts hydrogen atoms from 1,4-cyclohexadiene to yield the corresponding anilido complex, demonstrating competency for C-H activation.

Reversible Formation of Alkyl Radicals at [Fe4S4] Clusters and Its Implications for Selectivity in Radical SAM Enzymes.

All kingdoms of life use the transient 5'-deoxyadenosyl radical (5'-dAdo•) to initiate a wide range of difficult chemical reactions. Because of its high reactivity, the 5'-dAdo• must be generated in a controlled manner to abstract a specific H atom and avoid unproductive reactions. In radical S-adenosylmethionine (SAM) enzymes, the 5'-dAdo• is formed upon reduction of SAM by an [Fe4S4] cluster. An organometallic precursor featuring an Fe-C bond between the [Fe4S4] cluster and the 5'-dAdo group was recently characterized and shown to be competent for substrate radical generation, presumably via Fe-C bond homolysis. Such reactivity is without precedent for Fe-S clusters. Here, we show that synthetic [Fe4S4]-alkyl clusters undergo Fe-C bond homolysis when the alkylated Fe site has a suitable coordination number, thereby providing support for the intermediacy of organometallic species in radical SAM enzymes. Addition of pyridine donors to [(IMes)3Fe4S4-R]+ clusters (R = alkyl or benzyl; IMes = 1,3-dimesitylimidazol-2-ylidene) generates R•, ultimately forming R-R coupled hydrocarbons. This process is facile at room temperature and allows for the generation of highly reactive radicals including primary carbon radicals. Mechanistic studies, including use of the 5-hexenyl radical clock, demonstrate that Fe-C bond homolysis occurs reversibly. Using these experimental insights and kinetic simulations, we evaluate the circumstances in which an organometallic intermediate can direct the 5'-dAdo• toward productive H-atom abstraction. Our findings demonstrate that reversible homolysis of even weak M-C bonds is a feasible protective mechanism for the 5'-dAdo• that can allow selective X-H bond activation in both radical SAM and adenosylcobalamin enzymes.

An [Fe4S4]3+-Alkyl Cluster Stabilized by an Expanded Scorpionate Ligand.

Alkyl-ligated iron-sulfur clusters in the [Fe4S4]3+ charge state have been proposed as short-lived intermediates in a number of enzymatic reactions. To better understand the properties of these intermediates, we have prepared and characterized the first synthetic [Fe4S4]3+-alkyl cluster. Isolation of this highly reactive species was made possible by the development of an expanded scorpionate ligand suited to the encapsulation of cuboidal clusters. Like the proposed enzymatic intermediates, this synthetic [Fe4S4]3+-alkyl cluster adopts an S = 1/2 ground state with giso > 2. Mössbauer spectroscopic studies reveal that the alkylated Fe has an unusually low isomer shift, which reflects the highly covalent Fe-C bond and the localization of Fe3+ at the alkylated site in the solid state. Paramagnetic 1H NMR studies establish that this valence localization persists in solution at physiologically relevant temperatures, an effect that has not been observed for [Fe4S4]3+ clusters outside of a protein. These findings establish the unusual electronic-structure effects imparted by the strong-field alkyl ligand and lay the foundation for understanding the electronic structures of [Fe4S4]3+-alkyl intermediates in biology.

A Synthetic Model of Enzymatic [Fe4S4]-Alkyl Intermediates.

Although alkyl complexes of [Fe4S4] clusters have been invoked as intermediates in a number of enzymatic reactions, obtaining a detailed understanding of their reactivity patterns and electronic structures has been difficult owing to their transient nature. To address this challenge, we herein report the synthesis and characterization of a 3:1 site-differentiated [Fe4S4]2+-alkyl cluster. Whereas [Fe4S4]2+ clusters typically exhibit pairwise delocalized electronic structures in which each Fe has a formal valence of 2.5+, Mössbauer spectroscopic and computational studies suggest that the highly electron-releasing alkyl group partially localizes the charge distribution within the cubane, an effect that has not been previously observed in tetrahedrally coordinated [Fe4S4] clusters.

Controlling Substrate Binding to Fe4S4 Clusters through Remote Steric Effects.

The extraordinary reactivity exhibited by many Fe-S enzymes is due in large part to the influence of the protein scaffold on substrate binding and activation. In principle, the coordination chemistry of synthetic Fe-S clusters could similarly be controlled through remote steric effects. Toward this end, we report the synthesis of 3:1 site-differentiated [Fe4S4] clusters ligated by N -heterocyclic carbene (NHC) ligands with variable steric profiles: IMes (1,3-dimesitylimidazol-2-ylidene) and I iPrMe (1,3-diisopropyl-4,5-dimethylimidazol-2-ylidene). Treatment of (IMes)3Fe4S4Cl with NaBArF4 in ethereal solvents (Et2O and THF) leads to the formation of an ether adduct, [(IMes)3Fe4S4(solv)][BArF4]; solvent can be displaced by addition of tBuNC to form the unusual monoisocyanide adduct [(IMes)3Fe4S4(CN tBu)][BArF4]. Carrying out the same reactions with the less sterically encumbered cluster (I iPrMe)3Fe4S4Cl results in more typical reactivity: undesired ligand redistribution to form the homoleptic cluster [(I iPrMe)4Fe4S4][BArF4] and generation of the triisocyanide adduct [(I iPrMe)3Fe4S4(CN tBu)3][BArF4]. The increased steric profile of the IMes ligands disfavors ligand redistribution and defines a binding pocket at the apical Fe, thereby enabling the generation of a coordinatively unsaturated and substitutionally labile Fe site. This method of controlling the coordination chemistry at the apical Fe site by modifying the sterics of ligands bound to adjacent Fe sites complements existing strategies for generating site-differentiated Fe-S clusters and provides new opportunities to direct reactivity at cuboidal metalloclusters.

Selective Synthesis of Site-Differentiated Fe4S4 and Fe6S6 Clusters.

Obtaining rational control over the structure and nuclearity of metalloclusters is an ongoing challenge in synthetic Fe-S cluster chemistry. We report a new family of tridentate imidazolin-2-imine ligands L(NImR)3 that can bind [Fe4S4]2+ or [Fe6S6]3+ clusters, depending on the steric profile of the ligand and the reaction stoichiometry. A high-yielding synthetic route to L(NImR)3 ligands (where R is the imidazolyl N substituents) from trianiline and 2-chloroimidazolium precursors is described. For L(NImMe)3 (tris(1,3,5-(3-( N, N-dimethyl-4,5-diphenylimidazolin-2-imino)phenylmethyl))benzene), metalation with 1 equiv of [Ph4P]2[Fe4S4Cl4] and 3 equiv of NaBPh4 furnishes a mixture of products, but adjusting the stoichiometry to 1.5 equiv of [Ph4P]2[Fe4S4Cl4] provides (L(NImMe)3)Fe6S6Cl6 in high yield. Formation of an [Fe6S6]3+ cluster using L(NImTol)3 (tris(1,3,5-(3-( N, N-bis(4-methylphenyl)-4,5-diphenylimidazolin-2-imino)phenylmethyl))benzene) is not observed; instead, the [Fe4S4]2+ cluster [(L(NImTol)3)(Fe4S4Cl)][BPh4] is cleanly generated when 1 equiv of [Ph4P]2[Fe4S4Cl4] is employed. The selectivity for cluster nuclearity is rationalized by the orientation of the imidazolyl rings whereby long N-imidazolyl substituents preclude formation of [Fe6S6]3+ clusters but not [Fe4S4]2+ clusters. Thus, the structure and nuclearity of L(NImR)3-bound Fe-S clusters may be selectively controlled through rational modification the ligand's substituents.