Chemistry ETDs

Author

Chao Dong

Publication Date

6-23-2015

Abstract

In this dissertation spectroscopic methods such as electronic absorption (EAS), resonance Raman (rRaman), electron paramagnetic resonance (EPR), and MCD spectroscopies have been used in combination with DFT calculations for the interpretation of experimental results. These methods have been used to investigate the molybdenum coordination environment in enzymes and model complexes. Xanthine oxidase (XO), one of the canonical molybdenum enzyme families, hydroxylizes heterocyclic substrates by inserting an oxygen atom from molybdenum-activated water into a substrate C-H bond. This generates reducing equivalents that pass through two iron sulfur clusters and then to FAD, where oxidation of the FAD takes place. In this catalytic cycle there are ongoing arguments about whether a proton or hydride is transferred to the terminal sulfide during the reductive half reaction. Additionally, in the reductive half reaction the pyranotperin dithiolene cofactor (PDT) is proposed to modulate the transfer of electron out of molybdenum center to the proximal iron sulfur cluster. Due to the interference from the Fe-S and FAD chromophores, the observance of vibrational modes inherent to the PDT-Mo has not been achieved. Such evidence would support the involvement of the PDT as an integral component of the enzyme electron transfer regeneration pathway. Finally, no spectroscopic data has been obtained that directly probes amino acids around the active site of XO, although kinetic data are reported to show their role in substrate orientation and lowering activation energy of transition state. Here, the observation of an intense metal to ligand charge transfer (MLCT) band from the reduced molybdenum enzyme/product complex, combined to DFT calculations of the transition state, are presented and discussed. The results indicate that the PDT is intimately involved in one-electron transfer, and the Mo-O-Cproduct linkage in two-electron transfer to oxidize substrate and reduce the molybdenum center. Reduced XOthione-violapteirn complexes shift the MLCT band to the near-infrared region of the spectrum, and this removes the interference from FAD and FeS clusters, resulting in the appearance of high quality resonance Raman spectra that reveal the low frequency vibrational region. Heavy atom substitution on these new substrates affects both the electronic absorption and resonance Raman spectra. Resonance Raman spectra of variants that are anticipated to affect the PDT through hydrogen bonding show small perturbations on the Raman frequencies of PDT-Mo vibrational modes. EPR studies on aldehyde inhibited' XO, a Mo(V) species formed during XO catalysis, display second coordination sphere effects from amino acids that interact with the active site of XO. The new hybrid substrates, incuding analogs of the drug FYX051(C), display an intense MLCT band after the enzyme reacting with substrates. This potentially provides an opportunity to probe the effects of second coordination sphere perturbations in the substrate-binding pocket using resonance Raman spectroscopy. Arsenite Oxidase, a member of the dimethyl sulfoxide reductase (DMSOr) enzyme family, differs from other DMSOR family members. The Mo site in arsenite oxidase is not coordinated by any amino acid residues. This enzyme facilitates the transfer of an oxygen atom from water to the arsenite substrate. Electronic structure studies of dioxomolybdenum (VI) complexes with ene-1, 2-dithiolate ligands have been performed using a combination of electronic absorption and resonance Raman spectroscopies, and bonding calculations. The similar frequencies of molybdenum—sulfur vibrational modes and the similar origin of the lowest energy ligand to metal charge transfer band indicate these complexes possess the similar catalytic activities, and this is supported by their oxygen atom transfer (OAT) rates. The active site of sulfite oxidase (SO) catalyzes the oxidation of sulfite to sulfate through oxygen atom transfer for detoxifying the excessive sulfite or in the final step of degrading the sulfur containing amino acids. A conserved cysteine thiolate ligand is proposed to modulate the reduction potential of redox orbital by control the covalency of Mo-S(cysteine) bond through the Ooxo-Mo-S-C dihedral angle. An \u223c90\xba Ooxo-Mo-S-C dihedral angle results in poor Mo(dX2-Y2)-S covalency, while an \u223c0 or180\xba Ooxo-Mo-S-C dihedral angle leads to appreciable Mo(dX2-Y2)-S orbital mixing. Our spectroscopic and computational studies of molybdenum model complexes with chalcogen ligands not only confirms prior assignments of the S \u2192 Mo charge transfer occuring at 22500cm-1 in chicken sulfite oxidase (CSO) enzyme, but also supports a hypothesis that the Ooxo-Mo-Se/S-C varies between \u223c90\xba and 65\xba in selenocysteine substituted Human sulfite oxidase (HSO) enzyme. EPR and MCD spectra for small molecule model complexes that mimic this interaction if SO family enzymes is discussed.

Project Sponsors

National Institutes of Health

Language

English

Keywords

Molybdenum Enzymes, Model Complexes, Electronic Structure, Spectroscopic and Computational, Pyranopterin Dithiolene Cofactor

Document Type

Dissertation

Degree Name

Chemistry

Level of Degree

Doctoral

Department Name

Department of Chemistry and Chemical Biology

First Advisor

Kirk, Martin

First Committee Member (Chair)

Guo, Hua

Second Committee Member

Wang, Wei

Third Committee Member

Feng, Changjian

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