Access Type

Open Access Dissertation

Date of Award

January 2012

Degree Type


Degree Name




First Advisor

Jeremy J. Kodanko


Oxidative inactivation of proteins (carbonic anhydrase–I, trypsin, chymotrypsin and 20S proteasome) by non–heme iron complexes, and glutathionylation of non–heme cobalt complexes mimicking the N5 coordination environment like that of biologically important cofactor cobalamin or B12 (Cbl) are reported. Different non–heme ligand sets or inhibitors were used to inactivate different proteins. Carbonic anhydrase–I (CA–I) and 20S proteasome were inactivated by iron complexes in the presence of O2 and a reductant (DTT), consistent with a pathway involving the reductive activation of O2, whereas serine proteases trypsin and chymotrypsin were inactivated by ferryls (single turnover), and by an iron complex in presence of biologically relevant oxidant, H2O2 (catalytic conditions). Analysis of the CA–I inactivation products by SDS–PAGE, ESI–MS and LC/MS/MS confirmed that the protein is inactivated by oxidation of amino acid side chains (His, Trp and Met) rather than fragmentation of the protein backbone. On the other hand, amino acid analysis of serine proteases inactivation products confirmed that residues Cys, Tyr, and Trp were oxidized under single turnover condition while the residue tyrosine was oxidized selectively under catalytic conditions. Control experiments preclude the role of ROS, and supported the role of a metal-based oxidant responsible for protein inactivation in all the cases.

A low spin cobalt (III) cobalamin model complex derived from a polypyridyl pentadentate N5 non–heme ligand was synthesized and characterized fully by X–ray crystallography, UV–vis, IR, 1H–NMR and 13C–NMR spectroscopies, and mass spectrometry (HRMS). Kinetic and thermodynamic studies on the reaction of this cobalt complex along with another related congener with glutathione were performed in aqueous buffer to generate biomimetic species of glutathionylcobalamin, an important form of cobalamin found in nature. The reaction follows second order kinetic, with both the rate constants and the observed equilibrium constants smaller than the rate constants and equilibrium constant of the reaction of aquacobalamin and GSH to give glutathionylcobalamin. Glutathionylation showed significant pH dependence, where rates increased with pH. Taken together, these results suggest that glutathionylation is a general reaction for Co(III) complexes related to Cbl.

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