The C-cluster of carbon monoxide dehydrogenase (CODH) appears to be the active site for the oxidation of CO to CO 2 . We have studied with EPR and Mössbauer spectroscopy the enzymes from Rhodospirillum rubrum (CODH Rr ; ∼8 Fe atoms and 1 Ni atom per R) and Clostridium thermoaceticum (CODH Ct ; ∼12 Fe atoms and 2 Ni atoms per R ). The study of CODH Rr offers two advantages. First, the enzyme lacks the A-cluster responsible for the synthase activity of CODH Ct . Second, a Ni-deficient protein (Ni-deficient CODH Rr ) containing all Fe components of the holoenzyme can be isolated. The holoenzymes of both species can be prepared in a state for which the C-cluster exhibits the so-called g av ) 1.82 EPR signal (C red1 ); the spectra of Ni-deficient CODH Rr do not exhibit this signal. Our results are as follows: The Mössbauer data show that all iron atoms of Ni-deficient CODH Rr belong to two [Fe 4 S 4 ] 1+/2+ clusters. The so-called B-cluster, which functions in electron transfer, is diamagnetic in the [Fe 4 S 4 ] 2+ state, B ox , and exhibits an S ) 1/2 (g ) 1.94) EPR signal in the [Fe 4 S 4 ] + state, B red . The spectroscopic properties of the B-cluster are the same in Ni-deficient, holo-CODH Rr and CODH Ct . The precursor to the C-cluster of Nideficient CODH Rr , labeled C*, is diamagnetic in the [Fe 4 S 4 ] 2+ state, but has an S ) 3/2 spin in the [Fe 4 S 4 ] + form. Upon incorporation of Ni, the properties of the C*-cluster change substantially. At E′ m ) -110 mV, the C-cluster undergoes a 1-electron reduction from the oxidized state, C ox , to the reduced state, C red1 , which exhibits the g av ) 1.82 EPR signal. A study of a sample poised at -300 mV shows that this signal originates from an S ) 1/2 [Fe 4 S 4 ] + cluster. In this state, the cluster has a distinct subsite, ferrous component II (FCII), having ∆E Q ) 2.82 mm/s and δ ) 0.82 mm/s; these parameters suggest a pentacoordinate site somewhat similar to subsite Fe a of the Fe 4 S 4 cluster of active aconitase. The same values for ∆E Q and δ were observed for CODH Ct . Upon addition of CN -, a potent inhibitor of CO oxidation, the ∆E Q of FCII of CODH Ct changes from 2.82 to 2.53 mm/s, suggesting that CNbinds to the FCII iron. The Mössbauer studies of CODH Rr showed that only ∼60% of the C-clusters were capable of attaining the C red1 state; the remainder were C ox (or C* ox ). For the Mössbauer sample, the EPR spin concentration of the g av ) 1.82 signal was ∼65% of that determined for the g ) 1.94 signal of B red of the fully reduced sample, a result consistent with the ∼60% obtained from Mössbauer spectroscopy. When CODH Rr was reduced with CO or dithionite, a fraction of the C-clusters developed a signal similar to the g av ) 1.86 signal (C red2 ) of CODH Ct . The Mössbauer and EPR spectra of dithionite-reduced CODH Rr show that a large fraction of the C-centers are in a state for which the [Fe 4 S 4 ] + cluster has S ) 3/2. While the assumption of an [Fe 4 S 4 ] + cluster with an aconitase-type subsite electronically isolated from the Ni site can explai...

Of the three known low-nuclearity iron−sulfur clusters in metallobiomolecules with the core units Fe2S2, Fe4S4, and Fe3S4, the last has not been obtained in stable form outside a protein environment. We describe a direct route to such clusters in the [Fe3S4]0 oxidation state, and demonstrate an effective stereochemical and electronic structural congruence with the native cluster. The synthesis is based on iron-site-differentiated clusters. Reaction of [Fe4S4(LS3)(SEt)]2- with (Et3NH)(OTf) affords [Fe4S4(LS3)(OTf)]2-, whose unique site is activated toward terminal ligand substitution. Treatment with 1 equiv of (Et4N)2(Meida) affords [Fe4S4(LS3)(Meida)]3-, which is readily converted to [Fe3S4(LS3)]3- with 1−2 equiv of additional reactant. The trinuclear cluster is formed by abstraction of Fe2+ from a precursor cubane-type [Fe4S4]2+ core and complexation as [Fe(Meida)2]2-. An analogous procedure starting with [Fe4Se4(LS3)(SEt)]2- yields [Fe3Se4(LS3)]3-. The compound (Et4N)3[Fe3S4(LS3)]·MeCN crystallizes in orthorhombic space group P212121 with no imposed symmetry. An X-ray structure solution demonstrates the presence of the desired cuboidal [Fe3(μ3-S)(μ2-S)3]0 core in a complex of absolute configuration Δ. Property comparisons support the cuboidal structure for [Fe3Se4(LS3)]3-. A series of reactions in the systems [Fe4S4(SEt)4]2-/(Et4N)2(Meida) and [Fe3S4(LS3)]3-/NaSEt in Me2SO disclose that, while cuboidal [Fe3S4(SEt)3]3- is formed in both systems, it is one of several cluster products and tends to decay with time. [Fe3S4(LS3)]3- is completely stable in anaerobic solutions at ambient temperature. Consequently, the semirigid cavitand ligand LS3 is conspicuously superior to a simple monodentate thiolate in stabilizing the [Fe3S4]0 core. The cuboidal core is metrically very similar in structure to the cubane core of [Fe4S4(LS3)Cl]2- and to protein-bound Fe3S4 clusters. Voltammetry of [Fe3S4(LS3)]3- reveals a reversible three-membered electron transfer series which includes the core states [Fe3S4]1+,0,1-. The electronic structures of [Fe3S4(LS3)]3- and [Fe3Se4(LS3)]3- were investigated by Mössbauer and EPR spectroscopies. These studies reveal that the synthetic clusters, like the protein-bound clusters, have an electronic ground state with cluster spin S = 2 that arises from an interplay of Heisenberg and double exchange between the sites of a delocalized Fe2+Fe3+ pair and an Fe3+ site. The zero-field splittings of the S = 2 multiplet and the entire set of 57Fe hyperfine parameters of the synthetic clusters match those of the protein-bound clusters. Evidently, protein structure is not required to sustain the cuboidal geometry nor the spin-quintet ground state and its attendant electron distribution and magnetic interactions. We conclude that the clusters [Fe3Q4(LS3)]3- (Q = S, Se) are accurate structural and electronic analogues of the cuboidal sites in native and selenide-reconstituted proteins. No cluster containing a discrete cuboidal Fe3S4 core has previously been isolated in substance.