Julie A. Kovacs

Nitrile Hydration by Thiolate–and Alkoxide–Ligated Co-NHase Analogues. Isolation of Co(III)-Amidate and Co(III)–Iminol Intermediates

Nitrile hydratases (NHases) are thiolate-ligated Fe(III)- or Co(III)-containing enzymes, which convert nitriles to the corresponding amide under mild conditions. Proposed NHase mechanisms involve M(III)-NCR, M(III)-OH, M(III)-iminol, and M(III)-amide intermediates. There have been no reported crystallographically characterized examples of these key intermediates. Spectroscopic and kinetic data support the involvement of a M(III)-NCR intermediate. A H-bonding network facilitates this enzymatic reaction. Herein we describe two biomimetic Co(III)-NHase analogues that hydrate MeCN, and four crystallographically characterized NHase intermediate analogues, [Co(III)(S(Me2)N(4)(tren))(MeCN)](2+) (1), [Co(III)(S(Me2)N(4)(tren))(OH)](+) (3), [Co(III)(S(Me2)N(4)(tren))(NHC(O)CH(3))](+) (2), and [Co(III)(O(Me2)N(4)(tren))(NHC(OH)CH(3))](2+) (5). Iminol-bound 5 represents the first example of a Co(III)-iminol compound in any ligand environment. Kinetic parameters (k(1)(298 K) = 2.98(5) M(-1) s(-1), ΔH(‡) = 12.65(3) kcal/mol, ΔS(‡) = -14(7) e.u.) for nitrile hydration by 1 are reported, and the activation energy E(a) = 13.2 kcal/mol is compared with that (E(a) = 5.5 kcal/mol) of the NHase enzyme. A mechanism involving initial exchange of the bound MeCN for OH- is ruled out by the fact that nitrile exchange from 1 (k(ex)(300 K) = 7.3(1) × 10(-3) s(-1)) is 2 orders of magnitude slower than nitrile hydration, and that hydroxide bound 3 does not promote nitrile hydration. Reactivity of an analogue that incorporates an alkoxide as a mimic of the highly conserved NHase serine residue shows that this moiety facilitates nitrile hydration under milder conditions. Hydrogen-bonding to the alkoxide stabilizes a Co(III)-iminol intermediate. Comparison of the thiolate versus alkoxide intermediate structures shows that C≡N bond activation and C═O bond formation proceed further along the reaction coordinate when a thiolate is incorporated into the coordination sphere.

Structural and Spectroscopic Characterization of Metastable Thiolate-Ligated Manganese(III)–Alkylperoxo Species

Metastable Mn-peroxo species are proposed to form as key intermediates in biological oxidation reactions involving O(2) and C-H bond activation. The majority of these have yet to be spectroscopically characterized, and their inherent instability, in most cases, precludes structural characterization. Cysteinate-ligated metal-peroxos have been shown to form as reactive intermediates in both heme and nonheme iron enzymes. Herein we report the only examples of isolable Mn(III)-alkylperoxo species, and the first two examples of structurally characterized synthetic thiolate-ligated metal-peroxos. Spectroscopic data, including electronic absorption and IR spectra, and ESI mass spectra for (16)O vs (18)O-labeled metastable Mn(III)-OOR (R = (t)Bu, Cm) are discussed, as well as preliminary reactivity.

S K-Edge X-Ray Absorption Spectroscopy and Density Functional Theory Studies of High and Low Spin {FeNO}7 Thiolate Complexes: Exchange Stabilization of Electron Delocalization in {FeNO}7 and {FeO2}8

S K-edge X-ray absorption spectroscopy (XAS) is a direct experimental probe of metal ion electronic structure as the pre-edge energy reflects its oxidation state, and the energy splitting pattern of the pre-edge transitions reflects its spin state. The combination of sulfur K-edge XAS and density functional theory (DFT) calculations indicates that the electronic structures of {FeNO}(7) (S = 3/2) (S(Me2)N4(tren)Fe(NO), complex I) and {FeNO}(7) (S = 1/2) ((bme-daco)Fe(NO), complex II) are Fe(III)(S = 5/2)-NO(-)(S = 1) and Fe(III)(S = 3/2)-NO(-)(S = 1), respectively. When an axial ligand is computationally added to complex II, the electronic structure becomes Fe(II)(S = 0)-NO•(S = 1/2). These studies demonstrate how the ligand field of the Fe center defines its spin state and thus changes the electron exchange, an important factor in determining the electron distribution over {FeNO}(7) and {FeO2}(8) sites.

Reactivities and biological functions of iron-sulfur clusters

Iron-sulfur clusters are prevalent in biological systems. Through studies of iron-sulfur proteins and synthetic model clusters, it was realized early on that these clusters functioned as facile electron transfer agents. Until recently it was widely thought that they served exclusively in that capacity. However, in the last decade, it has become clear that their reactivities and biological functions are much more diverse. It is now apparent that these clusters can serve as the active sites of enzymes, as well as in the regulation of enzymatic activity. Synthetic clusters, which have been shown to undergo a variety of core rearrangements or structural changes, have provided insight into possible mechanisms of cluster formation or activity regulation in enzymes. Rigid tripodal ligands have been constructed which capture synthetic iron-sulfur clusters in a cavity which permits controlled reactivity studies. In this article, we review these recent developments and suggest some future directions the field may take.

Properties of Square-Pyramidal Alkyl-Thiolate FeIII-Complexes, Including an Analogue of the Unmodified Form of Nitrile Hydratase

The syntheses and structures of three new coordinatively unsaturated, monomeric, square-pyramidal thiolate-ligated Fe(III) complexes are described, [Fe(III)((tame-N(3))S(2)(Me2))](+) (1), [Fe(III)(Et-N(2)S(2)(Me2))(py)](1-) (3), and [Fe(III)((tame-N(2)S)S(2)(Me2))](2-) (15). The anionic bis-carboxamide, tris-thiolate N(2)S(3) coordination sphere of 15 is potentially similar to that of the yet-to-be characterized unmodified form of NHase. Comparison of the magnetic and reactivity properties of these reveals how anionic charge build up (from cationic 1 to anionic 3 and dianionic 15) and spin-state influence apical ligand affinity. For all of the ligand-field combinations examined, an intermediate S = 3/2 spin state was shown to be favored by a strong N(2)S(2) basal plane ligand field, and this was found to reduce the affinity for apical ligands, even when they are built in. This is in contrast to the post-translationally modified NHase active site, which is low spin and displays a higher affinity for apical ligands. Cationic 1 and its reduced Fe(II) precursor are shown to bind NO and CO, respectively, to afford [Fe(III)((tame-N(3))S(2)(Me))(NO)](+) (18, nu(NuO) = 1865 cm(-1)), an analogue of NO-inactivated NHase, and [Fe(II)((tame-N(3))S(2)(Me))(CO)] (16; nu(CO) stretch (1895 cm(-1)). Anions (N(3)(-), CN(-)) are shown to be unreactive toward 1, 3, and 15 and neutral ligands unreactive toward 3 and 15, even when present in 100-fold excess and at low temperatures. The curtailed reactivity of 15, an analogue of the unmodified form of NHase, and its apical-oxygenated S = 3/2 derivative [Fe(III)((tame-N(2)SO(2))S(2)(Me2))](2-) (20) suggests that regioselective post-translational oxygenation of the basal plane NHase cysteinate sulfurs plays an important role in promoting substrate binding. This is supported by previously reported theoretical (DFT) calculations.

Characterization and Dioxygen Reactivity of a New Series of Coordinatively Unsaturated Thiolate-Ligated Manganese(II) Complexes

The synthesis, structural, and spectroscopic characterization of four new coordinatively unsaturated mononuclear thiolate-ligated manganese(II) complexes ([Mn(II)(S(Me2)N(4)(6-Me-DPEN))](BF(4)) (1), [Mn(II)(S(Me2)N(4)(6-Me-DPPN))](BPh(4))·MeCN (3), [Mn(II)(S(Me2)N(4)(2-QuinoPN))](PF(6))·MeCN·Et(2)O (4), and [Mn(II)(S(Me2)N(4)(6-H-DPEN)(MeOH)](BPh(4)) (5)) is described, along with their magnetic, redox, and reactivity properties. These complexes are structurally related to recently reported [Mn(II)(S(Me2)N(4)(2-QuinoEN))](PF(6)) (2) (Coggins, M. K.; Kovacs, J. A. J. Am. Chem. Soc.2011, 133, 12470). Dioxygen addition to complexes 1-5 is shown to result in the formation of five new rare examples of Mn(III) dimers containing a single, unsupported oxo bridge: [Mn(III)(S(Me2)N(4)(6-Me-DPEN)](2)-(μ-O)(BF(4))(2)·2MeOH (6), [Mn(III)(S(Me2)N(4)(QuinoEN)](2)-(μ-O)(PF(6))(2)·Et(2)O (7), [Mn(III)(S(Me2)N(4)(6-Me-DPPN)](2)-(μ-O)(BPh(4))(2) (8), [Mn(III)(S(Me2)N(4)(QuinoPN)](2)-(μ-O)(BPh(4))(2) (9), and [Mn(III)(S(Me2)N(4)(6-H-DPEN)](2)-(μ-O)(PF(6))(2)·2MeCN (10). Labeling studies show that the oxo atom is derived from (18)O(2). Ligand modifications, involving either the insertion of a methylene into the backbone or the placement of an ortho substituent on the N-heterocyclic amine, are shown to noticeably modulate the magnetic and reactivity properties. Fits to solid-state magnetic susceptibility data show that the Mn(III) ions of μ-oxo dimers 6-10 are moderately antiferromagnetically coupled, with coupling constants (2J) that fall within the expected range. Metastable intermediates, which ultimately convert to μ-oxo bridged 6 and 7, are observed in low-temperature reactions between 1 and 2 and dioxygen. Complexes 3-5, on the other hand, do not form observable intermediates, thus illustrating the effect that relatively minor ligand modifications have upon the stability of metastable dioxygen-derived species.

How does cyanide inhibit superoxide reductase?Insight from synthetic FeIIIN4S model complexes

Superoxide reductases (SORs) are nonheme iron-containing enzymes that reduce HO2 to H2O2. Exogenous substrates such as N\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}_{3}^{-}\end{equation*}\end{document} and CN− have been shown to bind to the catalytic iron site of SOR, and cyanide acts as an inhibitor. To understand how these exogenous ligands alter the physical and reactivity properties of the SOR iron site, acetate-, azide-, and cyanide-ligated synthetic models of SOR have been prepared. The x-ray crystal structures of azide-ligated [FeIII(SMe2N4(tren))(N3)]+ (3), dimeric cyanide-bridged ([FeIII(SMe2N4(tren))]2-μ-CN)3+ (5), and acetate-ligated [FeIII(SMe2N4(tren))(OAc)]+ (6) are described, in addition to x-ray absorption spectrum-derived and preliminary crystallographic structures of cyanide-ligated [FeIII(SMe2N4(tren))(CN)]+ (4). Cyanide coordination to our model (4) causes the redox potential to shift anodically by 470 mV relative to acetate-ligated 6 and 395 mV relative to azide-ligated 3. If cyanide coordination were to cause a similar shift in redox potential with SOR, then the reduction potential of the catalytically active Fe3+ center would fall well below that of its biological reductants. These results suggest therefore that cyanide inhibits SOR activity by making the Fe2+ state inaccessible and thus preventing the enzyme from turning over. Cyanide inhibits activity in the metalloenzyme superoxide dismutase via a similar mechanism. The reduced five-coordinate precursor to 3, 4, and 6 [FeII(SMe2N4(tren))]+ (1) was previously shown by us to react with superoxide to afford H2O2 via an [FeIII(SMe2N4(tren))(OOH)]+ intermediate. Cyanide and azide do not bind to 1 and do not prevent 1 from reducing superoxide.

Synthesis and structure of a thiolate-ligated Ni cluster which contains an unusual thiolate bridging mode and an exposed Ni site

Abstract The synthesis and structure of an aromatic thiolate-ligated trinuclear cluster Ni3((μ2)2-bdt)((μ2,μ′2)-bdt)2(PPh3)2 (6) is reported, which contains two potentially reactive sites; a weak bridge that is susceptible to cleavage, and an ‘exposed’ four-coordinate Ni site. Cluster 6 crystallizes in the triclinic space group P 1 with a = 11.106(2), b = 11.406(2), c = 25.638(5) A , α = 85.16(2), β = 85.80(2), γ = 64.46(2)° and Z = 2. Both the solid state and solution properties indicate that 6 consists of two weakly connected charged fragments, dicationic [Ni2((μ2)2-bdt)(PPh3)2]2+ and dianionic [Ni(bdt)2]2−. The weak nature of the bridge which connects these fragments is probably a consequence of strain introduced by an unusual μ2,μ2′-bdt bridging mode. Cluster 6 remains intact in THF, toluence and CHCl3, but cluster fragmentation occurs in polar coordinating solvents DMF and DMSO.

Iron L2,3-Edge X-ray Absorption and X-ray Magnetic Circular Dichroism Studies of Molecular Iron Complexes with Relevance to the FeMoco and FeVco Active Sites of Nitrogenase

Herein, a systematic study of a series of molecular iron model complexes has been carried out using Fe L2,3-edge X-ray absorption (XAS) and X-ray magnetic circular dichroism (XMCD) spectroscopies. This series spans iron complexes of increasing complexity, starting from ferric and ferrous tetrachlorides ([FeCl4]−/2–), to ferric and ferrous tetrathiolates ([Fe(SR)4]−/2–), to diferric and mixed-valent iron–sulfur complexes [Fe2S2R4]2–/3–. This test set of compounds is used to evaluate the sensitivity of both Fe L2,3-edge XAS and XMCD spectroscopy to oxidation state and ligation changes. It is demonstrated that the energy shift and intensity of the L2,3-edge XAS spectra depends on both the oxidation state and covalency of the system; however, the quantitative information that can be extracted from these data is limited. On the other hand, analysis of the Fe XMCD shows distinct changes in the intensity at both L3 and L2 edges, depending on the oxidation state of the system. It is also demonstrated that the XMCD intensity is modulated by the covalency of the system. For mononuclear systems, the experimental data are correlated with atomic multiplet calculations in order to provide insights into the experimental observations. Finally, XMCD is applied to the tetranuclear heterometal–iron–sulfur clusters [MFe3S4]3+/2+ (M = Mo, V), which serve as structural analogues of the FeMoco and FeVco active sites of nitrogenase. It is demonstrated that the XMCD data can be utilized to obtain information on the oxidation state distribution in complex clusters that is not readily accessible for the Fe L2,3-edge XAS data alone. The advantages of XMCD relative to standard K-edge and L2,3-edge XAS are highlighted. This study provides an important foundation for future XMCD studies on complex (bio)inorganic systems.

Metal-Carbon Bonds in Nature

In contrast to most metabolic processes, but similar to some industrial catalysts, vitamin B12 contains a cobalt-active species and is sometimes referred to as “natures organometallic catalyst.” Kovacs et al. provide background and commentary to accompany the research report by Kumar et al. (p. 628) describing a second organometallic intermediate in biology—an active nickel species that serves as a precursor to acetic acid through its reaction with carbon monoxide and acetyl-CoA synthase.