Spectroscopic and Magnetic Studies of Co(II) Scorpionate Complexes: Is There a Halide Effect on Magnetic Anisotropy?

The observation of single-molecule magnetism in transition-metal complexes relies on the phenomenon of zero-field splitting (ZFS), which arises from the interplay of spin-orbit coupling (SOC) with ligand-field-induced symmetry lowering. Previous studies have demonstrated that the magnitude of ZFS in complexes with 3d metal ions is sometimes enhanced through coordination with heavy halide ligands (Br and I) that possess large free-atom SOC constants. In this study, we systematically probe this "heavy-atom effect" in high-spin cobalt(II)-halide complexes supported by substituted hydrotris(pyrazol-1-yl)borate ligands (TptBu,Me and TpPh,Me). Two series of complexes were prepared: [CoIIX(TptBu,Me)] (1-X; X = F, Cl, Br, and I) and [CoIIX(TpPh,Me)(HpzPh,Me)] (2-X; X = Cl, Br, and I), where HpzPh,Me is a monodentate pyrazole ligand. Examination with dc magnetometry, high-frequency and -field electron paramagnetic resonance, and far-infrared magnetic spectroscopy yielded axial (D) and rhombic (E) ZFS parameters for each complex. With the exception of 1-F, complexes in the four-coordinate 1-X series exhibit positive D-values between 10 and 13 cm-1, with no dependence on halide size. The five-coordinate 2-X series exhibit large and negative D-values between -60 and -90 cm-1. Interpretation of the magnetic parameters with the aid of ligand-field theory and ab initio calculations elucidated the roles of molecular geometry, ligand-field effects, and metal-ligand covalency in controlling the magnitude of ZFS in cobalt-halide complexes.

Reversible Dioxygen Binding to Co(II) Complexes with Noninnocent Ligands.

A series of mononuclear Co(II) complexes with noninnocent (redox-active) ligands are prepared that exhibit metal-ligand cooperativity during the reversible binding of O2. The complexes have the general formula, [CoII(LS,N)(TpR2)] (R = Me, Ph), where LS,N is a bidentate o-aminothiophenolate and TpR2 is a hydrotris(pyrazol-1-yl)borate scorpionate with R-substituents at the 3- and 5-positions. Exposure to O2 at room temperature results in one-electron oxidation and deprotonation of LS,N. The oxidized derivatives possess substantial "singlet diradical" character arising from antiferromagnetic coupling between an iminothiosemiquinonate (ITSQ•-) ligand radical and a low-spin Co(II) ion. The [CoII(TpMe2)(X2ITSQ)] complexes, where X = H or tBu, coordinate O2 reversibly at reduced temperatures to provide Co/O2 adducts. The O2 binding reactions closely resemble those previously reported by our group (Kumar et al., J. Am. Chem. Soc. 2019,141, 10984-10987) for the related complexes [CoII(TpMe2)(tBu2SQ)] and [CoII(TpMe2)(tBu2ISQ)], where tBu2(I)SQ represents 4,6-di-tert-butyl-(2-imino)semiquinonate radicals. In each case, the oxygenation reaction proceeds via the addition of O2 to both the cobalt ion and the ligand radical, generating metallocyclic cobalt(III)-alkylperoxo structures. Thermodynamic measurements elucidate the relationship between O2 affinity and redox potentials of the (imino)(thio)semiquinonate radicals, as well as energetic differences between these reactions and conventional metal-based oxygenations. The results highlight the utility and versatility of noninnocent ligands in the design of O2-absorbing compounds.

Identification of an Intermediate Species along the Nitrile Hydratase Reaction Pathway by EPR Spectroscopy.

A new method to trap catalytic intermediate species was employed with Fe-type nitrile hydratase from Rhodococcus equi TG328-2 (ReNHase). ReNHase was incubated with substrates in a 23% (w/w) NaCl/H2O eutectic system that remained liquid at -20 °C, thereby permitting the observation of transient species that were present at electron paramagnetic resonance (EPR)-detectable levels in samples frozen while in the steady state. FeIII-EPR signals from the resting enzyme were unaffected by the presence of 23% NaCl, and the catalytic activity was ∼55% that in the absence of NaCl at the optimum pH of 7.5. The reaction of ReNHase in the eutectic system at -20 °C with the substrates acetonitrile or benzonitrile induced significant changes in the EPR spectra. A previously unobserved signal with highly rhombic g-values (g1 = 2.31) was observed during the steady state but did not persist beyond the exhaustion of the substrate, indicating that it arises from a catalytically competent intermediate. Distinct signals due to product complexes provide a detailed mechanism for product release, the rate-limiting step of the reaction. Assignment of the observed EPR signals was facilitated by density functional theory calculations, which provided candidate structures and g-values for various proposed ReNHase intermediates. Collectively, these results provide new insights into the catalytic mechanism of NHase and offer a new approach for isolating and characterizing EPR-active intermediates in metalloenzymes.

Probing the Magnetic Anisotropy of Co(II) Complexes Featuring Redox-Active Ligands.

Coordination complexes that possess large magnetic anisotropy (otherwise known as zero-field splitting, ZFS) have possible applications in the field of magnetic materials, including single molecule magnets (SMMs). Previous studies have explored the role of coordination number and geometry in controlling the magnetic anisotropy and SMM behavior of high-spin (S = 3/2) Co(II) complexes. Building upon these efforts, the present work examines the impact of ligand oxidation state and structural distortions on the spin states and ZFS parameters of pentacoordinate Co(II) complexes. The five complexes included in this study (1-5) have the general formula, [Co(TpPh2)(LX,Y)]n+ (X = O, S; Y = N, O; n = 0 or 1), where TpPh2 is the scorpionate ligand hydrotris(3,5-diphenyl-pyrazolyl)borate(1-) and LX,Y are bidentate dioxolene-type ligands that can access multiple oxidation states. The specific LX,Y ligands used herein are 4,6-di-tert-butyl substituted o-aminophenolate and o-aminothiophenolate (1 and 2, respectively), o-iminosemiquinonate and o-semiquinonate radicals (3 and 4, respectively), and o-iminobenzoquinone (5). Each complex exhibits a distorted trigonal bipyramidal geometry, as revealed by single-crystal X-ray diffraction. Direct current (dc) magnetic susceptibility experiments confirmed that the complexes with closed-shell ligands (1, 2, and 5) possess S = 3/2 ground states with negative D-values (easy-axis anisotropy) of -41, -78, and -30 cm-1, respectively. For 3 and 4, antiferromagnetic coupling between the Co(II) center and o-(imino)semiquinonate radical ligand results in S = 1 ground states that likewise exhibit very large and negative anisotropy (-100 > D > -140 cm-1). Notably, ZFS was measured directly for each complex using far-infrared magnetic spectroscopy (FIRMS). In combination with high-frequency and -field electron paramagnetic resonance (HFEPR) studies, these techniques provided precise spin-Hamiltonian parameters for complexes 1, 2, and 5. Multireference ab initio calculations, using the CASSCF/NEVPT2 approach, indicate that the strongly negative anisotropies of these Co(II) complexes arise primarily from distortions in the equatorial plane due to constrictions imposed by the TpPh2 ligand. This effect is further amplified by cobalt(II)-radical exchange interactions in 3 and 4.

Cobalt Superoxo and Alkylperoxo Complexes Derived from Reaction of Ring-Cleaving Dioxygenase Models with O2.

The syntheses and O2 reactivities of active-site models of cobalt-substituted ring-cleaving dioxygenases are presented. The pentacoordinate cobalt(II)-aminophenolate complex, [Co(TpMe2)(tBu2APH)], gives rise to two distinct dioxygen adducts at reduced temperatures. The first is a paramagnetic (S = 1/2) cobalt(III)-superoxo species that was characterized with spectroscopic and computational techniques. The identity of the second Co/O2 adduct was elucidated by X-ray crystallography, which revealed an unprecedented cobalt(III)-alkylperoxo structure generated by O2 addition to the metal ion and ligand. These results provide synthetic precedents for proposed intermediates in the catalytic cycles of O2-activating cobalt enzymes.

Spectroscopic and Computational Comparisons of Thiolate-Ligated Ferric Nonheme Complexes to Cysteine Dioxygenase: Second-Sphere Effects on Substrate (Analogue) Positioning.

Parallel spectroscopic and computational studies of iron(III) cysteine dioxygenase (CDO) and synthetic models are presented. The synthetic complexes utilize the ligand tris(4,5-diphenyl-1-methylimidazol-2-yl)phosphine (Ph2TIP), which mimics the facial three-histidine triad of CDO and other thiol dioxygenases. In addition to the previously reported [FeII(CysOEt)(Ph2TIP)]BPh4 (1; CysOEt is the ethyl ester of anionic l-cysteine), the formation and crystallographic characterization of [FeII(2-MTS)(Ph2TIP)]BPh4 (2) is reported, where the methyl 2-thiosalicylate anion (2-MTS) resembles the substrate of 3-mercaptopropionate dioxygenase (MDO). One-electron chemical oxidation of 1 and 2 yields ferric species that bind cyanide and azide anions, which have been used as spectroscopic probes of O2 binding in prior studies of FeIII-CDO. The six-coordinate FeIII-CN and FeIII-N3 adducts are examined with UV-vis absorption, electron paramagnetic resonance (EPR), and resonance Raman (rRaman) spectroscopies. In addition, UV-vis and rRaman studies of cysteine- and cyanide-bound FeIII-CDO are reported for both the wild-type (WT) enzyme and C93G variant, which lacks the Cys-Tyr cross-link that is present in the second coordination sphere of the WT active site. Density functional theory (DFT) and ab initio calculations are employed to provide geometric and electronic structure descriptions of the synthetic and enzymatic FeIII adducts. In particular, it is shown that the complete active space self-consistent field (CASSCF) method, in tandem with n-electron valence state second-order perturbation theory (NEVPT2), is capable of elucidating the structural basis of subtle shifts in EPR g values for low-spin FeIII species.

Multiple States of Nitrile Hydratase from Rhodococcus equi TG328-2: Structural and Mechanistic Insights from Electron Paramagnetic Resonance and Density Functional Theory Studies.

Iron-type nitrile hydratases (NHases) contain an Fe(III) ion coordinated in a characteristic "claw setting" by an axial cysteine thiolate, two equatorial peptide nitrogens, the sulfur atoms of equatorial cysteine-sulfenic and cysteine-sulfinic acids, and an axial water/hydroxyl moiety. The cysteine-sulfenic acid is susceptible to oxidation, and the enzyme is traditionally prepared using butyric acid as an oxidative protectant. The as-prepared enzyme exhibits a complex electron paramagnetic resonance (EPR) spectrum due to multiple low-spin (S = 1/2) Fe(III) species. Four distinct signals can be assigned to the resting active state, the active state bound to butyric acid, an oxidized Fe(III)-bis(sulfinic acid) form, and an oxidized complex with butyric acid. A combination of comparison with earlier work, development of methods to elicit individual signals, and design and application of a novel density functional theory method for reproducing g tensors to unprecedentedly high precision was used to assign the signals. These species account for the previously reported EPR spectra from Fe-NHases, including spectra observed upon addition of substrates. Completely new EPR signals were observed upon addition of inhibitory boronic acids, and the distinctive g1 features of these signals were replicated in the steady state with the slow substrate acetonitrile. This latter signal constitutes the first EPR signal from a catalytic intermediate of NHase and is assigned to a key intermediate in the proposed catalytic cycle. Earlier, apparently contradictory, electron nuclear double resonance reports are reconsidered in the context of this work.

Oxygen activation by mononuclear Mn, Co, and Ni centers in biology and synthetic complexes.

The active sites of metalloenzymes that catalyze O2-dependent reactions generally contain iron or copper ions. However, several enzymes are capable of activating O2 at manganese or nickel centers instead, and a handful of dioxygenases exhibit activity when substituted with cobalt. This minireview summarizes the catalytic properties of oxygenases and oxidases with mononuclear Mn, Co, or Ni active sites, including oxalate-degrading oxidases, catechol dioxygenases, and quercetin dioxygenase. In addition, recent developments in the O2 reactivity of synthetic Mn, Co, or Ni complexes are described, with an emphasis on the nature of reactive intermediates featuring superoxo-, peroxo-, or oxo-ligands. Collectively, the biochemical and synthetic studies discussed herein reveal the possibilities and limitations of O2 activation at these three "overlooked" metals.

Bimetallic Cooperativity in Proton Reduction with an Amido-Bridged Cobalt Catalyst.

The bimetallic catalyst [CoII2 (L1 )(bpy)2 ]ClO4 (1), in which L1 is an [NN'2 O2 ] fused ligand, efficiently reduced H+ to H2 in CH3 CN in the presence of 100 equiv of HOAc with a turnover number of 18 and a Faradaic efficiency of 94 % after 3 h of bulk electrolysis at -1.6 V (vs. Ag/AgCl). This observation allowed the proposal that this bimetallic cooperativity is associated with distance, angle, and orbital alignment of the two Co centers, as promoted by the unique Co-Namido -Co environment offered by L1 . Experimental results revealed that the parent [CoII CoII ] complex undergoes two successive metal-based 1 e- reductions to generate the catalytically active species [CoI CoI ], and DFT calculations suggested that addition of a proton to one CoI triggers a cooperative 1 e- transfer by each of these CoI centers. This 2 e- transfer is an alternative route to generate a more reactive [CoII (CoII -H- )] hydride, thus avoiding the CoIII -H- required in monometallic species. This [CoII (CoII -H- )] species then accepts another H+ to release H2 .

Synthesis, X-ray Structures, Electronic Properties, and O2/NO Reactivities of Thiol Dioxygenase Active-Site Models.

Mononuclear non-heme iron complexes that serve as structural and functional mimics of the thiol dioxygenases (TDOs), cysteine dioxygenase (CDO) and cysteamine dioxygenase (ADO), have been prepared and characterized with crystallographic, spectroscopic, kinetic, and computational methods. The high-spin Fe(II) complexes feature the facially coordinating tris(4,5-diphenyl-1-methylimidazol-2-yl)phosphine (Ph2TIP) ligand that replicates the three histidine (3His) triad of the TDO active sites. Further coordination with bidentate l-cysteine ethyl ester (CysOEt) or cysteamine (CysAm) anions yielded five-coordinate (5C) complexes that resemble the substrate-bound forms of CDO and ADO, respectively. Detailed electronic-structure descriptions of the [Fe(Ph2TIP)(LS,N)]BPh4 complexes, where LS,N = CysOEt (1) or CysAm (2), were generated through a combination of spectroscopic techniques [electronic absorption, magnetic circular dichroism (MCD)] and density functional theory (DFT). Complexes 1 and 2 decompose in the presence of O2 to yield the corresponding sulfinic acid (RSO2H) products, thereby emulating the reactivity of the TDO enzymes and related complexes. Rate constants and activation parameters for the dioxygenation reactions were measured and interpreted with the aid of DFT calculations for O2-bound intermediates. Treatment of the TDO models with nitric oxide (NO)-a well-established surrogate of O2-led to a mixture of high-spin and low-spin {FeNO}7 species at low temperature (-70 °C), as indicated by electron paramagnetic resonance (EPR) spectroscopy. At room temperature, these Fe/NO adducts convert to a common species with EPR and infrared (IR) features typical of cationic dinitrosyl iron complexes (DNICs). To complement these results, parallel spectroscopic, computational, and O2/NO reactivity studies were carried out using previously reported TDO models that feature an anionic hydrotris(3-phenyl-5-methyl-pyrazolyl)borate (Ph,MeTp-) ligand. Though the O2 reactivities of the Ph2TIP- and Ph,MeTp-based complexes are quite similar, the supporting ligand perturbs the energies of Fe 3d-based molecular orbitals and modulates Fe-S bond covalency, suggesting possible rationales for the presence of neutral 3His coordination in CDO and ADO.

Bimetallic Complexes Supported by a Redox-Active Ligand with Fused Pincer-Type Coordination Sites.

The remarkable chemistry of mononuclear complexes featuring tridentate, meridionally chelating "pincer" ligands has stimulated the development of ligand frameworks containing multiple pincer sites. Here, the coordination chemistry of a novel pentadentate ligand (L(N3O2)) that provides two closely spaced NNO pincer-type compartments fused together at a central diarylamido unit is described. The trianionic L(N3O2) chelate supports homobimetallic structures in which each M(II) ion (M = Co, Cu, Zn) is bound in a meridional fashion by the bridging diarylamido N atom and O,N-donors of the salicyaldimine arms. The metal centers are also coordinated by a mono- or bidentate auxiliary ligand (L(aux)), resulting in complexes with the general form [M2(L(N3O2))(L(aux))2](+) (where L(aux) = 1-methyl-benzimidazole (1MeBI), 2,2'-bipyridine (bpy), 4,4'-dibromo-2,2'-bipyridine (bpy(Br2)), or (S)-2-(4-isopropyl-4,5-dihydrooxazolyl)pyridine (S-(iPr)OxPy)). The fused nature of the NNO pincer sites results in short metal-metal distances ranging from 2.70 Å for [Co2(L(N3O2)) (bpy)2](+) to 3.28 Å for [Zn2(L(N3O2)) (bpy)2](+), as revealed by X-ray crystallography. The complexes possess C2 symmetry due to the twisting of the aryl rings of the µ-NAr2 core; spectroscopic studies indicate that chiral L(aux) ligands, such as S-(iPr)OxPy, are capable of controlling the helical sense of the L(N3O2) scaffold. Since the four- or five-coordinate M(II) centers are linked solely by the amido moiety, each features an open coordination site in the intermetallic region, allowing for the possibility of metal-metal cooperativity in small-molecule activation. Indeed, the dicobalt(II) complex [Co2(L(N3O2)) (bpy(Br2))2](+) reacts with O2 to yield a dicobalt(III) species with a µ-1,2-peroxo ligand. The bpy-containing complexes exhibit rich electrochemical properties due to multiple metal- and ligand-based redox events across a wide (3.0 V) potential window. Using electron paramagnetic resonance (EPR) spectroscopy and density functional theory (DFT), it was determined that one-electron oxidation of [Co2(L(N3O2)) (bpy)2](+) results in formation of a S = 1/2 species with a L(N3O2)-based radical coupled to low-spin Co(II) centers.

Di- and Trinuclear Mixed-Valence Copper Amidinate Complexes from Reduction of Iodine.

Molecular examples of mixed-valence copper complexes through chemical oxidation are rare but invoked in the mechanism of substrate activation, especially oxygen, in copper-containing enzymes. To examine the cooperative chemistry between two metals in close proximity to each other we began studying the reactivity of a dinuclear Cu(I) amidinate complex. The reaction of [(2,6-Me2C6H3N)2C(H)]2Cu2, 1, with I2 in tetrahydrofuran (THF), CH3CN, and toluene affords three new mixed-valence copper complexes [(2,6-Me2C6H3N)2C(H)]2Cu2(µ2-I3)(THF)2, 2, [(2,6-Me2C6H3N)2C(H)]2Cu2(µ2-I) (NCMe)2, 3, and [(2,6-Me2C6H3N)2C(H)]3Cu3(µ3-I)2, 4, respectively. The first two compounds were characterized by UV-vis and electron paramagnetic resonance spectroscopies, and their molecular structure was determined by X-ray crystallography. Both di- and trinuclear mixed-valence intermediates were characterized for the reaction of compound 1 to compound 4, and the molecular structure of 4 was determined by X-ray crystallography. The electronic structure of each of these complexes was also investigated using density functional theory.

One-electron oxidation of an oxoiron(IV) complex to form an [O═FeV═NR]+ center.

Oxoiron(V) species are postulated to be involved in the mechanisms of the arene cis-dihydroxylating Rieske dioxygenases and of bioinspired nonheme iron catalysts for alkane hydroxylation, olefin cis-dihydroxylation, and water oxidation. In an effort to obtain a synthetic oxoiron(V) complex, we report herein the one-electron oxidation of the S = 1 complex [Fe(IV)(O)(TMC)(NCCH(3))](2+) (1, where TMC is tetramethylcyclam) by treatment with tert -butyl hydroperoxide and strong base in acetonitrile to generate a metastable complex 2 at -44 °C, which has been characterized by UV-visible, resonance Raman, Mössbauer, and EPR methods. The defining spectroscopic characteristic of 2 is the unusual x/y anisotropy observed for the (57)Fe and (17)O A tensors associated with the high-valent Fe═O unit and for the (14)N A tensor of a ligand derived from acetonitrile. As shown by detailed density functional theory (DFT) calculations, the unusual x/y anisotropy observed can only arise from an iron center with substantially different spin populations in the d(xz) and d(yz) orbitals, which cannot correspond to an Fe(IV)═O unit but is fully consistent with an Fe(V) center, like that found for [Fe(V)(O)(TAML)](-) (where TAML is tetraamido macrocyclic ligand), the only well-characterized oxoiron(V) complex reported. Mass spectral analysis shows that the generation of 2 entails the addition of an oxygen atom to 1 and the loss of one positive charge. Taken together, the spectroscopic data and DFT calculations support the formulation of 2 as an iron(V) complex having axial oxo and acetylimido ligands, namely [Fe(V)(O)(TMC)(NC(O)CH(3))](+).

Synthesis and spectroscopic characterization of high-spin mononuclear iron(II) p-semiquinonate complexes.

Two mononuclear iron(II) p-semiquinonate (pSQ) complexes have been generated via one-electron reduction of precursor complexes containing a substituted 1,4-naphthoquinone ligand. Detailed spectroscopic and computational analysis confirmed the presence of a coordinated pSQ radical ferromagnetically coupled to the high-spin Fe(II) center. The complexes are intended to model electronic interactions between (semi)quinone and iron cofactors in biology.

Dioxygen reactivity of biomimetic Fe(II) complexes with noninnocent catecholate, o-aminophenolate, and o-phenylenediamine ligands.

This study describes the O2 reactivity of a series of high-spin mononuclear Fe(II) complexes each containing the facially coordinating tris(4,5-diphenyl-1-methylimidazol-2-yl)phosphine ((Ph2)TIP) ligand and one of the following bidentate, redox-active ligands: 4-tert-butylcatecholate ((tBu)CatH(-)), 4,6-di-tert-butyl-2-aminophenolate ((tBu2)APH(-)), or 4-tert-butyl-1,2-phenylenediamine ((tBu)PDA). The preparation and X-ray structural characterization of [Fe(2+)((Ph2)TIP)((tBu)CatH)]OTf, [3]OTf and [Fe(2+)((Ph2)TIP)((tBu)PDA)](OTf)2, [4](OTf)2 are described here, whereas [Fe(2+)((Ph2)TIP)((tBu2)APH)]OTf, [2]OTf was reported in our previous paper [Bittner et al., Chem.-Eur. J. 2013, 19, 9686-9698]. These complexes mimic the substrate-bound active sites of nonheme iron dioxygenases, which catalyze the oxidative ring-cleavage of aromatic substrates like catechols and aminophenols. Each complex is oxidized in the presence of O2, and the geometric and electronic structures of the resulting complexes were examined with spectroscopic (absorption, EPR, Mössbauer, resonance Raman) and density functional theory (DFT) methods. Complex [3]OTf reacts rapidly with O2 to yield the ferric-catecholate species [Fe(3+)((Ph2)TIP)((tBu)Cat)](+) (3(ox)), which undergoes further oxidation to generate an extradiol cleavage product. In contrast, complex [4](2+) experiences a two-electron (2e(-)), ligand-based oxidation to give [Fe(2+)((Ph2)TIP)((tBu)DIBQ)](2+) (4(ox)), where DIBQ is o-diiminobenzoquinone. The reaction of [2](+) with O2 is also a 2e(-) process, yet in this case both the Fe center and (tBu2)AP ligand are oxidized; the resulting complex (2(ox)) is best described as [Fe(3+)((Ph2)TIP)((tBu2)ISQ)](+), where ISQ is o-iminobenzosemiquinone. Thus, the oxidized complexes display a remarkable continuum of electronic structures ranging from [Fe(3+)(L(2-))](+) (3(ox)) to [Fe(3+)(L(•-))](2+) (2(ox)) to [Fe(2+)(L(0))](2+) (4(ox)). Notably, the O2 reaction rates vary by a factor of 10(5) across the series, following the order [3](+) > [2](+) > [4](2+), even though the complexes have similar structures and Fe(3+/2+) redox potentials. To account for the kinetic data, we examined the relative abilities of the title complexes to bind O2 and participate in H-atom transfer reactions. We conclude that the trend in O2 reactivity can be rationalized by accounting for the role of proton transfer(s) in the overall reaction.

Role of second-sphere arginine residues in metal binding and metallocentre assembly in nitrile hydratases.

Two conserved second-sphere ßArg (R) residues in nitrile hydratases (NHase), that form hydrogen bonds with the catalytically essential sulfenic and sulfinic acid ligands, were mutated to Lys and Ala residues in the Co-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) and the Fe-type NHase from Rhodococcus equi TG328-2 (ReNHase). Only five of the eight mutants (PtNHase ßR52A, ßR52K, ßR157A, ßR157K and ReNHase ßR61A) were successfully expressed and purified. Apart from the PtNHase ßR52A mutant that exhibited no detectable activity, the kcat values obtained for the PtNHase and ReNHase ßR mutant enzymes were between 1.8 and 12.4 s-1 amounting to <1% of the kcat values observed for WT enzymes. The metal content of each mutant was also significantly decreased with occupancies ranging from ∼10 to ∼40%. UV-Vis spectra coupled with EPR data obtained on the ReNHase mutant enzyme, suggest a decrease in the Lewis acidity of the active site metal ion. X-ray crystal structures of the four PtNHase ßR mutant enzymes confirmed the mutation and the low active site metal content, while also providing insight into the active site hydrogen bonding network. Finally, DFT calculations suggest that the equatorial sulfenic acid ligand, which has been shown to be the catalytic nucleophile, is protonated in the mutant enzyme. Taken together, these data confirm the necessity of the conserved second-sphere ßR residues in the proposed subunit swapping process and post-translational modification of the α-subunit in the α activator complex, along with stabilizing the catalytic sulfenic acid in its anionic form.

Observation of oxygenated intermediates in functional mimics of aminophenol dioxygenase.

Aminophenol dioxygenases (APDO) are mononuclear nonheme iron enzymes that utilize dioxygen (O2) to catalyze the conversion of o-aminophenols to 2-picolinic acid derivatives in metabolic pathways. This study describes the synthesis and O2 reactivity of two synthetic models of substrate-bound APDO: [FeII(TpMe2)(tBu2APH)] (1) and [FeII(TpMe2)(tBuAPH)] (2), where TpMe2 = hydrotris(3,5-dimethylpyrazole-1-yl)borate, tBu2APH = 4,6-di-tert-butyl-2-aminophenolate, and tBuAPH2 = 4-tert-butyl-2-aminophenolate. Both Fe(II) complexes behave as functional APDO mimics, as exposure to O2 results in oxidative CC bond cleavage of the o-aminophenolate ligand. The ring-cleaved products undergo spontaneous cyclization to give substituted 2-picolinic acids, as verified by 1H NMR spectroscopy, mass spectrometry, and X-ray crystallography. Reaction of the APDO models with O2 at low temperature reveals multiple intermediates, which were probed with UV-vis absorption, electron paramagnetic resonance (EPR), Mössbauer (MB), and resonance Raman (rRaman) spectroscopies. The most stable intermediate at -70 °C in THF exhibits multiple isotopically-sensitive features in rRaman samples prepared with 16O2 and 18O2, confirming incorporation of O2-derived atom(s) into its molecular structure. Insights into the geometric structures, electronic properties, and spectroscopic features of the observed intermediates were obtained from density functional theory (DFT) calculations. Although functional APDO models have been previously reported, this is the first time that an oxygenated ligand-based radical has been detected and spectroscopically characterized in the ring-cleaving mechanism of a relevant synthetic system.

Synthetic, spectroscopic, and DFT studies of iron complexes with iminobenzo(semi)quinone ligands: implications for o-aminophenol dioxygenases.

The oxidative C-C bond cleavage of o-aminophenols by nonheme Fe dioxygenases is a critical step in both human metabolism (the kynurenine pathway) and the microbial degradation of nitroaromatic pollutants. The catalytic cycle of o-aminophenol dioxygenases (APDOs) has been proposed to involve formation of an Fe(II)/O2/iminobenzosemiquinone complex, although the presence of a substrate radical has been called into question by studies of related ring-cleaving dioxygenases. Recently, we reported the first synthesis of an iron(II) complex coordinated to an iminobenzosemiquinone (ISQ) ligand, namely, [Fe((Ph2)Tp)((tBu)ISQ)] (2a; where (Ph2)Tp=hydrotris(3,5-diphenylpyrazol-1-yl)borate and (tBu)ISQ is the radical anion derived from 2-amino-4,6-di-tert-butylphenol). In the current manuscript, density functional theory (DFT) calculations and a wide variety of spectroscopic methods (electronic absorption, Mössbauer, magnetic circular dichroism, and resonance Raman) were employed to obtain detailed electronic-structure descriptions of 2a and its one-electron oxidized derivative [3a](+). In addition, we describe the synthesis and characterization of a parallel series of complexes featuring the neutral supporting ligand tris(4,5-diphenyl-1-methylimidazol-2-yl)phosphine ((Ph2)TIP). The isomer shifts of about 0.97 mm s(-1) obtained through Mössbauer experiments confirm that 2a (and its (Ph2)TIP-based analogue [2b](+)) contain Fe(II) centers, and the presence of an ISQ radical was verified by analysis of the absorption spectra in light of time-dependent DFT calculations. The collective spectroscopic data indicate that one-electron oxidation of the Fe(II)-ISQ complexes yields complexes ([3a](+) and [3b](2+)) with electronic configurations between the Fe(III)-ISQ and Fe(II)-IBQ limits (IBQ=iminobenzoquinone), highlighting the ability of o-amidophenolates to access multiple oxidation states. The implications of these results for the mechanism of APDOs and other ring-cleaving dioxygenases are discussed.

Hydrogen-bonding effects on the reactivity of [X-Fe(III)-O-Fe(IV)═O] (X = OH, F) complexes toward C-H bond cleavage.

Complexes 1-OH and 1-F are related complexes that share similar [X-Fe(III)-O-Fe(IV)═O](3+) core structures with a total spin S of ½, which arises from antiferromagnetic coupling of an S = 5/2 Fe(III)-X site and an S = 2 Fe(IV)═O site. EXAFS analysis shows that 1-F has a nearly linear Fe(III)-O-Fe(IV) core compared to that of 1-OH, which has an Fe-O-Fe angle of ~130° due to the presence of a hydrogen bond between the hydroxo and oxo groups. Both complexes are at least 1000-fold more reactive at C-H bond cleavage than 2, a related complex with a [OH-Fe(IV)-O-Fe(IV)═O](4+) core having individual S = 1 Fe(IV) units. Interestingly, 1-F is 10-fold more reactive than 1-OH. This raises an interesting question about what gives rise to the reactivity difference. DFT calculations comparing 1-OH and 1-F strongly suggest that the H-bond in 1-OH does not significantly change the electrophilicity of the reactive Fe(IV)═O unit and that the lower reactivity of 1-OH arises from the additional activation barrier required to break its H-bond in the course of H-atom transfer by the oxoiron(IV) moiety.

A synthetic model of the putative Fe(II)-iminobenzosemiquinonate intermediate in the catalytic cycle of o-aminophenol dioxygenases.

The oxidative ring cleavage of aromatic substrates by nonheme Fe dioxygenases is thought to involve formation of a ferrous-(substrate radical) intermediate. Here we describe the synthesis of the trigonal-bipyramdial complex Fe((Ph2)Tp)(ISQ(tBu)) (2), the first synthetic example of an iron(II) center bound to an iminobenzosemiquinonate (ISQ) radical. The unique electronic structure of this S = 3/2 complex and its one-electron oxidized derivative ([3](+)) have been established on the basis of crystallographic, spectroscopic, and computational analyses. These findings further demonstrate the viability of Fe(2+)-ISQ intermediates in the catalytic cycles of o-aminophenol dioxygenases.