Thomas C. Brunold

Spectroscopic and computational characterization of substrate-bound mouse cysteine dioxygenase: nature of the ferrous and ferric cysteine adducts and mechanistic implications.

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 enzymes 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 superoxide dismutase: structural and functional roles of Cys2 and Cys6

Nickel superoxide dismutase (NiSOD) is unique among the family of superoxide dismutase enzymes in that it coordinates Cys residues (Cys2 and Cys6) to the redox-active metal center and exhibits a hexameric quaternary structure. To assess the role of the Cys residues with respect to the activity of NiSOD, mutations of Cys2 and Cys6 to Ser (C2S-NiSOD, C6S-NiSOD, and C2S/C6S-NiSOD) were carried out. The resulting mutants do not catalyze the disproportionation of superoxide, but retain the hexameric structure found for wild-type NiSOD and bind Ni(II) ions in a 1:1 stoichiometry. X-ray absorption spectroscopic studies of the Cys mutants revealed that the nickel active-site structure for each mutant resembles that of C2S/C6S-NiSOD and demonstrate that mutation of either Cys2 or Cys6 inhibits coordination of the remaining Cys residue. Mutation of one or both Cys residue(s) in NiSOD induces the conversion of the low-spin Ni(II) site in the native enzyme to a high-spin Ni(II) center in the mutants. This result indicates that coordination of both Cys residues is required to generate the native low-spin configurations and maintain catalytic activity. Analysis of the quaternary structure of the Cys mutants by differential scanning calorimetry, mass spectrometry, and size-exclusion chromatography revealed that the Cys ligands, particularly Cys2, are also important for stabilizing the hexameric quaternary structure of the native enzyme.

Absorption and luminescence spectroscopy of Zn2SiO4 willemite crystals doped with Co2

The polarized absorption spectra of Zn2SiO4 willemite crystals doped with Co2+ consist of three band systems in the near-infrared and visible spectra region centered at 3800, 7000, and 17000 cm−1, respectively. In the tetrahedral approximation they are assigned to the d → d transitions 4A2 → 4T2, 4T1(4F), and 4T1(4P), respectively. The crystal field parameter 10Dq is 4000 cm−1, and the Racah parameters B and C are 740 and 3330 cm−1, respectively. From the fine structure in the origin region of the 4A2 → 4T2 absorption band it follows that the 4A2 ground-state splittings of Co2+ occupying the two crystallographically inequivalent Zn2+ sites of C1 symmetry are 13 and 15 cm−1, respectively. Only a weak emission originating at 15335 cm−1 is observed upon 4A2 → 4T1(4P) photoexcitation. It is assigned to the 2E → 4A2 sharp-line luminescence of Co2+ located in a minority site. No luminescence is observed from regularly incorporated Co2+ ions.

Hydrogen Sulfide Oxidation by Myoglobin

Enzymes in the sulfur network generate the signaling molecule, hydrogen sulfide (H2S), from the amino acids cysteine and homocysteine. Since it is toxic at elevated concentrations, cells are equipped to clear H2S. A canonical sulfide oxidation pathway operates in mitochondria, converting H2S to thiosulfate and sulfate. We have recently discovered the ability of ferric hemoglobin to oxidize sulfide to thiosulfate and iron-bound hydropolysulfides. In this study, we report that myoglobin exhibits a similar capacity for sulfide oxidation. We have trapped and characterized iron-bound sulfur intermediates using cryo-mass spectrometry and X-ray absorption spectroscopy. Further support for the postulated intermediates in the chemically challenging conversion of H2S to thiosulfate and iron-bound catenated sulfur products is provided by EPR and resonance Raman spectroscopy in addition to density functional theory computational results. We speculate that the unusual sensitivity of skeletal muscle cytochrome c oxidase to sulfide poisoning in ethylmalonic encephalopathy, resulting from the deficiency in a mitochondrial sulfide oxidation enzyme, might be due to the concentration of H2S by myoglobin in this tissue.

Spectroscopic and Computational Investigation of Second-Sphere Contributions to Redox Tuning in Escherichia coli Iron Superoxide Dismutase

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, Fe(3+/2+)reduction potentials that vary by approximately 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 Fe(3+/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 p K of the Fe-bound solvent ligand and, thus, drastically affect the proton-coupled metal ion reduction potential.

Absorption and luminescence spectroscopy of ferrate (VI) doped into crystals of K2MO4(M = S, Se, Cr, Mo)

The absorption spectra of the ferrate (VI) ion (FeO2-4) in K2MO4 (M = S, Se, Cr, Mo) host lattices consist of a series of relatively weak bands at low energy, which can be assigned to transitions within the partially filled 3d shell and some intense bands at higher energy, which are assigned to ligand-to-metal charge transfer transitions (LMCT). In the near-infrared (NIR) region sharp lines are observed belonging to the spin-forbidden spin-flip transitions 3A2 → 1E and 3A2 → 1A1. The lowest excited state is the 1E state, serving as initial state for 1E → 3A2 sharp-line luminescence at around 6200 cm-1. Another luminescence is observed centered at 9000 cm-1, which is assigned to the 3T2 → 3A2 transition. It is rather broad and three orders of magnitude weaker than the 1E luminescence at 30K as a result of efficient non-radiative relaxation processes to the 1E state. The temperature dependence of the total intensity and the lifetime of the 1E → 3A2 luminescence is understood within a complex scheme of radiative and non-radiative processes.

Spectroscopic and computational insights into the geometric and electronic properties of the A-cluster of acetyl-coenzyme A synthase

For the last two decades, the bifunctional enzyme acetyl-coenzyme A synthase/carbon monoxide dehydrogenase (ACS/CODH) from Moorella thermoacetica has been the subject of considerable research aimed at elucidating the geometric and electronic properties of the A-cluster, which serves as the active site for ACS catalysis. While the recent success in obtaining high-resolution X-ray structures of this enzyme solved many of the mysteries regarding the number, identities, and coordination environments of the metal centers of the A-cluster, fundamental questions concerning the catalytic mechanism of this highly elaborate polynuclear active site have yet to be answered. This Commentary summarizes relevant information obtained from spectroscopic and computational studies on the oxidized, reduced, and CO-bound forms of the A-cluster and highlights some of the key issues regarding the electronic properties and reactivity of this cluster that need to be addressed in future studies.

Spectroscopic and Computational Studies of Glutathionylcobalamin: Nature of Co–S Bonding and Comparison to Co–C Bonding in Coenzyme B12

Glutathionylcobalamin (GSCbl) is a unique, biologically relevant cobalamin featuring an axial Co-S bond that distinguishes it from the enzymatically active forms of vitamin B(12), 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.

Excited state properties of ferrate(VI) doped crystals of K2SO4 and K2CrO4

Abstract The 15 K polarized single-crystal absorption spectra of Fe6+ doped K2SO4 consist of two sharp bands below 10 000 cm−1 assigned to the spin-flip transitions 3 A 2 → 1 E, 1 A 1 and a series of broad bands in the near-infrared (NIR) and visible region assigned to 3 A 2 → 3 T 2 , 3 T 1 d → d transitions and O → Fe charge transfer transitions. From the excited state absorption (ESA) spectrum we derive that the 1T2 state lies 19 300 cm−1 above the 3A2 ground state. This allows a clear assignment of all the ligand field states below 33 000 cm−1, which is in good agreement with an angular overlap model (AOM) calculation. Differences in the temperature dependence of the 1 E → 3 A 2 luminescence intensity upon broadband and 3 A 2 → 1 a 1 excitation are shown to be due to direct nonradiative relaxation processes from 3T2 to the 3A2 ground state.

Spectroscopic and Computational Characterization of the NO Adduct of Substrate-Bound Fe(II) Cysteine Dioxygenase: Insights into the Mechanism of O2 Activation

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.