Synthesis, X-ray Structures, Electronic Properties, and O\u3csub\u3e2\u3c/sub\u3e/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

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. Synopsis The geometric and electronic structures of thiolate-ligated FeIII complexes of relevance to the active sites of thiol dioxygenases have been elucidated with spectroscopic and computational methods. Data collected for the synthetic models are compared to those previously obtained for the analogous enzymatic species, and newly collected resonance Raman spectra of Cys- and CN-bound FeIII-CDO are presented. The combined enzymatic/synthetic approach reveals that second-sphere residues perturb the positions of substrate (analogues) coordinated to the nonheme iron site of CDO

Combined Spectroscopic/Computational Studies of Metal Centers in Proteins and Cofactors: Application to Coenzyme B12

This article illustrates how the combined computational/spectroscopic methodology used in our studies of metal centers in proteins and cofactors can be applied to vitamin B12 and its biologically relevant derivatives. The B12 cofactors have long fascinated chemists because of their complex structures and unusual reactivities in biological systems; however, their electronic absorption (Abs) spectra have remained largely unassigned. In this study, Abs, circular dichroism (CD), magnetic CD (MCD), and resonance Raman spectroscopic techniques are used to probe the electronic excited states of various Co3+ Cbl species that differ with respect to their upper axial ligand. Spectroscopic data for each species are analyzed within the framework of time-dependent density functional theory (TD-DFT) to assign the major spectral features and to generate experimentally validated bonding descriptions. A simple model is presented that explains why the identity of the upper axial ligand has a major effect on the Co–Nax bond strength, whereas the lower axial ligand does not appreciably modulate the nature of the Co–C bond. Impli- cations of these results with respect to enzymatic Co–C bond activation are discussed

Vibronic Coupling in Vitamin B₁₂: A Combined Spectroscopic and Computational Study

Understanding the diverse reactivities of vitamin B12 and its derivatives, collectively called cobalamins, requires detailed knowledge of their geometric and electronic structures. Electronic absorption (Abs) and resonance Raman (rR) spectroscopies have proven invaluable in this area, particularly when used in concert with computational techniques such as density functional theory (DFT). There remain, however, lingering uncertainties in the computational description of electronic excited states of cobalamins, particularly surrounding the vibronic coupling that impacts the Abs bandshapes and gives rise to rR enhancement of vibrational modes. Past computational analyses of the vibrational spectra of cobalamins have either neglected rR enhancement or calculated rR enhancement for only a small number of modes. In the present study, we used the recently developed ORCA_ASA computational tool in conjunction with the popular B3LYP and BP86 functionals to predict Abs bandshapes and rR spectra for vitamin B12. The ORCA_ASA/B3LYP-computed Abs envelope in the visible spectral region and rR spectra of vitamin B12 agree remarkably well with our experimental data, while BP86 fails to reproduce both. This finding represents a significant advance in our understanding of how these two commonly used density functionals differently model the electronic properties of cobalamins. Guided by the computed frequencies for the Co–C stretching and Co–C–N bending modes, we identified, for the first time, isotope-sensitive features in our rR spectra of 12CNCbl and 13CNCbl that can be assigned to these modes. A normal coordinate analysis of the experimentally determined Co–C stretching and Co–C–N bending frequencies indicates that the Co–C force constant for vitamin B12 is 2.67 mdyn/Å, considerably larger than the Co–C force constants reported for alkylcobalamins

Combined Spectroscopic and Computational Analysis of the Vibrational Properties of Vitamin B₁₂ in its Co³⁺, Co²⁺, and Co¹⁺ Oxidation States

While the geometric and electronic structures of vitamin B₁₂ (cyanocobalamin, CNCbl) and its reduced derivatives Co²⁺cobalamin (Co²⁺Cbl) and Co¹⁺cobalamin (Co¹⁺Cbl^–) are now reasonably well established, their vibrational properties, in particular their resonance Raman (rR) spectra, have remained quite poorly understood. The goal of this study was to establish definitive assignments of the corrin-based vibrational modes that dominate the rR spectra of vitamin B₁₂ in its Co³⁺, Co²⁺, and Co¹⁺ oxidation states. rR spectra were collected for all three species with laser excitation in resonance with the most intense corrin-based π → π* transitions. These experimental data were used to validate the computed vibrational frequencies, eigenvector compositions, and relative rR intensities of the normal modes of interest as obtained by density functional theory (DFT) calculations. Importantly, the computational methodology employed in this study successfully reproduces the experimental observation that the frequencies and rR excitation profiles of the corrin-based vibrational modes vary significantly as a function of the cobalt oxidation state. Our DFT results suggest that this variation reflects large differences in the degree of mixing between the occupied Co 3d orbitals and empty corrin π* orbitals in CNCbl, Co²⁺Cbl, and Co¹⁺Cbl^–. As a result, vibrations mainly involving stretching of conjugated C–C and C–N bonds oriented along one axis of the corrin ring may, in fact, couple to a perpendicularly polarized electronic transition. This unusual coupling between electronic transitions and vibrational motions of corrinoids greatly complicates an assignment of the corrin-based normal modes of vibrations on the basis of their rR excitation profiles

Spectroscopic and Computational Studies of a Small-Molecule Functional Mimic of Iron Superoxide Dismutase, Iron 2,6-Diacetylpyridinebis(semioxamazide)

Iron 2,6-diacetylpyridinebis­(semioxamazide) (Fe­(dapsox)) is a heptacoordinate pentagonal bipyramidal, functional mimic of iron-dependent superoxide dismutase that has been well-characterized on the basis of kinetics and mechanistic studies; however, prior to our studies, its electronic structure had yet to be examined. This paper details our initial characterization of Fe­(dapsox) in both its reduced and oxidized states, by electronic absorption (Abs) and low-temperature magnetic circular dichroism spectroscopies. Density functional theory (DFT) geometry optimizations have yielded models in good agreement with the published crystal structures. Time-dependent DFT and INDO/S-CI calculations performed on these models successfully reproduce the experimental Abs spectra and identify intense, low-energy transitions in the reduced complex (Fe^II(H₂dapsox)) as metal-to-ligand charge transfer transitions, suggesting the presence of π-backbonding in this complex. This backbonding, along, with the proton uptake accompanying metal ion reduction, provides a compelling mechanism by which the metal-centered redox potential is correctly tuned for catalytic superoxide disproportionation