The Nitrogenase FeMo‐Cofactor Precursor Formed by NifB Protein: A Diamagnetic Cluster Containing Eight Iron Atoms

The biological activation of N₂ occurs at the FeMo‐cofactor, a 7Fe–9S–Mo–C–homocitrate cluster. FeMo‐cofactor formation involves assembly of a Fe_6–8–S_X–C core precursor, NifB‐co, which occurs on the NifB protein. Characterization of NifB‐co in NifB is complicated by the dynamic nature of the assembly process and the presence of a permanent [4Fe–4S] cluster associated with the radical SAM chemistry for generating the central carbide. We have used the physiological carrier protein, NifX, which has been proposed to bind NifB‐co and deliver it to the NifEN protein, upon which FeMo‐cofactor assembly is ultimately completed. Preparation of NifX in a fully NifB‐co‐loaded form provided an opportunity for Mössbauer analysis of NifB‐co. The results indicate that NifB‐co is a diamagnetic (S=0) 8‐Fe cluster, containing two spectroscopically distinct Fe sites that appear in a 3:1 ratio. DFT analysis of the ⁵⁷Fe electric hyperfine interactions deduced from the Mössbauer analysis suggests that NifB‐co is either a 4Fe²⁺–4Fe³⁺ or 6Fe²⁺–2Fe³⁺ cluster having valence‐delocalized states.     

Extended X-ray absorption fine structure and nuclear resonance vibrational spectroscopy reveal that NifB-co, a FeMo-co precursor, comprises a 6Fe core with an interstitial light atom

NifB-co, an Fe-S cluster produced by the enzyme NifB, is an intermediate on the biosynthetic pathway to the iron molybdenum cofactor (FeMo-co) of nitrogenase. We have used Fe K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy together with (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to probe the structure of NifB-co while bound to the NifX protein from Azotobacter vinelandii. The spectra have been interpreted in part by comparison with data for the completed FeMo-co attached to the NafY carrier protein: the NafY:FeMo-co complex. EXAFS analysis of the NifX:NifB-co complex yields an average Fe-S distance of 2.26 A and average Fe-Fe distances of 2.66 and 3.74 A. Search profile analyses reveal the presence of a single Fe-X (X = C, N, or O) interaction at 2.04 A, compared to a 2.00 A Fe-X interaction found in the NafY:FeMo-co EXAFS. This suggests that the interstitial light atom (X) proposed to be present in FeMo-co has already inserted at the NifB-co stage of biosynthesis. The NRVS exhibits strong bands from Fe-S stretching modes peaking around 270, 315, 385, and 408 cm(-1). Additional intensity at approximately 185-200 cm(-1) is interpreted as a set of cluster "breathing" modes similar to those seen for the FeMo-cofactor. The strength and location of these modes also suggest that the FeMo-co interstitial light atom seen in the crystal structure is already in place in NifB-co. Both the EXAFS and NRVS data for NifX:NifB-co are best simulated using a Fe 6S 9X trigonal prism structure analogous to the 6Fe core of FeMo-co, although a 7Fe structure made by capping one trigonal 3S terminus with Fe cannot be ruled out. The results are consistent with the conclusion that the interstitial light atom is already present at an early stage in FeMo-co biosynthesis prior to the incorporation of Mo and R-homocitrate.     

Control of the Transition between Ni‐C and Ni‐SIa States by the Redox State of the Proximal Fe?S Cluster in the Catalytic Cycle of [NiFe] Hydrogenase

[NiFe] hydrogenase catalyzes the reversible cleavage of H₂. The electrons produced by the H₂ cleavage pass through three Fe–S clusters in [NiFe] hydrogenase to its redox partner. It has been reported that the Ni‐SI_a, Ni‐C, and Ni‐R states of [NiFe] hydrogenase are involved in the catalytic cycle, although the mechanism and regulation of the transition between the Ni‐C and Ni‐SI_a states remain unrevealed. In this study, the FT‐IR spectra under light irradiation at 138–198 K show that the Ni‐L state of [NiFe] hydrogenase is an intermediate between the transition of the Ni‐C and Ni‐SI_a states. The transition of the Ni‐C state to the Ni‐SI_a state occurred when the proximal [Fe₄S₄]_p^2+/+ cluster was oxidized, but not when it was reduced. These results show that the catalytic cycle of [NiFe] hydrogenase is controlled by the redox state of its [Fe₄S₄]_p^2+/+ cluster, which may function as a gate for the electron flow from the NiFe active site to the redox partner.     

Spectroscopic Characterization of an Eight‐Iron Nitrogenase Cofactor Precursor that Lacks the “9th Sulfur”

Nitrogenases catalyze the reduction of N₂ to NH₄⁺ at its cofactor site. Designated the M‐cluster, this [MoFe₇S₉C(R‐homocitrate)] cofactor is synthesized via the transformation of a [Fe₄S₄] cluster pair into an [Fe₈S₉C] precursor (designated the L‐cluster) prior to insertion of Mo and homocitrate. We report the characterization of an eight‐iron cofactor precursor (designated the L*‐cluster), which is proposed to have the composition [Fe₈S₈C] and lack the “9^th sulfur” in the belt region of the L‐cluster. Our X‐ray absorption and electron spin echo envelope modulation (ESEEM) analyses strongly suggest that the L*‐cluster represents a structural homologue to the l ‐cluster except for the missing belt sulfur. The absence of a belt sulfur from the L*‐cluster may prove beneficial for labeling the catalytically important belt region, which could in turn facilitate investigations into the reaction mechanism of nitrogenases.     

Protonation and Proton‐Coupled Electron Transfer at S‐Ligated [4Fe‐4S] Clusters

Biological [Fe‐S] clusters are increasingly recognized to undergo proton‐coupled electron transfer (PCET), but the site of protonation, mechanism, and role for PCET remains largely unknown. Here we explore this reactivity with synthetic model clusters. Protonation of the arylthiolate‐ligated [4Fe‐4S] cluster [Fe₄S₄(SAr)₄]^2? ( 1 , SAr=S‐2,4‐6‐(iPr)₃C₆H₂) leads to thiol dissociation, reversibly forming [Fe₄S₄(SAr)₃L]^1? ( 2 ) and ArSH (L=solvent, and/or conjugate base). Solutions of 2 +ArSH react with the nitroxyl radical TEMPO to give [Fe₄S₄(SAr)₄]^1? ( 1_ox ) and TEMPOH. This reaction involves PCET coupled to thiolate association and may proceed via the unobserved protonated cluster [Fe₄S₄(SAr)₃(HSAr)]^1? ( 1‐H ). Similar reactions with this and related clusters proceed comparably. An understanding of the PCET thermochemistry of this cluster system has been developed, encompassing three different redox levels and two protonation states.     

Structural and Mechanistic Insights into CO2 Activation by Nitrogenase Iron Protein

The Fe protein of nitrogenase catalyzes the ambient reduction of CO₂ when its cluster is present in the all-ferrous, [Fe₄S₄]⁰ oxidation state. Here, we report a combined structural and theoretical study that probes the unique reactivity of the all-ferrous Fe protein toward CO₂. Structural comparisons of the Azotobacter vinelandii Fe protein in the [Fe₄S₄]⁰ and [Fe₄S₄]⁺ states point to a possible asymmetric functionality of a highly conserved Arg pair in CO₂ binding and reduction. Density functional theory (DFT) calculations provide further support for the asymmetric coordination of O by the “proximal” Arg and binding of C to a unique Fe atom of the all-ferrous cluster, followed by donation of protons by the proximate guanidinium group of Arg that eventually results in the scission of a C−O bond. These results provide important mechanistic and structural insights into CO₂ activation by a surface-exposed, scaffold-held [Fe₄S₄] cluster.     

Synthesis, crystal structures, Mössbauer spectra, and redox properties of binuclear and tetranuclear iron-sulfur nitrosyl clusters

The iron-sulfur nitrosyl complexes A[Fe₄S₃(NO)₇], where A=Na⁺, NH₄ ⁺, or N(Bu ⁿ )₄ ⁺, and B₂[Fe₂S₂(NO)₄], where B=Na⁺, Cs⁺, or N(Buⁿ)₄ ⁺, were synthesized. Their structures and properties were studied by X-ray diffraction analysis, Mössbauer spectroscopy, and cyclic voltammetry. The effect of the crystal packing on the geometry of the tetranuclear NH₄[Fe₄S₃(NO)₇]·H₂O and binuclear Cs₂[Fe₂S₂(NO)₄]·2H₂O complexes was analyzed. The changes in the Fe⁵⁷ Mössbauer spectral parameters of the anion in the B₂[Fe₂S₂(NO)₄] series depend on the size of the B cation and agree with variations in the structural parameters of the Fe[S₂(NO)₂] chromophores as well as in the stretching vibrations of the NO groups caused by changes in intermolecular contacts. The presence of electronic states delocalized through the Fe?Fe bonds explains the fact that the electronic states of the Fe_a(S₃NO) and Fe_b(S₂(NO)₂) chromophores in the [Fe₄S₃(NO)₇]^? anion are nearly identical. The binuclear clusters are unstable upon storage in the solid phase and decompose in solutions to form the tetranuclear [Fe₄S₃(NO)₇]^? complexes, sulfur, and nitrogen oxides. The redox properties of the [Fe₄S₃(NO)₇]^? and [Fe₂S₂(NO₄)]^2? anions in CH₃CN and THF solutions were studied. The mechanism of reduction of the anion in the tetranuclear cluster is proposed.     

Control of the Transition between Ni‐C and Ni‐SIa States by the Redox State of the Proximal FeS Cluster in the Catalytic Cycle of [NiFe] Hydrogenase

[NiFe] hydrogenase catalyzes the reversible cleavage of H₂. The electrons produced by the H₂ cleavage pass through three Fe–S clusters in [NiFe] hydrogenase to its redox partner. It has been reported that the Ni‐SI_a, Ni‐C, and Ni‐R states of [NiFe] hydrogenase are involved in the catalytic cycle, although the mechanism and regulation of the transition between the Ni‐C and Ni‐SI_a states remain unrevealed. In this study, the FT‐IR spectra under light irradiation at 138–198 K show that the Ni‐L state of [NiFe] hydrogenase is an intermediate between the transition of the Ni‐C and Ni‐SI_a states. The transition of the Ni‐C state to the Ni‐SI_a state occurred when the proximal [Fe₄S₄]_p^2+/+ cluster was oxidized, but not when it was reduced. These results show that the catalytic cycle of [NiFe] hydrogenase is controlled by the redox state of its [Fe₄S₄]_p^2+/+ cluster, which may function as a gate for the electron flow from the NiFe active site to the redox partner.     

Electronic structure of the Fe3S4 cluster and its quasi-aromaticity

The localized molecular orbitals and their energy levels for the clusters [Fe₃S₄(SH)₃]^2–, [(HS)₃Fe₃S₄·Ni(PH₃)]^2–, [Mo₃S₄(OH₂)₉]⁴⁺, and [Mo₃S₄·Ni]⁴⁺ have been calculated by mean of the Edmiston-Ruedenberg energy localization technique under the CNDO/2 approximation in order to reveal the resemblance between [Fe₃S₄]⁺ and [Mo₃S₄]⁴⁺ in the geometrical configurations and the addition reactivities with heterometal atoms. It is shown that in these two cluster species with core {M ₃(₃-S)(-S)₃} of similar structure (M = Mo, Fe) there exist three synergically connected three-centered two-electron (M-S-M) -bonds around the puckered six-membered {M₃S₃} rings on account of delocalization of a lone electron pair on each bridging S atom; these (M-S-M) -bonds are thus capable of forming cubane-like heterometal clusters with intruder metal atoms through the ( M) bonding. It is therefore seen that unlike the [Mo₃S₄]⁴⁺ with appreciable bonding between the Mo atoms, the extra d-electrons on the metal atoms in the [Fe₃S₄]⁺ cluster are localized on the Fe atoms, exhibiting an electronic structure significantly different from that of the [Mo₃S₄]⁴⁺ cluster.     

Ligand exchange processes in some iron-sulphur-carbonyl and -nitrosyl complexes

The complex [Fe₂(SMe)₂(CO)₆] undergoes stepwise exchange with Et₂S₂ to yield successively [Fe₂(SMe)(SEt)(CO)₆] and [Fe₂(SEt)₂(CO)₆]. Carbonyl complexes [Fe₂(SR)₂(CO)₆] are efficiently converted to the nitrosyls [Fe₂(SR)₂(NO)₄] by the action either of NO gas or of methanolic sodium nitrite: the analogous species [Fe₂S₂(CO)₆], [Fe₂S₂(CO)₆]^2?, and [Fe₃S₂(CO)₉] all, with methanolic nitrite, yield [Fe₄S₃(NO)₇]^?. This anion, [Fe₄S₃(NO)₇]^?, reacts with sulphur to give the cubane-like [Fe₄S₄(NO)₄]: the synthesis of its selenium analogue, [Fe₄Se₃(NO)₇]^? is described. The complexes [Fe₂(SR)₂(NO)₄] (R = Me, Et, Prⁿ, Prⁱ, Bu^t, PhCH₂) all consist of two isomers in solution, presumed to have structures of C_2h and C_2v, symmetry: activation parameters for the C_2h?C_2v reaction are reported.     

Redox potential of iron–sulfur clusters as a model of the Desulfovibrio vulgaris center

A theoretical study of Heisenberg exchange and double exchange effects in clusters with four and six iron ions has been performed for [Fe₄ S₃ O] ^m+, [Fe₄ S₄]^m+ (where m = 3, 2), and [Fe₆ S₆] ⁿ⁺ (where n = 5, 4) ions as models of the Desulfovibrio vulgaris iron–sulfur centers. Assuming that the redox potential mostly depends on the Heisenberg spin coupling and the resonance delocalization, we performed an analysis of the reduction process for the [Fe₄ S₃ O] ^3+/2+, [Fe₄ S₄] ^3+/2+, and [Fe₆ S₆] ^5+/4+ ions and showed that the redox potential can be calculated as a difference between average spin energies of the tetravalent and pentavalent double cubane superclusters. For the Heisenberg parameter of J₁ = 20 cm^-1, the redox potential amounts to about 0.03 V.It complies with close to zero experimental values of the redox potential.Electronic Supplementary Material: Supplementary material is available in the online version of this article at     

Transition-Metal-Induced Rearrangement of [(PhSn)4S6] Towards Ternary CuI/Sn/S or CuII/Sn/S Clusters

Direct access of ternary copper-tin sulfide clusters by reactions of a binary organotin sulfide cluster, [(PhSn)₄S₆] ( A ), with transition metal complexes was achieved for the first time without extra addition of further chalcogenide sources. This indicates that an in situ rearrangement of the inorganic core takes place even without initial formation of anionic fragments. The use of [Cu(PPh₃)₃Cl] or [Cu(PPh₃)₂Cl₂] as reactants yielded the ternary clusters [(CuPPh₃)₄(PhSn)₁₈Cu₆S₃₁Cl₂] ( 1 ) and [{Cu(PPh₃)₂}₂(PhSn)₃(SnCl)S₈] ( 2 ), respectively. Whereas 1 represents the largest neutral Cu/Sn/S cluster known to date, compound 2 , which is the first example of a ternary Cu/Sn/E (E=S, Se) cluster containing copper in the +II oxidation state, may be viewed as a very early stage of cluster formation. Apparently, the presence of Cu^II inhibits effective cluster growth, which rationalizes the lack of such species so far. The two ternary clusters exhibit very similar optical absorption energies despite their markedly different cluster sizes. According to time-dependent DFT calculations, this is due to different characters of the electronic excitation in the triplet compound 2 , as compared to the excitation of the closed shell cluster 1 , which serve to compensate for the different extensions of the clusters.     

Synthesis and structures of new heteronuclear cluster complexes [PPh4][Fe4Rh3Se2(CO)16] and [PPh4]2[Fe3Rh4Te2(CO)15]

The reaction of the K₂[Fe₃Q(CO)₉] clusters (Q = Se or Te) with Rh₂(CO)₄Cl₂ under mild conditions is accompanied by complicated fragmentation of cores of the starting clusters to form large heteronuclear cluster anions. The [PPh₄][Fe₄Rh₃Se₂(CO)₁₆] and [PPh₄]₂[Fe₃Rh₄Te₂(CO)₁₅] compounds were isolated by treatment of the reaction products with tetraphenylphosphonium bromide. The structures of the products were established by X-ray diffraction. In both compounds, the core of the heteronuclear cluster consists of two octahedra fused via a common Rh₃ face. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 5, pp. 775–778, May, 2006.     

Electronic structure of paramagnetic clusters of transition metal ions. 3. Magnetic properties and scattered wave description of the electronic structure of the hexanuclear octahedral cluster [Fe6(μ3−S)8(PEt3)6] (BPh4)2

Spin-polarized Xα–SW calculations of [Fe₆(μ₃?S)₈(PH₃)₆]²⁺ as a model of the cluster [Fe₆(μ₃?S)₈(PEt₃)₆] (BPh₄)₂ have been performed. The highest occupied energy levels are well separated from empty levels, and up to a maximum of eight electrons can be unpaired, giving a maximum spin state with S = 4. This electronic state is consistent with the magnetic data of [Fe₆(μ₃?S)₈(PEt₃)₆](BPh ₄)₂, which have been interpreted using the Heisenberg–Dirac–Van Vleck exchange spin Hamiltonian. The S = 4 state arises from the magnetic coupling between five low-spin (S_i = 1/2) and one intermediate-spin (S = 3/2) iron(III) center. © 1994 John Wiley & Sons, Inc.     

NaAg3S2, ein Thioargentat mit dem Clusteranion [Ag6S4]2−

NaAg₃S₂, a Thioargentate Containing the Anionic Cluster [Ag₆S₄]^2? . Dark-red octahedrally shaped crystals of NaAg₃S₂ could be obtained by the reaction of NaAg(CN)₂ and NaCN in a stream of hydrogen sulfide at 630 K. NaAg₃S₂ crystallizes cubic, a=12.358(1) Å, space group Fd3 m, Z=16. The structure was determined from four-circle diffractometer data. NaAg₃S₂ contains the anionic cluster [Ag₆S₄]^2?. The structure can be traced back to the spinel structure typ. An extended Hückel calculation for the cluster anion, which is considered to be isolated, shows weak bonding silver-silver interactions. NaAg₃S₂ is diamagnetic at room temperature.     

Chemical transformations of a SiO2-supported [Fe5RhC(CO)16]− cluster and catalysis of propylene hydroformylation

Chemical transformations of SiO₂-supported [Fe₅RhC(CO)₁₆]^– and [Fe₄RhC(CO)₁₄]^– clusters in Ar, CO, and synthesis gas are studied by IR spectroscopy, Mössbauer spectroscopy, and transmission electron microscopy. It is shown that partial transformation of the [Fe₅RhC(CO)₁₆]^– cluster to the [Fe₄RhC(CO)₁₄]^– cluster occurs immediately after its deposition on the substrate surface with the simultaneous formation of Fe²⁺ ions. The complete conversion of the supported [Fe₅RhC(CO)₁₆]^– cluster to [Fe₄RhC(CO)₁₄]^– is observed at 323 K in the synthesis gas. At 373 to 423 K [Fe₅RhC(CO)₁₆]^– transforms into a mixture of Fe₄Rh₂C(CO)₁₆, [Fe₄RhC(CO)₁₄]^–, and [Fe₅₃Rh₃C(CO)₁₅]^– clusters. In the 523 to 623 K range, the supported [Fe₅RhC(CO)₁₆]^– cluster decarbonylates completely to form bimetallic species Å 5 Å in size. Silica-supported FeRh clusters are active in propylene hydroformylation at 423 to 473 K and form a mixture of butyl alcohols and butyraldehydes.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 4, pp. 632–641, April, 1995.This work was financially supported by the Krasnoyarsk Region Scince Foundation (Grant No. 1F0020).     

A novel water-soluble sulfide cluster of iron: synthesis,properties, and intercalation into molybdenum disulfide

A novel water-soluble sulfide cluster of iron, {Fe₆S₈[P(CH₂OH)₃]₆}²⁺, was obtained from ferrous chloride tetrahydrate, tris(hydroxymethyl)phosphine, and H₂S in methanol. A reaction of the cluster in solution with dispersed molybdenum disulfide gave an inclusion compound, MoS₂({Fe₆S₈[P(CH₂OH)₃]₆}Cl₂)_0.06. The products obtained were characterized by IR, NMR, and ESR spectroscopy, electronic absorption spectroscopy, mass spectrometry, and elemental analysis. The magnetic susceptibility of the cluster was studied in solution and in a molybdenum disulfide matrix.     

A [3Cu:2S] cluster provides insight into the assembly and function of the CuZ site of nitrous oxide reductase

Nitrous oxide reductase (N₂OR) is the only known enzyme reducing environmentally critical nitrous oxide (N₂O) to dinitrogen (N₂) as the final step of bacterial denitrification. The assembly process of its unique catalytic [4Cu:2S] cluster Cu_Z remains scarcely understood. Here we report on a mutagenesis study of all seven histidine ligands coordinating this copper center, followed by spectroscopic and structural characterization and based on an established, functional expression system for Pseudomonas stutzeri N₂OR in Escherichia coli. While no copper ion was found in the Cu_Z binding site of variants H129A, H130A, H178A, H326A, H433A and H494A, the H382A variant carried a catalytically inactive [3Cu:2S] center, in which one sulfur ligand, S_Z2, had relocated to form a weak hydrogen bond to the sidechain of the nearby lysine residue K454. This link provides sufficient stability to avoid the loss of the sulfide anion. The UV-vis spectra of this cluster are strikingly similar to those of the active enzyme, implying that the flexibility of S_Z2 may have been observed before, but not recognized. The sulfide shift changes the metal coordination in Cu_Z and is thus of high mechanistic interest.

Variants of all seven histidine ligands of the [4Cu:2S] active site of nitrous oxide reductase mostly result in loss of the metal site. However, a H382A variant retains a [3Cu:2S] cluster that hints towards a structural flexibility also present in the intact site.     

Anionic Copolymerization of Elemental Sulfur with Propylene Sulfide. Equilibrium Sulfur Concentration

In the anionic copolymerization of elemental sulfur (S₈) with propylene sulfide (P) below the floor temperature of elemental sulfur homopolymerization (T_f = 159°C), there is a certain concentration of elemental sulfur left when copolymerization is completed ([S₈]_eq). The dependence of [S₈]_eq on the feed ratio [P]₀/[S₈]₀ and temperature was determined by using laser Raman spectroscopy, and this enabled us to distinguish between the S-S bonds in elemental sulfur and in the linear polysulfide. [S₈]_eq was found to decrease with increasing temperature and with an increasing [P₀]/[S₈]₀ ratio. The experimental dependence of the average enthalpy and entropy of polymerization (δ[Hbar]_[Xbar], and δ [Sbar]_[Xbar]) on [Xbar], as described by the equilibrium …-CH₂CH(CH₃)S_X ^? + S₈ = …-CH₂CH(CH₃)S_X+8 ^?, has shown that at [Xbar] ?; 9 the experimental δ[Hbar]_[Xbar] and δ [Sbar]_[Xbar] approach values determined earlier for the free radical homopolymerization of elemental sulfur …-S_n ^? + S₈ ≤ …-S^? _n+8&; The corresponding values are 3.1 kcal/mol and 4.76 cal/mol · degree.     

Non-planar Nest-like [Fe2S2] Cluster Sites for Efficient Oxygen Reduction Catalysis

Metal-nitrogen-carbon catalysts, as promising alternative to platinum-based catalysts for oxygen reduction reaction (ORR), are still highly expected to achieve better performance by modulating the composition and spatial structure of active site. Herein, we constructed the non-planar nest-like [Fe₂S₂] cluster sites in N-doped carbon plane. Adjacent double Fe atoms effectively weaken the O−O bond by forming a peroxide bridge-like adsorption configuration, and the introduction of S atoms breaks the planar coordination of Fe resulting in greater structural deformation tension, lower spin state, and downward shifted Fe d-band center, which together facilitate the release of OH* intermediate. Hence, the non-planar [Fe₂S₂] cluster catalyst, with a half-wave potential of 0.92 V, displays superior ORR activity than that of planar [FeN₄] or [Fe₂N₆]. This work provides insights into the co-regulation of atomic composition and spatial configuration for efficient oxygen reduction catalysis.