From Neutral Diarsenes to Diarsene Radical Ions and Diarsene Dications.

Diarsene [L(MeO)GaAs]2 (L=HC[C(Me)N(Ar)]2, Ar=2,6-iPr2C6H3, 4) reacts with MeOTf and MeNHC (MeNHC=1,3,4,5-tetra-methylimidazol-2-ylidene) to the diarsene [L(TfO)GaAs]2 (5) and the carbene-coordinated diarsene [L(MeO)GaAsAs(MeNHC)Ga(OMe)L] (6). The NHC-coordination results in an inversion of the redox properties of the diarsene 4, which shows only a reversible reduction event at E1/2=-2.06 V vs Fc0/+1, whereas the carbene-coordinated diarsene 6 shows a reversible oxidation event at E1/2=-1.31 V vs Fc0/+1. Single electron transfer reactions of 4 and 6 yielded [K[2.2.2.]cryp][L(MeO)GaAs]2 (8) and [L(MeO)GaAsAs(MeNHC)-Ga(OMe)L][B(C6F5)4] (9) containing the radical anion [L(MeO)GaAs]2⋅- (8⋅-) and the NHC-coordinated radical cation [L(MeO)GaAsAs(MeNHC)Ga(OMe)L]⋅+ (9⋅+), respectively, while the salt-elimination reaction of the triflate-coordinated diarsene 5 with Na[B(C6F5)4] gave [LGaAs]2[B(C6F5)4]2 (11) containing the dication [LGaAs]2 2+ (112+). Compounds 1-11 were characterized by 1H and 13C NMR, EPR (8, 9), IR, and UV-Vis spectroscopy and by single crystal X-ray diffraction (sc-XRD). DFT calculations provided a detailed understanding of the electronic nature of the diarsenes (4, 6) and the radical ions (8⋅-, 9⋅+), respectively.

Cu4S Cluster in "0-Hole" and "1-Hole" States: Geometric and Electronic Structure Variations for the Active CuZ* Site of N2O Reductase.

The active site of nitrous oxide reductase (N2OR), a key enzyme in denitrification, features a unique µ4-sulfido-bridged tetranuclear Cu cluster (the so-called CuZ or CuZ* site). Details of the catalytic mechanism have remained under debate and, to date, synthetic model complexes of the CuZ*/CuZ sites are extremely rare due to the difficulty in building the unique {Cu4(µ4-S)} core structure. Herein, we report the synthesis and characterization of [Cu4(µ4-S)]n+ (n = 2, 2; n = 3, 3) clusters, supported by a macrocyclic {py2NHC4} ligand (py = pyridine, NHC = N-heterocyclic carbene), in both their 0-hole (2) and 1-hole (3) states, thus mimicking the two active states of the CuZ* site during enzymatic N2O reduction. Structural and electronic properties of these {Cu4(µ4-S)} clusters are elucidated by employing multiple methods, including X-ray diffraction (XRD), nuclear magnetic resonance (NMR), UV/vis, electron paramagnetic resonance (EPR), Cu/S K-edge X-ray emission spectroscopy (XES), and Cu K-edge X-ray absorption spectroscopy (XAS) in combination with time-dependent density functional theory (TD-DFT) calculations. A significant geometry change of the {Cu4(µ4-S)} core occurs upon oxidation from 2 (τ4(S) = 0.46, seesaw) to 3 (τ4(S) = 0.03, square planar), which has not been observed so far for the biological CuZ(*) site and is unprecedented for known model complexes. The single electron of the 1-hole species 3 is predominantly delocalized over two opposite Cu ions via the central S atom, mediated by a π/π superexchange pathway. Cu K-edge XAS and Cu/S K-edge XES corroborate a mixed Cu/S-based oxidation event in which the lowest unoccupied molecular orbital (LUMO) has a significant S-character. Furthermore, preliminary reactivity studies evidence a nucleophilic character of the central µ4-S in the fully reduced 0-hole state.

Syntheses and Structures of 5-Membered Heterocycles Featuring 1,2-Diphospha-1,3-Butadiene and Its Radical Anion.

LGa(P2 OC)cAAC 2 features a 1,2-diphospha-1,3-butadiene unit with a delocalized π-type HOMO and a π*-type LUMO according to DFT calculations. [LGa(P2 OC)cAAC][K(DB-18-c-6)] 3[K(DB-18-c-6] containing the 1,2-diphospha-1,3-butadiene radical anion 3⋅- was isolated from the reaction of 2 with KC8 and dibenzo-18-crown-6. 3 reacted with [Fc][B(C6 F5 )4 ] (Fc=ferrocenium) to 2 and with TEMPO to [L-H Ga(P2 OC)cAAC][K(DB-18-c-6)] 4[K(DB-18-c-6] containing the 1,2-diphospha-1,3-butadiene anion 4- . The solid state structures of 2, 3K(DB-18-c-6], and 4[K(DB-18-c-6] were determined by single crystal X-ray diffraction (sc-XRD).

Combining Valence-to-Core X-ray Emission and Cu K-edge X-ray Absorption Spectroscopies to Experimentally Assess Oxidation State in Organometallic Cu(I)/(II)/(III) Complexes.

A series of organometallic copper complexes in formal oxidation states ranging from +1 to +3 have been characterized by a combination of Cu K-edge X-ray absorption (XAS) and Cu Kß valence-to-core X-ray emission spectroscopies (VtC XES). Each formal oxidation state exhibits distinctly different XAS and VtC XES transition energies due to the differences in the Cu Zeff, concomitant with changes in physical oxidation state from +1 to +2 to +3. Herein, we demonstrate the sensitivity of XAS and VtC XES to the physical oxidation states of a series of N-heterocyclic carbene (NHC) ligated organocopper complexes. We then extend these methods to the study of the [Cu(CF3)4]- ion. Complemented by computational methods, the observed spectral transitions are correlated with the electronic structure of the complexes and the Cu Zeff. These calculations demonstrate that a contraction of the Cu 1s orbitals to deeper binding energy upon oxidation of the Cu center manifests spectroscopically as a stepped increase in the energy of both XAS and Kß2,5 emission features with increasing formal oxidation state within the [Cun+(NHC2)]n+ series. The newly synthesized Cu(III) cation [CuIII(NHC4)]3+ exhibits spectroscopic features and an electronic structure remarkably similar to [Cu(CF3)4]-, supporting a physical oxidation state assignment of low-spin d8 Cu(III) for [Cu(CF3)4]-. Combining XAS and VtC XES further demonstrates the necessity of combining multiple spectroscopies when investigating the electronic structures of highly covalent copper complexes, providing a template for future investigations into both synthetic and biological metal centers.

Determination of the iron(IV) local spin states of the Q intermediate of soluble methane monooxygenase by Kß X-ray emission spectroscopy.

Soluble methane monooxygenase (sMMO) facilitates the conversion of methane to methanol at a non-heme FeIV2 intermediate MMOHQ, which is formed in the active site of the sMMO hydroxylase component (MMOH) during the catalytic cycle. Other biological systems also employ high-valent FeIV sites in catalysis; however, MMOHQ is unique as Nature's only identified FeIV2 intermediate. Previous 57Fe Mössbauer spectroscopic studies have shown that MMOHQ employs antiferromagnetic coupling of the two FeIV sites to yield a diamagnetic cluster. Unfortunately, this lack of net spin prevents the determination of the local spin state (Sloc) of each of the irons by most spectroscopic techniques. Here, we use Fe Kß X-ray emission spectroscopy (XES) to characterize the local spin states of the key intermediates of the sMMO catalytic cycle, including MMOHQ trapped by rapid-freeze-quench techniques. A pure XES spectrum of MMOHQ is obtained by subtraction of the contributions from other reaction cycle intermediates with the aid of Mössbauer quantification. Comparisons of the MMOHQ spectrum with those of known Sloc = 1 and Sloc = 2 FeIV sites in chemical and biological models reveal that MMOHQ possesses Sloc = 2 iron sites. This experimental determination of the local spin state will help guide future computational and mechanistic studies of sMMO catalysis.

Sequential Reduction of Borylstibane to an Electronically Nonsymmetric Diboryldistibene Radical Anion.

Understanding the formation of metal-metal bonds and their electronic structures is still a scientific task. We herein report on the stepwise synthesis of boryl-substituted antimony compounds in which the antimony atoms adopt four different oxidation states (+III, +II, +I, +I/0). Sb-C bond homolysis of Cp*[(HCNDip)2B]SbCl (1; Cp* = C5Me5; Dip = 2,6-iPr2C6H3) gave diboryldichlorodistibane [(HCNDip)2BSbCl]2 (2), which reacted with KC8 to form diboryldistibene [(HCNDip)2BSb]2 (3) and traces of cyclotetrastibane [(HCNDip)2B]3Sb4Cl (5). One-electron reduction of 3 yielded the potassium salt of the diboryldistibene radical anion [(HCNDip)2BSb]2̇-, [K(18-c-6)(OEt2)][{(HCNDip)2BSb}2] (4), which exhibits an unprecedented inequivalent spin localization on the Sb-Sb bond and hence an unsymmetric electronic structure. Compounds 1-4 were characterized by heteronuclear nuclear magnetic resonance (NMR) (1H, 13C, 11B), infrared (IR), ultraviolet-visible (UV-vis) spectroscopy (3, 4), and single crystal X-ray diffraction (sc-XRD, 1-5), while the bonding nature of 3 and 4 was analyzed by quantum chemical calculations. EPR spectroscopy resolves the dissimilar Sb hyperfine tensors of 4, reflecting the inequivalent spin distribution, setting 4 uniquely apart from all previously characterized dipnictene radical anions.

Single-Electron Oxidation of Carbene-Coordinated Pnictinidenes-Entry into Heteroleptic Radical Cations and Metalloid Clusters.

Stable heavy main group element radicals are challenging synthetic targets. Although several strategies have been developed to stabilize such odd-electron species, the number of heavier pnictogen-centered radicals is limited. We report on a series of two-coordinated pnictogen-centered radical cations [(MecAAC)EGa(Cl)L][B(C6F5)4] (MecAAC = [H2C(CMe2)2NDipp]C; Dipp = 2,6-i-Pr2C6H3; E = As 1, Sb 2, Bi 3; L = HC[C(Me)NDipp]2) synthesized by one-electron oxidation of L(Cl)Ga-substituted pnictinidenes (MecAAC)EGa(Cl)L (E = As I, Sb II, Bi III). 1-3 were characterized by electron paramagnetic resonance (EPR) spectroscopy and single crystal X-ray diffraction (sc-XRD) (1, 2), while quantum chemical calculations support their description as carbene-coordinated pnictogen-centered radical cations. The low thermal stability of 3 enables access to metalloid bismuth clusters as shown by formation of [{LGa(Cl)}3Bi6][B(C6F5)4] (4).

The Spectroscopy of Nitrogenases.

Nitrogenases are responsible for biological nitrogen fixation, a crucial step in the biogeochemical nitrogen cycle. These enzymes utilize a two-component protein system and a series of iron-sulfur clusters to perform this reaction, culminating at the FeMco active site (M = Mo, V, Fe), which is capable of binding and reducing N2 to 2NH3. In this review, we summarize how different spectroscopic approaches have shed light on various aspects of these enzymes, including their structure, mechanism, alternative reactivity, and maturation. Synthetic model chemistry and theory have also played significant roles in developing our present understanding of these systems and are discussed in the context of their contributions to interpreting the nature of nitrogenases. Despite years of significant progress, there is still much to be learned from these enzymes through spectroscopic means, and we highlight where further spectroscopic investigations are needed.

Observation of Discrete Valence Tautomers in Crystalline Cyclopentadienyl Radicals.

Single crystal X-ray (sc XRD) analyses of three symmetrically substituted cyclopentadienyl radicals (1, 2, 5) containing sterically demanding aryl groups showed that they crystallize as discrete valence tautomers (Jahn-Teller distortion) in the solid state with the unpaired electron either located in the b1 orbital (type I, state 2B1), resulting in a localized radical with two adjacent double bonds, or the a2 orbital (type II, state 2A2), leading to an allyl-type radical. Their properties in solution were examined by EPR spectroscopy as well as cyclovoltammetry and UV/vis spectroscopy including two additional cyclopentadienyl radicals (1-5). The electronic nature of 1-5 was further investigated by quantum chemical calculations.

The Asp1 pyrophosphatase from S. pombe hosts a [2Fe-2S]2+ cluster in vivo.

The Schizosaccharomyces pombe Asp1 protein is a bifunctional kinase/pyrophosphatase that belongs to the highly conserved eukaryotic diphosphoinositol pentakisphosphate kinase PPIP5K/Vip1 family. The N-terminal Asp1 kinase domain generates specific high-energy inositol pyrophosphate (IPP) molecules, which are hydrolyzed by the C-terminal Asp1 pyrophosphatase domain (Asp1365-920). Thus, Asp1 activities regulate the intracellular level of a specific class of IPP molecules, which control a wide number of biological processes ranging from cell morphogenesis to chromosome transmission. Recently, it was shown that chemical reconstitution of Asp1371-920 leads to the formation of a [2Fe-2S] cluster; however, the biological relevance of the cofactor remained under debate. In this study, we provide evidence for the presence of the Fe-S cluster in Asp1365-920 inside the cell. However, we show that the Fe-S cluster does not influence Asp1 pyrophosphatase activity in vitro or in vivo. Characterization of the as-isolated protein by electronic absorption spectroscopy, mass spectrometry, and X-ray absorption spectroscopy is consistent with the presence of a [2Fe-2S]2+ cluster in the enzyme. Furthermore, we have identified the cysteine ligands of the cluster. Overall, our work reveals that Asp1 contains an Fe-S cluster in vivo that is not involved in its pyrophosphatase activity.

Ligand Protonation Triggers H2 Release from a Dinickel Dihydride Complex to Give a Doubly "T"-Shaped Dinickel(I) Metallodiradical.

The dinickel(II) dihydride complex (1K ) of a pyrazolate-based compartmental ligand with ß-diketiminato (nacnac) chelate arms (L- ), providing two pincer-type {N3 } binding pockets, has been reported to readily eliminate H2 and to serve as a masked dinickel(I) species. Discrete dinickel(I) complexes (2Na , 2K ) of L- are now synthesized via a direct reduction route. They feature two adjacent T-shaped metalloradicals that are antiferromagnetically coupled, giving an S=0 ground state. The two singly occupied local d x 2 - y 2 type magnetic orbitals are oriented into the bimetallic cleft, enabling metal-metal cooperative 2 e- substrate reductions as shown by the rapid reaction with H2 or O2 . X-ray crystallography reveals distinctly different positions of the K+ in 1K and 2K , suggesting a stabilizing interaction of K+ with the dihydride unit in 1K . H2 release from 1K is triggered by peripheral γ-C protonation at the nacnac subunits, which DFT calculations show lowers the barrier for reductive H2 elimination from the bimetallic cleft.

A Mechanistic Study on Reactions of Group 13 Diyls LM with Cp*SbX2 : From Stibanyl Radicals to Antimony Hydrides.

Oxidative addition of Cp*SbX2 (X=Cl, Br, I; Cp*=C5 Me5 ) to group 13 diyls LM (M=Al, Ga, In; L=HC[C(Me)N (Dip)]2 , Dip=2,6-iPr2 C6 H3 ) yields elemental antimony (M=Al) or the corresponding stibanylgallanes [L(X)Ga]Sb(X)Cp* (X=Br 1, I 2) and -indanes [L(X)In]Sb(X)Cp* (X=Cl 5, Br 6, I 7). 1 and 2 react with a second equivalent of LGa to eliminate decamethyl-1,1'-dihydrofulvalene (Cp*2 ) and form stibanyl radicals [L(X)Ga]2 Sb. (X=Br 3, I 4), whereas analogous reactions of 5 and 6 with LIn selectively yield stibanes [L(X)In]2 SbH (X=Cl 8, Br 9) by elimination of 1,2,3,4-tetramethylfulvene. The reactions are proposed to proceed via formation of [L(X)M]2 SbCp* as reaction intermediate, which is supported by the isolation of [L(Cl)Ga]2 SbCp (11, Cp=C5 H5 ). The reaction mechanism was further studied by computational calculations using two different models. The energy values for the Ga- and the In-substituted model systems showing methyl groups instead of the very bulky Dip units are very similar, and in both cases the same products are expected. Homolytic Sb-C bond cleavage yields van der Waals complexes from the as-formed radicals ([L(Cl)M]2 Sb. and Cp*. ), which can be stabilized by hydrogen atom abstraction to give the corresponding hydrides, whereas the direct formation of Sb hydrides starting from [L(Cl)M]2 SbCp* via concerted ß-H elimination is unlikely. The consideration of the bulky Dip units reveals that the amount of the steric overload in the intermediate I determines the product formation (radical vs. hydride).

Ligand Effects on the Electronic Structure of Heteroleptic Antimony-Centered Radicals.

We report on the structures of three unprecedented heteroleptic Sb-centered radicals [L(Cl)Ga](R)Sb. (2-R, R=B[N(Dip)CH]2 2-B, 2,6-Mes2 C6 H3 2-C, N(SiMe3 )Dip 2-N) stabilized by one electropositive metal fragment [L(Cl)Ga] (L=HC[C(Me)N(Dip)]2 , Dip=2,6-i-Pr2 C6 H3 ) and one bulky B- (2-B), C- (2-C), or N-based (2-N) substituent. Compounds 2-R are predominantly metal-centered radicals. Their electronic properties are largely influenced by the electronic nature of the ligands R, and significant delocalization of unpaired-spin density onto the ligands was observed in 2-B and 2-N. Cyclic voltammetry (CV) studies showed that 2-B undergoes a quasi-reversible one-electron reduction, which was confirmed by the synthesis of [K([2.2.2]crypt)][L(Cl)GaSbB[N(Dip)CH]2 ] ([K([2.2.2]crypt)][2-B]) containing the stibanyl anion [2-B]- , which was shown to possess significant Sb-B multiple-bonding character.

An Adaptable N-Heterocyclic Carbene Macrocycle Hosting Copper in Three Oxidation States.

A neutral hybrid macrocycle with two trans-positioned N-heterocyclic carbenes (NHCs) and two pyridine donors hosts copper in three oxidation states (+I-+III) in a series of structurally characterized complexes (1-3). Redox interconversion of [LCu]+/2+/3+ is electrochemically (quasi)reversible and occurs at moderate potentials (E1/2 =-0.45 V and +0.82 V (vs. Fc/Fc+ )). A linear CNHC -Cu-CNHC arrangement and hemilability of the two pyridine donors allows the ligand to adapt to the different stereoelectronic and coordination requirements of CuI versus CuII /CuIII . Analytical methods such as NMR, UV/Vis, IR, electron paramagnetic resonance, and Cu Kß high-energy-resolution fluorescence detection X-ray absorption spectroscopies, as well as DFT calculations, give insight into the geometric and electronic structures of the complexes. The XAS signatures of 1-3 are textbook examples for CuI , CuII , and CuIII species. Facile 2-electron interconversion combined with the exposure of two basic pyridine N sites in the reduced CuI form suggest that [LCu]+/2+/3+ may operate in catalysis via coupled 2 e- /2 H+ transfer.

Synthesis of a Ga-Stabilized As-Centered Radical and a Gallastibene by Tailoring Group 15 Element-Carbon Bond Strengths.

A convenient synthetic route to Ga-stabilized pnictogen-centered radicals and gallapnictenes by manipulation of pnictogen-carbon bond strengths is presented. Two equivalents of LGa (L = HC[C(Me)N(Dip)]2, Dip = 2,6-i-Pr2C6H3) react with CpArAsCl2 (CpAr = C5(4-t-BuC6H4)5) with formation of the arsenic-centered radical [L(Cl)Ga]2As·1. In contrast, the analogous reaction with TerSbCl2 (Ter = 2,6-Mes2C6H3; Mes = 2,4,6-Me3C6H2) yields the gallastibene LGa═SbTer (2) containing a Ga-Sb double bond, whereas reactions of DipSbCl2 with one and two equivalents of LGa give the monoinsertion and bisinsertion products L(Cl)GaSb(ClDip) (3) and [L(Cl]Ga]2SbDip (4), respectively. 1-4 were structurally characterized by single crystal X-ray diffraction. The description of 1 as an arsenic-centered radical is consistent with results of electron paramagnetic resonance and density functional theory (DFT) studies. The π-bonding in LGa═SbTer (2) is estimated to 10.68 kcal mol-1 by variable-temperature (VT) NMR spectroscopy, and DFT studies reveal a significant π-bonding interaction between Sb and Ga.

Spectroscopic X-ray and Mössbauer Characterization of M6 and M5 Iron(Molybdenum)-Carbonyl Carbide Clusters: High Carbide-Iron Covalency Enhances Local Iron Site Electron Density Despite Cluster Oxidation.

The present study employs a suite of spectroscopic techniques to evaluate the electronic and bonding characteristics of the interstitial carbide in a set of iron-carbonyl-carbide clusters, one of which is substituted with a molybdenum atom. The M6C and M5C clusters are the dianions (Et4N)2[Fe6(µ6-C)(µ2-CO)2(CO)14] (1), [K(benzo-18-crown-6)]2[Fe5(µ5-C)(µ2-CO)1(CO)13] (2), and [K(benzo-18-crown-6)]2[Fe5Mo(µ6-C)(µ2-CO)2(CO)15] (3). Because 1 and 2 have the same overall cluster charge (2-) but different numbers of iron sites (1: 6 sites → 2: 5 sites), the metal atoms of 2 are formally oxidized compared to those in 1. Despite this, Mössbauer studies indicate that the iron sites in 2 possess significantly greater electron density (lower spectroscopic oxidation state) compared with those in 1. Iron K-edge X-ray absorption and valence-to-core X-ray emission spectroscopy measurements, paired with density functional theory spectral calculations, revealed the presence of significant metal-to-metal and carbide 2p-based character in the filled valence and low-lying unfilled electronic manifolds. In all of the above experiments, the presence of the molybdenum atom in 3 (Fe5Mo) results in somewhat unremarkable spectroscopic properties that are essentially a "hybrid" of 1 (Fe6) and 2 (Fe5). The overall electronic portrait that emerges illustrates that the central inorganic carbide ligand is essential for distributing charge and maximizing electronic communication throughout the cluster. It is evident that the carbide coordination environment is quite flexible and adaptive: it can drastically modify the covalency of individual Fe-C bonds based on local structural changes and redox manipulation of the clusters. In light of these findings, our data and calculations suggest a potential role for the central carbon atom in FeMoco, which likely performs a similar function in order to maintain cluster integrity through multiple redox and ligand binding events.

Valence-to-Core X-ray Emission Spectroscopy as a Probe of O-O Bond Activation in Cu2 O2 Complexes.

Valence-to-Core (VtC) X-ray emission spectroscopy (XES) was used to directly detect the presence of an O-O bond in a complex comprising the [CuII2 (µ-η2 :η2 -O2 )]2+ core relative to its isomer with a cleaved O-O bond having a [CuIII2 (µ-O)2 ]2+ unit. The experimental studies are complemented by DFT calculations, which show that the unique VtC XES feature of the [CuII2 (µ-η2 :η2 -O2 )]2+ core corresponds to the copper stabilized in-plane 2p π peroxo molecular orbital. These calculations illustrate the sensitivity of VtC XES for probing the extent of O-O bond activation in µ-η2 :η2 -O2 species and highlight the potential of this method for time-resolved studies of reaction mechanisms.

High-Resolution Extended X-ray Absorption Fine Structure Analysis Provides Evidence for a Longer Fe···Fe Distance in the Q Intermediate of Methane Monooxygenase.

Despite decades of intense research, the core structure of the methane C-H bond breaking diiron(IV) intermediate, Q, of soluble methane monooxygenase remains controversial, with conflicting reports supporting either a "diamond" diiron core structure or an open core structure. Early extended X-ray absorption fine structure (EXAFS) data assigned a short 2.46 Å Fe-Fe distance to Q (Shu et al. Science 1997, 275, 515 ) that is inconsistent with several theoretical studies and in conflict with our recent high-resolution Fe K-edge X-ray absorption spectroscopy (XAS) studies (Castillo et al. J. Am. Chem. Soc. 2017, 139, 18024 ). Herein, we revisit the EXAFS of Q using high-energy resolution fluorescence-detected extended X-ray absorption fine structure (HERFD-EXAFS) studies. The present data show no evidence for a short Fe-Fe distance, but rather a long 3.4 Å diiron distance, as observed in open core synthetic model complexes. The previously reported 2.46 Å feature plausibly arises from a background metallic iron contribution from the experimental setup, which is eliminated in HERFD-EXAFS due to the increased selectivity. Herein, we explore the origin of the short diiron feature in partial-fluorescent yield EXAFS measurements and discuss the diagnostic features of background metallic scattering contribution to the EXAFS of dilute biological samples. Lastly, differences in sample preparation and resultant sample inhomogeneity in rapid-freeze quenched samples for EXAFS analysis are discussed. The presented approaches have broad implications for EXAFS studies of all dilute iron-containing samples.

13C ENDOR Spectroscopy of Lipoxygenase-Substrate Complexes Reveals the Structural Basis for C-H Activation by Tunneling.

In enzymatic C-H activation by hydrogen tunneling, reduced barrier width is important for efficient hydrogen wave function overlap during catalysis. For native enzymes displaying nonadiabatic tunneling, the dominant reactive hydrogen donor-acceptor distance (DAD) is typically ca. 2.7 Å, considerably shorter than normal van der Waals distances. Without a ground state substrate-bound structure for the prototypical nonadiabatic tunneling system, soybean lipoxygenase (SLO), it has remained unclear whether the requisite close tunneling distance occurs through an unusual ground state active site arrangement or by thermally sampling conformational substates. Herein, we introduce Mn2+ as a spin-probe surrogate for the SLO Fe ion; X-ray diffraction shows Mn-SLO is structurally faithful to the native enzyme. 13C ENDOR then reveals the locations of 13C10 and reactive 13C11 of linoleic acid relative to the metal; 1H ENDOR and molecular dynamics simulations of the fully solvated SLO model using ENDOR-derived restraints give additional metrical information. The resulting three-dimensional representation of the SLO active site ground state contains a reactive (a) conformer with hydrogen DAD of ∼3.1 Å, approximately van der Waals contact, plus an inactive (b) conformer with even longer DAD, establishing that stochastic conformational sampling is required to achieve reactive tunneling geometries. Tunneling-impaired SLO variants show increased DADs and variations in substrate positioning and rigidity, confirming previous kinetic and theoretical predictions of such behavior. Overall, this investigation highlights the (i) predictive power of nonadiabatic quantum treatments of proton-coupled electron transfer in SLO and (ii) sensitivity of ENDOR probes to test, detect, and corroborate kinetically predicted trends in active site reactivity and to reveal unexpected features of active site architecture.

Advanced paramagnetic resonance spectroscopies of iron-sulfur proteins: Electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM).

The advanced electron paramagnetic resonance (EPR) techniques, electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) spectroscopies, provide unique insights into the structure, coordination chemistry, and biochemical mechanism of nature's widely distributed iron-sulfur cluster (FeS) proteins. This review describes the ENDOR and ESEEM techniques and then provides a series of case studies on their application to a wide variety of FeS proteins including ferredoxins, nitrogenase, and radical SAM enzymes. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.