The EutT enzyme from Salmonella enterica, a member of the family of ATP:cobalt(I) corrinoid adenosyltransferase (ACAT) enzymes, requires a divalent transition metal ion for catalysis, with Fe(II) yielding the highest activity. EutT contains a unique cysteine-rich HX11CCX2C(83) motif (where H and the last C occupy the 67th and 83rd positions, respectively, in the amino acid sequence) not found in other ACATs and employs an unprecedented mechanism for the formation of adenosylcobalamin. Recent kinetic and spectroscopic studies of this enzyme revealed that residues in the HX11CCX2C(83) motif are required for the tight binding of the divalent metal ion and are critical for the formation of a four-coordinate (4c) cob(II)alamin [Co(II)Cbl] intermediate in the catalytic cycle. However, it remained unknown which, if any, of the residues in the HX11CCX2C(83) motif bind the divalent metal ion. To address this issue, we have characterized Co(II)-substituted wild-type EutT (EutTWT/Co) by using electronic absorption, electron paramagnetic resonance, and magnetic circular dichroism (MCD) spectroscopies. Our results indicate that the reduced catalytic activity of EutTWT/Co relative to that of the Fe(II)-containing enzyme arises from the incomplete incorporation of Co(II) ions and, thus, a decrease in the relative population of 4c Co(II)Cbl. Our MCD data for EutTWT/Co also reveal that the Co(II) ions reside in a distorted tetrahedral coordination environment with direct cysteine sulfur ligation. Additional spectroscopic studies of EutT/Co variants possessing a single alanine substitution of either His67, His75, Cys79, Cys80, or Cys83 indicate that Cys80 coordinates to the Co(II) ion, while the additional residues are important for maintaining the structural integrity and/or high affinity of the metal binding site.

EutT from Salmonella enterica is a member of a class of enzymes termed ATP:Co(I)rrinoid adenosyltransferases (ACATs), implicated in the biosynthesis of adenosylcobalamin (AdoCbl). In the presence of cosubstrate ATP, ACATs raise the Co(II)/Co(I) reduction potential of their cob(II)alamin [Co(II)Cbl] substrate by >250 mV via the formation of a unique four-coordinate (4c) Co(II)Cbl species, thereby facilitating the formation of a “supernucleophilic” cob(I)alamin intermediate required for the formation of the AdoCbl product. Previous kinetic studies of EutT revealed the importance of a HX11CCX2C(83) motif for catalytic activity and have led to the proposal that residues in this motif serve as the binding site for a divalent transition metal cofactor [e.g., Fe(II) or Zn(II)]. This motif is absent in other ACAT families, suggesting that EutT employs a distinct mechanism for AdoCbl formation. To assess how metal ion binding to the HX11CCX2C(83) motif affects the relative yield of 4c Co(II)Cbl generated in the EutT active site, we have characterized several enzyme variants by using electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopies. Our results indicate that Fe(II) or Zn(II) binding to the HX11CCX2C(83) motif of EutT is required for promoting the formation of 4c Co(II)Cbl. Intriguingly, our spectroscopic data also reveal the presence of an equilibrium between five-coordinate “base-on” and “base-off” Co(II)Cbl species bound to the EutT active site at low ATP concentrations, which shifts in favor of “base-off” Co(II)Cbl in the presence of excess ATP, suggesting that the base-off species serves as a precursor to 4c Co(II)Cbl.

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.

5′-deoxyadenosylcobalamin (coenzyme B12, AdoCbl) serves as the cofactor for several enzymes that play important roles in fermentation and catabolism. All of these enzymes initiate catalysis by promoting homolytic cleavage of the cofactor’s Co–C bond in response to substrate binding to their active sites. Despite considerable research efforts, the role of the lower axial ligand in facilitating Co–C bond homolysis remains incompletely understood. In the present study, we characterized several derivatives of AdoCbl and its one-electron reduced form, Co(II)Cbl, by using electronic absorption and magnetic circular dichroism spectroscopies. To complement our experimental data, we performed computations on these species, as well as additional Co(II)Cbl analogues. The geometries of all species investigated were optimized using a quantum mechanics/molecular mechanics method, and the optimized geometries were used to compute absorption spectra with time-dependent density functional theory. Collectively, our results indicate that a reduction in the basicity of the lower axial ligand causes changes to the cofactor’s electronic structure in the Co(II) state that replicate the effects seen upon binding of Co(II)Cbl to Class I isomerases, which replace the lower axial dimethylbenzimidazole ligand of AdoCbl with a protein-derived histidine (His) residue. Such a reduction of the basicity of the His ligand in the enzyme active site may be achieved through proton uptake by the catalytic triad of conserved residues, DXHXGXK, during Co–C bond homolysis.

Cysteine dioxygenase (CDO) is a mononuclear, non-heme iron(II)-dependent enzyme that utilizes molecular oxygen to catalyze the oxidation of l-cysteine (Cys) to cysteinesulfinic acid. Although the kinetic consequences of various outer-sphere amino acid substitutions have previously been assessed, the effects of these substitutions on the geometric and electronic structures of the active site remained largely unexplored. In this work, we have performed a spectroscopic and computational characterization of the H155A CDO variant, which was previously shown to display a rate of Cys oxidation ∼100-fold decreased relative to that of wild-type (WT) CDO. Magnetic circular dichroism and electron paramagnetic resonance spectroscopic data indicate that the His155 → Ala substitution has a significant effect on the electronic structure of the Cys-bound Fe(II)CDO active site. An analysis of these data within the framework of density functional theory calculations reveals that Cys-bound H155A Fe(II)CDO possesses a six-coordinate Fe(II) center, differing from the analogous WT CDO species in the presence of an additional water ligand. The enhanced affinity of the Cys-bound Fe(II) center for a sixth ligand in the H155A CDO variant likely stems from the increased level of conformational freedom of the cysteine–tyrosine cross-link in the absence of the H155 imidazole ring. Notably, the nitrosyl adduct of Cys-bound Fe(II)CDO [which mimics the (O2/Cys)–CDO intermediate] is essentially unaffected by the H155A substitution, suggesting that the primary role played by the H155 side chain in CDO catalysis is to discourage the binding of a water molecule to the Cys-bound Fe(II)CDO active site.

Nitrosylcobalamin (NOCbl) is readily formed when Co(II)balamin reacts with nitric oxide (NO) gas. NOCbl has been implicated in the inhibition of various B12-dependent enzymes, as well as in the modulation of blood pressure and of the immunological response. Previous studies revealed that among the known biologically relevant cobalamin species, NOCbl possesses the longest bond between the Co ion and the axially bound 5,6-dimethylbenzimidazole base, which was postulated to result from a strong trans influence exerted by the NO ligand. In this study, various spectroscopic (electronic absorption, circular dichroism, magnetic circular dichroism, and resonance Raman) and computational (density functional theory (DFT) and time-dependent DFT) techniques were used to generate experimentally validated electronic structure descriptions for the “base-on” and “base-off” forms of NOCbl. Further insights into the principal Co–ligand bonding interactions were obtained by carrying out natural bond orbital analyses. Collectively, our results indicate that the formally unoccupied Co 3dz2 orbital engages in a highly covalent bonding interaction with the filled NO π* orbital and that the Co–NO bond is strengthened further by sizable π-backbonding interactions that are not present in any other Co(III)Cbl characterized to date. Because of the substantial NO– to Co(III) charge donation, NOCbl is best described as a hybrid of Co(III)–NO– and Co(II)–NO• resonance structures. In contrast, our analogous computational characterization of a related species, superoxocobalamin, reveals that in this case a Co(III)–O2– description is adequate due to the larger oxidizing power of O2 versus NO. The implications of our results with respect to the unusual structural features and thermochromism of NOCbl and the proposed inhibition mechanisms of B12-dependent enzymes by NOCbl are discussed.

CobA from Salmonella enterica (SeCobA) is a member of the family of ATP:Co(I)rrinoid adenosyltransferase (ACAT) enzymes that participate in the biosynthesis of adenosylcobalamin by catalyzing the transfer of the adenosyl group from an ATP molecule to a reactive Co(I)rrinoid species transiently generated in the enzyme active site. This reaction is thermodynamically challenging, as the reduction potential of the Co(II)rrinoid precursor in solution is far more negative than that of available reducing agents in the cell (e.g., flavodoxin), precluding nonenzymic reduction to the Co(I) oxidation state. However, in the active sites of ACATs, the Co(II)/Co(I) redox potential is increased by >250 mV via the formation of a unique four-coordinate (4c) Co(II)rrinoid species. In the case of the SeCobA ACAT, crystallographic and kinetic studies have revealed that the phenylalanine 91 (F91) and tryptophan 93 (W93) residues are critical for in vivo activity, presumably by blocking access to the lower axial ligand site of the Co(II)rrinoid substrate. To further assess the importance of the F91 and W93 residues with respect to enzymatic function, we have characterized various SeCobA active-site variants using electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopies. Our data provide unprecedented insight into the mechanism by which SeCobA converts the Co(II)rrinoid substrate to 4c species, with the hydrophobicity, size, and ability to participate in offset π-stacking interactions of key active-site residues all being critical for activity. The structural changes that occur upon Co(II)rrinoid binding also appear to be crucial for properly orienting the transiently generated Co(I) “supernucleophile” for rapid reaction with cosubstrate ATP.

Cysteine dioxygenase (CDO) is a mononuclear, non-heme iron-dependent enzyme that converts exogenous cysteine (Cys) to cysteine sulfinic acid using molecular oxygen. Although the complete catalytic mechanism is not yet known, several recent reports presented evidence for an Fe(III)-superoxo reaction intermediate. In this work, we have utilized spectroscopic and computational methods to investigate the as-isolated forms of CDO, as well as Cys-bound Fe(III)CDO, both in the absence and presence of azide (a mimic of superoxide). An analysis of our electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance data of the azide-treated as-isolated forms of CDO within the framework of density functional theory (DFT) computations reveals that azide coordinates directly to the Fe(III), but not the Fe(II) center. An analogous analysis carried out for Cys-Fe(III)CDO provides compelling evidence that at physiological pH, the iron center is six coordinate, with hydroxide occupying the sixth coordination site. Upon incubation of this species with azide, the majority of the active sites retain hydroxide at the iron center. Nonetheless, a modest perturbation of the electronic structure of the Fe(III) center is observed, indicating that azide ions bind near the active site. Additionally, for a small fraction of active sites, azide displaces hydroxide and coordinates directly to the Cys-bound Fe(III) center to generate a low-spin (S = 1/2) Fe(III) complex. In the DFT-optimized structure of this complex, the central nitrogen atom of the azide moiety lies within 3.12 Å of the cysteine sulfur. A similar orientation of the superoxide ligand in the putative Fe(III)-superoxo reaction intermediate would promote the attack of the distal oxygen atom on the sulfur of substrate Cys.

We have prepared and thoroughly characterized, using X-ray crystallographic, spectroscopic, and computational methods, the diazide adduct of [FeIII(dapsox)(H2O)2]+ [dapsox = 2,6-diacetylpyridinebis(semioxamazide)], (1), a low-molecular weight, functional analogue of iron superoxide dismutase (FeSOD). The X-ray crystal structure of the dimeric form of 1, (Na[FeIII(dapsox)(N3)2]·DMF)2 (2) shows two axially coordinated, symmetry inequivalent azides with differing Fe–N3 bond lengths and Fe–N–N2 bond angles. This inequivalence of the azide ligands likely reflects the presence of an interdimer hydrogen bonding interaction between a dapsox NH group and the coordinated nitrogen of one of the two azide ligands. Resonance Raman (rR) data obtained for frozen aqueous solution and solid-state samples of 2 indicate that the azides remain inequivalent in solution, suggesting that one of the azide ligands of 1 engages in an intermolecular hydrogen bonding interaction with a water molecule. Density functional theory (DFT) and time-dependent DFT calculations have been used to study two different computational models of 1, one using coordinates taken from the X-ray crystal structure of 2, and the other generated via DFT geometry optimization. An evaluation of these models on the basis of electronic absorption, magnetic circular dichroism, and rR data indicates that the crystal structure based model yields a more accurate electronic structure description of 1, providing further support for the proposed intermolecular hydrogen bonding of 1 in the solid state and in solution. An analysis of the experimentally validated DFT results for this model reveals that the azides have both σ- and π-bonding interactions with the FeIII center and that more negative charge is located on the Fe-bound, rather than on the terminal, nitrogen atom of each azide. These observations are reminiscent of the results previously reported for the azide adduct of FeSOD and provide clues regarding the origin of the high catalytic activity of Fe-dapsox for superoxide disproportionation.

The active-site structures of the oxidized and reduced forms of manganese-substituted iron superoxide dismutase (Mn(Fe)SOD) are examined, for the first time, using a combination of spectroscopic and computational methods. On the basis of electronic absorption, circular dichroism (CD), magnetic CD (MCD), and variable-temperature variable-field MCD data obtained for oxidized Mn(Fe)SOD, we propose that the active site of this species is virtually identical to that of wild-type manganese SOD (MnSOD), with both containing a metal ion that resides in a trigonal bipyramidal ligand environment. This proposal is corroborated by quantum mechanical/molecular mechanical (QM/MM) computations performed on complete protein models of Mn(Fe)SOD in both its oxidized and reduced states and, for comparison, wild-type (WT) MnSOD. The major differences between the QM/MM optimized active sites of WT MnSOD and Mn(Fe)SOD are a smaller (His)N–Mn–N(His) equatorial angle and a longer (Gln146(69))NH···O(sol) H-bond distance in the metal-substituted protein. Importantly, these modest geometric differences are consistent with our spectroscopic data obtained for the oxidized proteins and high-field electron paramagnetic resonance spectra reported previously for reduced Mn(Fe)SOD and MnSOD. As Mn(Fe)SOD exhibits a reduction midpoint potential (Em) almost 700 mV higher than that of MnSOD, which has been shown to be sufficient for explaining the lack of SOD activity displayed by the metal-subtituted species (Vance, C. K.; Miller, A. F. Biochemistry2001, 40, 13079–13087), Em’s were computed for our experimentally validated QM/MM optimized models of Mn(Fe)SOD and MnSOD. These computations properly reproduce the experimental trend and reveal that the drastically elevated Em of the metal substituted protein stems from a larger separation between the second-sphere Gln residue and the coordinated solvent in Mn(Fe)SOD relative to MnSOD, which causes a weakening of the corresponding H-bond interaction in the oxidized state and alleviates steric crowding in the reduced state.

Cysteine dioxygenase (CDO) is a mononuclear nonheme iron(II)-dependent enzyme critical for maintaining appropriate cysteine (Cys) and taurine levels in eukaryotic systems. Because CDO possesses both an unusual 3-His facial ligation sphere to the iron center and a rare Cys–Tyr cross-link near the active site, the mechanism by which it converts Cys and molecular oxygen to cysteine sulfinic acid is of broad interest. However, as of yet, direct experimental support for any of the proposed mechanisms is still lacking. In this study, we have used NO as a substrate analogue for O2 to prepare a species that mimics the geometric and electronic structures of an early reaction intermediate. The resultant unusual S = 1/2 {FeNO}7 species was characterized by magnetic circular dichroism, electron paramagnetic resonance, and electronic absorption spectroscopies as well as computational methods including density functional theory and semiempirical calculations. The NO adducts of Cys- and selenocysteine (Sec)-bound Fe(II)CDO exhibit virtually identical electronic properties; yet, CDO is unable to oxidize Sec. To explore the differences in reactivity between Cys- and Sec-bound CDO, the geometries and energies of viable O2-bound intermediates were evaluated computationally, and it was found that a low-energy quintet-spin intermediate on the Cys reaction pathway adopts a different geometry for the Sec-bound adduct. The absence of a low-energy O2 adduct for Sec-bound CDO is consistent with our experimental data and may explain why Sec is not oxidized by CDO.

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.

Vitamin B12 (cyanocobalamin) and its biologically active derivatives, methylcobalamin and adenosylcobalamin, are members of the family of corrinoids, which also includes cobinamides. As biological precursors to cobalamins, cobinamides possess the same structural core, consisting of a low-spin Co3+ ion that is ligated equatorially by the four nitrogens of a highly substituted tetrapyrrole macrocycle (the corrin ring), but differ with respect to the lower axial ligation. Specifically, cobinamides possess a water molecule instead of the nucleotide loop that coordinates axially to Co3+cobalamins via its dimethylbenzimidazole (DMB) base. Compared to the cobalamin species, cobinamides have proven much more difficult to study experimentally, thus far eluding characterization by X-ray crystallography. In this study, we have utilized combined quantum mechanics/molecular mechanics (QM/MM) computations to generate complete structural models of a representative set of cobinamide species with varying upper axial ligands. To validate the use of this approach, analogous QM/MM geometry optimizations were carried out on entire models of the cobalamin counterparts for which high-resolution X-ray structural data are available. The accuracy of the cobinamide structures was assessed further by comparing electronic absorption spectra computed using time-dependent density functional theory to those obtained experimentally. Collectively, the results obtained in this study indicate that the DMB → H2O lower axial ligand switch primarily affects the energies of the Co 3dz2-based molecular orbital (MO) and, to a lesser extent, the other Co 3d-based MOs as well as the corrin π-based highest energy MO. Thus, while the energy of the lowest-energy electronic transition of cobalamins changes considerably as a function of the upper axial ligand, it is nearly invariant for the cobinamides.

The PduO-type adenosine 5′-triphosphate (ATP):corrinoid adenosyltransferase from Lactobacillus reuteri (LrPduO) catalyzes the transfer of the adenosyl-group of ATP to Co1+cobalamin (Cbl) and Co1+cobinamide (Cbi) substrates to synthesize adenosylcobalamin (AdoCbl) and adenosylcobinamide (AdoCbi+), respectively. Previous studies revealed that to overcome the thermodynamically challenging Co2+ → Co1+ reduction, the enzyme drastically weakens the axial ligand–Co2+ bond so as to generate effectively four-coordinate (4c) Co2+corrinoid species. To explore how LrPduO generates these unusual 4c species, we have used magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectroscopic techniques. The effects of active-site amino acid substitutions on the relative yield of formation of 4c Co2+corrinoid species were examined by performing eight single-amino acid substitutions at seven residues that are involved in ATP-binding, an intersubunit salt bridge, and the hydrophobic region surrounding the bound corrin ring. A quantitative analysis of our MCD and EPR spectra indicates that the entire hydrophobic pocket below the corrin ring, and not just residue F112, is critical for the removal of the axial ligand from the cobalt center of the Co2+corrinoids. Our data also show that a higher level of coordination among several LrPduO amino acid residues is required to exclude the dimethylbenzimidazole moiety of Co(II)Cbl from the active site than to remove the water molecule from Co(II)Cbi+. Thus, the hydrophilic interactions around and above the corrin ring are more critical to form 4c Co(II)Cbl than 4c Co(II)Cbi+. Finally, when ATP analogues were used as cosubstrate, only “unactivated” five-coordinate (5c) Co(II)Cbl was observed, disclosing an unexpectedly large role of the ATP-induced active-site conformational changes with respect to the formation of 4c Co(II)Cbl. Collectively, our results indicate that the level of control exerted by LrPduO over the timing for the formation of the 4c Co2+corrinoid intermediates is even more exquisite than previously anticipated.

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 (FeII(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.

Glutathionylcobalamin (GSCbl) is a unique, biologically relevant cobalamin featuring an axial Co–S bond that distinguishes it from the enzymatically active forms of vitamin B12, which possess axial Co–C bonds. GSCbl has been proposed to serve as an intermediate in cobalamin processing and, more recently, as a therapeutic for neurological disorders associated with oxidative stress. In this study, GSCbl and its close relative cysteinylcobalamin (CysCbl) were investigated using electronic absorption, circular dichroism, magnetic circular dichroism, and resonance Raman spectroscopies. The spectroscopic data were analyzed in the framework of density functional theory (DFT) and time-dependent DFT computations to generate experimentally validated electronic structure descriptions. Although the change in the upper axial ligand from an alkyl to a thiol group represents a major perturbation in terms of the size, basicity, and polarizability of the coordinating atom, our spectroscopic and computational results reveal striking similarities in electronic structure between methylcobalamin (MeCbl) and GSCbl, especially with regard to the σ donation from the alkyl/thiol ligand and the extent of mixing between the cobalt 3d and the ligand frontier orbitals. A detailed comparison of Co–C and Co–S bonding in MeCbl and GSCbl, respectively, is presented, and the implications of our results with respect to the proposed biological roles of GSCbl are discussed.

The electronic structures of a series of high-spin Ni(II)-thiolate complexes of the form [PhTttBu]Ni(SR) (R = CPh3, 2; C6F5, 3; C6H5, 4; PhTttBu = phenyltris((tert-butylthio)methyl)borate) have been characterized using a combined spectroscopic and computational approach. Resonance Raman (rR) spectroscopic data reveal that the νNi−SR vibrational feature occurs between 404 and 436 cm−1 in these species. The corresponding rR excitation profiles display a striking de-enhancement behavior because of interference effects involving energetically proximate electronic excited states. These data were analyzed in the framework of time-dependent Heller theory to obtain quantitative insight into excited state nuclear distortions. The electronic absorption and magnetic circular dichroism spectra of 2−4 are characterized by numerous charge transfer (CT) transitions. The dominant absorption feature, which occurs at ∼18,000 cm−1 in all three complexes, is assigned as a thiolate-to-Ni CT transition involving molecular orbitals that are of π-symmetry with respect to the Ni−S bond, reminiscent of the characteristic absorption feature of blue copper proteins. Density functional theory computational data provide molecular orbital descriptions for 2−4 and allow for detailed assignments of the key spectral features. A comparison of the results obtained in this study to those reported for similar Ni-thiolate species reveals that the supporting ligand plays a secondary role in determining the spectroscopic properties, as the electronic structure is primarily determined by the metal−thiolate bonding interaction.

In this study, a combined spectroscopic and computational approach has been employed to generate a detailed description of the electronic structure of a binuclear side-on disulfido (NiII)2 complex, [{(PhTttBu)Ni}2(μ-η2:η2-S2)] (1, where PhTttBu = phenyltris[(tert-butylthio)methyl]borate). The disulfido-to-NiII charge-transfer transitions that dominate the electronic absorption spectrum have been assigned on the basis of time-dependent density functional theory (DFT) calculations. Resonance Raman spectroscopic studies of 1 have revealed that the S−S stretching mode occurs at 446 cm−1, indicating that the S−S bond is weaker in 1 than in the analogous μ-η2:η2-S2 dicopper species. DFT computational data indicate that the steric bulk of PhTttBu stabilize the side-on core enough to prevent its conversion to the electronically preferred bis(μ-sulfido) (NiIII)2 structure. Hence, 1 provides an interesting contrast to its O2-derived analogue, [{(PhTttBu)Ni}2(μ-O)2], which was shown previously to assume a bis(μ-oxo) (NiIII)2 “diamond core”. By a comparison of 1 to analogous disulfidodicopper and peroxodinickel species, new insight has been obtained into the roles that the metal centers, bridging ligands, and supporting ligands play in determining the core structures and electronic properties of these dimers.

A powerful means of enhancing our understanding of the structures and functions of enzymes that contain nickel−sulfur bonds, such as Ni superoxide dismutase, acetyl-coenzyme A synthase/carbon monoxide dehydrogenase, [NiFe] hydrogenase, and methyl-CoM reductase, involves the investigation of model compounds with similar structural and/or electronic properties. In this study, we have characterized a trans-μ-1,2-disulfido-bridged dinickel(II) species, [{(tmc)Ni}2(S2)]2+ (1, tmc = 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane) by using electronic absorption, magnetic circular dichroism (MCD), and resonance Raman (rR) spectroscopic techniques, as well as density functional theory (DFT) and time-dependent DFT computational methods. Our computational results, validated on the basis of the experimental MCD data and previously reported 1H NMR spectra, reveal that 1 is best described as containing two antiferromagnetically coupled high-spin NiII centers. A normal coordinate analysis of the rR vibrational data was performed to quantify the core bond strengths, yielding force constants of kNi−S = 2.69 mdyn/Å and kS−S = 2.40 mdyn/Å. These values provide a useful basis for a comparison of metal−S/O bonding in 1 and related Ni2(O2), Cu2(O2), and Cu2(S2) dimers. In both the disulfido and the peroxo species, the lower effective nuclear charge of NiII as compared to CuII results in a decreased covalency, and thus relatively weaker metal−S/O bonding interactions in the Ni2 dimers than in the Cu2 complexes.

Cysteine dioxygenase (CDO) is a mononuclear non-heme Fe-dependent dioxygenase that catalyzes the initial step of oxidative cysteine catabolism. Its active site consists of an Fe(II) ion ligated by three histidine residues from the protein, an interesting variation on the more common 2-His-1-carboxylate motif found in many other non-heme Fe(II)-dependent enzymes. Multiple structural and kinetic studies of CDO have been carried out recently, resulting in a variety of proposed catalytic mechanisms; however, many open questions remain regarding the structure/function relationships of this vital enzyme. In this study, resting and substrate-bound forms of CDO in the Fe(II) and Fe(III) states, both of which are proposed to have important roles in this enzyme’s catalytic mechanism, were characterized by utilizing various spectroscopic methods. The nature of the substrate/active site interactions was also explored using the cysteine analogue selenocysteine (Sec). Our electronic absorption, magnetic circular dichroism, and resonance Raman data exhibit features characteristic of direct S (or Se) ligation to both the high-spin Fe(II) and Fe(III) active site ions. The resulting Cys- (or Sec-) bound species were modeled and further characterized using density functional theory computations to generate experimentally validated geometric and electronic structure descriptions. Collectively, our results yield a more complete description of several catalytically relevant species and provide support for a reaction mechanism similar to that established for many structurally related 2-His-1-carboxylate Fe(II)-dependent dioxygenases.

Nickel-dependent superoxide dismutase (NiSOD) is a member of a class of metalloenzymes that protect aerobic organisms from the damaging superoxide radical (O2·−). A distinctive and fascinating feature of NiSOD is the presence of active-site nickel–thiolate interactions involving the Cys2 and Cys6 residues. Mutation of one or both Cys residues to Ser prevents catalysis of O2·−, demonstrating that both residues are necessary to support proper enzymatic activity (Ryan et al., J Biol Inorg Chem, 2010). In this study, we have employed a combined spectroscopic and computational approach to characterize three Cys-to-Ser (Cys → Ser) mutants (C2S, C6S, and C2S/C6S NiSOD). Similar electronic absorption and magnetic circular dichroism spectra are observed for these mutants, indicating that they possess nearly identical active-site geometric and electronic structures. These spectroscopic data also reveal that the Ni2+ ion in each mutant adopts a high-spin (S = 1) configuration, characteristic of a five- or six-coordinate ligand environment, as opposed to the low-spin (S = 0) configuration observed for the four-coordinate Ni2+ center in the native enzyme. An analysis of the electronic absorption and magnetic circular dichroism data within the framework of density functional theory computations performed on a series of five- and six-coordinate C2S/C6S NiSOD models reveals that the active site of each Cys → Ser mutant possesses an essentially six-coordinate Ni2+ center with a rather weak axial bonding interaction. Factors contributing to the lack of catalytic activity displayed by the Cys → Ser NiSOD mutants are explored.

Heme oxygenases (HOs) are monooxygenases that catalyze the first step in heme degradation, converting heme to biliverdin with concomitant release of Fe(II) and CO from the porphyrin macrocycle. Two heme oxygenase isoforms, HO-1 and HO-2, exist that differ in several ways, including a complete lack of Cys residues in HO-1 and the presence of three Cys residues as part of heme-regulatory motifs (HRMs) in HO-2. HRMs in other heme proteins are thought to directly bind heme, or to otherwise regulate protein stability or activity; however, it is not currently known how the HRMs exert these effects on HO-2 function. To better understand the properties of this vital enzyme and to elucidate possible roles of its HRMs, various forms of HO-2 possessing distinct alterations to the HRMs were prepared. In this study, variants with Cys265 in a thiol form are compared with those with this residue in an oxidized (part of a disulfide bond or existing as a sulfenate moiety) form. Absorption and magnetic circular dichroism spectroscopic data of these HO-2 variants clearly demonstrate that a new low-spin Fe(III) heme species characteristic of thiolate ligation is formed when Cys265 is reduced. Additionally, absorption, magnetic circular dichroism, and resonance Raman data collected at different temperatures reveal an intriguing temperature dependence of the iron spin state in the heme–HO-2 complex. These findings are consistent with the presence of a hydrogen-bonding network at the heme’s distal side within the active site of HO-2 with potentially significant differences from that observed in HO-1.

Vitamin B12 and its biologically active derivatives, 5′-deoxyadenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl), have long fascinated chemists with their elaborate structures and unusual reactivities in enzymatic systems. Due to their large size and complex electronic structures, these cofactors have posed a major challenge for computational chemists. Yet, recent insights gained from kinetic, spectroscopic, and X-ray crystallographic studies, have established an excellent foundation for the successful completion of density functional theory studies aimed at elucidating the electronic structures of the isolated cofactors and the catalytic cycles of B12-dependent enzymes. This review summarizes important information obtained from experimentally validated computational studies of: (i) the free AdoCbl and MeCbl cofactors in their Co3+, Co2+, and Co1+ oxidation states; (ii) the mechanism by which enzymes involved in the biosynthesis of AdoCbl accomplish the thermodynamically challenging Co2+ → Co1+ reduction; (iii) the strategies employed by AdoCbl-dependent enzymes to achieve a trillion-fold rate acceleration for the homolytic cleavage of the cofactor's CoC(Ado) bond; and (iv) the means by which MeCbl-dependent methyltransferases accelerate the rate of methyl transfer via heterolytic CoC(Me) bond cleavage by as much as six orders of magnitude and reactivate the accidentally oxidized form of the cofactor.

The one-electron-reduced form of vitamin B12, cob(II)alamin (Co2+Cbl), is found in several essential human enzymes, including the cobalamin-dependent methionine synthase (MetH). In this work, experimentally validated electronic structure descriptions for two “base-off” Co2+Cbl species have been generated using a combined spectroscopic and computational approach, so as to obtain definitive clues as to how these and related enzymes catalyze the thermodynamically challenging reduction of Co2+Cbl to cob(I)alamin (Co1+Cbl). Specifically, electron paramagnetic resonance (EPR), electronic absorption (Abs), and magnetic circular dichroism (MCD) spectroscopic techniques have been employed as complementary tools to characterize the two distinct forms of base-off Co2+Cbl that can be trapped in the H759G variant of MetH, one containing a five-coordinate and the other containing a four-coordinate, square-planar Co2+ center. Accurate spin Hamiltonian parameters for these low-spin Co2+ centers have been determined by collecting EPR data using both X- and Q-band microwave frequencies, and Abs and MCD spectroscopic techniques have been employed to probe the corrin-centered π → π* and Co-based d → d excitations, respectively. By using these spectroscopic data to evaluate electronic structure calculations, we found that density functional theory provides a reasonable electronic structure description for the five-coordinate form of base-off Co2+Cbl. However, it was necessary to resort to a multireference ab initio treatment to generate a more realistic description of the electronic structure of the four-coordinate form. Consistent with this finding, our computational data indicate that, in the five-coordinate Co2+Cbl species, the unpaired spin density is primarily localized in the Co 3dz2-based molecular orbital, as expected, whereas in the four-coordinate form, extensive Co 3d orbital mixing, configuration interaction, and spin−orbit coupling cause the unpaired electron to delocalize over several Co 3d orbitals. These results provide important clues to the mechanism of enzymatic Co2+Cbl → Co1+Cbl reduction.

In this study, the mechanism by which second-sphere residues modulate the structural and electronic properties of substrate-analogue complexes of the Fe-dependent superoxide dismutase (FeSOD) has been explored. Both spectroscopic and computational methods were used to investigate the azide (N3−) adducts of Fe3+SOD (N3−Fe3+SOD) and its Q69E mutant, as well as Fe3+-substituted MnSOD (N3−Fe3+(Mn)SOD) and its Y34F mutant. Electronic absorption, circular dichroism, and magnetic circular dichroism spectroscopic data reveal that the energy of the dominant N3− → Fe3+ ligand-to-metal charge transfer (LMCT) transition decreases in the order N3−Fe3+(Mn)SOD > N3−Fe3+SOD > Q69E N3−Fe3+SOD. Intriguingly, the LMCT transition energies correlate almost linearly with the Fe3+/2+ reduction potentials of the corresponding Fe3+-bound SOD species in the absence of azide, which span a range of ∼1 V (see the preceding paper). To explore the origin of this correlation, combined quantum mechanics/molecular mechanics (QM/MM) geometry optimizations were performed on complete enzyme models. The INDO/S−CI computed electronic transition energies satisfactorily reproduce the experimental trend in LMCT transition energies, indicating that the QM/MM optimized active-site models are reasonable. Density functional theory calculations on these experimentally validated active-site models reveal that the differences in spectral and electronic properties among the four N3− adducts arise primarily from differences in the hydrogen-bond network involving the conserved second-sphere Gln (mutated to Glu in Q69E FeSOD) and the solvent ligand. The implications of our findings with respect to the mechanism by which the second-coordination sphere modulates substrate−analogue binding as well as the catalytic properties of FeSOD are discussed.

In Fe- and Mn-dependent superoxide dismutases (SODs), second-sphere residues have been implicated in precisely tuning the metal ion reduction potential to maximize catalytic activity (Vance, C. K.; Miller, A.-F. J. Am. Chem. Soc. 1998, 120, 461–467). In the present study, spectroscopic and computational methods were used to characterize three distinct Fe-bound SOD species that possess different second-coordination spheres and, consequently, Fe3+/2+reduction potentials that vary by ∼1 V, namely, FeSOD, Fe-substituted MnSOD (Fe(Mn)SOD), and the Q69E FeSOD mutant. Despite having markedly different metal ion reduction potentials, FeSOD, Fe(Mn)SOD, and Q69E FeSOD exhibit virtually identical electronic absorption, circular dichroism, and magnetic circular dichroism (MCD) spectra in both their oxidized and reduced states. Likewise, variable-temperature, variable-field MCD data obtained for the oxidized and reduced species do not reveal any significant electronic, and thus geometric, variations within the Fe ligand environment. To gain insight into the mechanism of metal ion redox tuning, complete enzyme models for the oxidized and reduced states of all three Fe-bound SOD species were generated using combined quantum mechanics/molecular mechanics (QM/MM) geometry optimizations. Consistent with our spectroscopic data, density functional theory computations performed on the corresponding active-site models predict that the three SOD species share similar active-site electronic structures in both their oxidized and reduced states. By using the QM/MM-optimized active-site models in conjunction with the conductor-like screening model to calculate the proton-coupled Fe3+/2+ reduction potentials, we found that different hydrogen-bonding interactions with the conserved second-sphere Gln (changed to Glu in Q69E FeSOD) greatly perturb the pK of the Fe-bound solvent ligand and, thus, drastically affect the proton-coupled metal ion reduction potential.

We have synthesized and characterized, using X-ray crystallographic, spectroscopic, and computational techniques, a six-coordinate diazide Fe3+ complex, LFe(N3)2 (where L is the tetradentate ligand 7-diisopropyl-1,4,7-triazacyclononane-1-acetic acid), that serves as a model of the azide adducts of Fe3+ superoxide dismutase (Fe3+SOD). While previous spectroscopic studies revealed that two distinct azide-bound Fe3+SOD species can be obtained at cryogenic temperatures depending on protein and azide concentrations, the number of azide ligands coordinated to the Fe3+ ion in each species has been the subject of some controversy. In the case of LFe(N3)2, the electronic absorption and magnetic circular dichroism spectra are dominated by two broad features centered at 21 500 cm−1 (ϵ ≈ 4000 M−1 cm−1) and ∼30 300 cm−1 (ϵ ≈ 7400 M−1 cm−1) attributed to N3− → Fe3+ charge transfer (CT) transitions. A normal coordinate analysis of resonance Raman (RR) data obtained for LFe(N3)2 indicates that the vibrational features at 363 and 403 cm−1 correspond to the Fe−N3 stretching modes (νFe−N3) associated with the two different azide ligands and yields Fe−N3 force constants of 1.170 and 1.275 mdyne/Å, respectively. RR excitation profile data obtained with laser excitation between 16 000 and 22 000 cm−1 reveal that the νFe−N3 modes at 363 and 403 cm−1 are preferentially enhanced upon excitation in resonance with the N3− → Fe3+ CT transitions at lower and higher energies, respectively. Consistent with this result, density functional theory electronic structure calculations predict a larger stabilization of the molecular orbitals of the more strongly bound azide due to increased σ-symmetry orbital overlap with the Fe 3d orbitals, thus yielding higher N3− → Fe3+ CT transition energies. Comparison of our data obtained for LFe(N3)2 with those reported previously for the two azide adducts of Fe3+SOD provides compelling evidence that a single azide is coordinated to the Fe3+ center in each protein species.

The PduO-type ATP:corrinoid adenosyltransferase from Lactobacillus reuteri (LrPduO) catalyzes the formation of the essential Co−C bond of adenosylcobalamin (coenzyme B12) by transferring the adenosyl group from cosubstrate ATP to a transient Co1+corrinoid species generated in the enzyme active site. While PduO-type enzymes have previously been believed to be capable of adenosylating only Co1+cobalamin (Co1+Cbl−), our kinetic data obtained in this study provide in vitro evidence that LrPduO can in fact also utilize the incomplete corrinoid Co1+cobinamide (Co1+Cbi) as an alternative substrate. To explore the mechanism by which LrPduO overcomes the thermodynamically challenging reduction of its Co2+corrinoid substrates, we have examined how the enzyme active site alters the geometric and electronic properties of Co2+Cbl and Co2+Cbi+ by using electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopic techniques. Our data reveal that upon binding to LrPduO that was preincubated with ATP, both Co2+corrinoids undergo a partial (∼40−50%) conversion to distinct paramagnetic Co2+ species. The spectroscopic signatures of these species are consistent with essentially four-coordinate, square-planar Co2+ complexes, based on a comparison with the results obtained in our previous studies of related enzymes. Consequently, it appears that the general strategy employed by adenosyltransferases for effecting Co2+ → Co1+ reduction involves the formation of an “activated” Co2+corrinoid intermediate that lacks any significant axial bonding interactions, to stabilize the redox-active, Co 3dz2-based molecular orbital.