Roby Kurian

Date of Award


Level of Access Assigned by Author

Campus-Only Thesis

Degree Name

Doctor of Philosophy (PhD)




Francois G. Amar

Second Committee Member

Mitchell R. M. Bruce

Third Committee Member

Alice E. Bruce


Thiolate-disulfide exchange reactions are involved in cellular processes like signal transduction, redox regulation and oxidative stress. Metals like zinc(II) and gold(I) alter the kinetics of thiolate-disulfide exchange reactions. Theoretical studies can provide insight into mechanistic details and provide valuable guidance for experimental studies.

Metallothioneins (MT) are low molecular weight, cysteine rich proteins that help regulate the concentration of metal ions in cells, especially zinc(II). Previous in vitro studies established that MT releases zinc under conditions of oxidative stress. The mechanism of zinc release from MT by glutathione disulfide, the oxidized form of the most abundant physiological thiol, glutathione, is investigated computationally using the hybrid QM/MM method called ONIOM that permits a higher level of theory on a reaction center while maintaining an adequate structural description of a protein.

The chemical exchange between glutathione (GSH) and Et3PAuCl, an analogue for the rheumatoid arthritis drug ‘auranofin’, is experimentally investigated by using NMR, mass spectroscopies, and computational studies. Experiments establish that free glutathione undergoes continuous exchange with Et3PAu(SG) in solution. Exchange rates, calculated by line broadening and 2D NMR techniques, increase with increasing pH, correlating with an increase in concentration of thiolate, GS_. At physiological pH, 7.4, the exchange rate is ~16 s'1, suggesting that glutathione may have a role in transport of gold(I) drugs.

Thioredoxins (Trx) are enzymes with a characteristic CXYC active-site motif that catalyze the reduction of disulfide bonds in proteins. Zinc(II) is not found bound to thioredoxin, and unlike other transition metals, zinc does not inhibit thioredoxin activity. In addition, during oxidative stress, Zn(II) is released from MT and may migrate to the surfaces of proteins. The effect of zinc(II) binding on Trx is studied by using DFT and hybrid QM/MM methods. Reactions of glutathione disulfide with Zn(II)-bound Trx and metal-free Trx are compared. The computational results suggest that Zn(II) thiolate disulfide exchange may be favorable due to both kinetic and thermodynamic reasons.

Glutathione, and its oxidized form GSSG, are central to all these studies, A computational investigation of various conformations of GSH and calculations of the four microscopic pK* values of this biologically important molecule are presented.