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UBC Theses and Dissertations

Investigation of cysteine and methionine oxidation using x-ray absorption spectroscopy Karunakaran, Anusha


Cysteine (Cys) and methionine (Met) are sulfur containing amino acids with various oxidation forms. Oxidation of Cys yields cysteinyl radicals that have been postulated as intermediates in several biological contexts including enzymatic catalysis, long-range electron transfer, peptide post-translational modification and cellular redox signaling. The challenges of detecting sulfur-based radicals with electron paramagnetic resonance (EPR) have led to the development of Sulfur K-edge X-ray absorption spectroscopy (S K-edge XAS) as a spectroscopic tool. The reactivity of sulfur-based radicals was studied in a Pseudomonas aeruginosa azurin protein system to probe the electronic structure of isolated cysteinyl radicals, which are characterized by S 3p ← 1s pre-edge transition. S K-edge XAS has shown to be a sensitive method in detecting these cysteinyl radicals in hydrophobic and hydrophilic protein environments. The pre-edge feature of the cysteinyl radicals in hydrophobic environments was lower in energy than their hydrophilic counterparts due to hydrogen bonding interactions. Additionally, S K-edge XAS was employed to study the redox photochemistry of Met and its oxidized forms methionine sulfoxide (MetSO) and methionine sulfone (MetSO₂). Met is easily photooxidized to MetSO and MetSO₂ in the presence of O₂. In the absence of O₂, photoirradiation leads to the one-electron-oxidized Met cation radical (MetS•⁺), suggesting an alternative mechanism for photooxidation of thioethers through direct oxidation. The photoirradiation of MetSO leads back to Met under both aerobic and anaerobic conditions while MetSO₂ is photochemically inert. These findings provide new insights into the formation of age-related cataracts. Finally, the metal-induced Met oxidation in amyloid-β (Aβ) peptide was investigated. Much of the research to date has focused on the redox chemistry of Cu²⁺ in Aβ peptide with inconsistent findings with regards to the role of Met₃₅ and the oxidation state of the Met₃₅. Findings reported here indicate that in the presence of Cu²⁺ alone, Met₃₅ was oxidized to MetSO, but surprisingly Fe³⁺ failed to oxidize the Met. These differences in the oxidation behaviour lead to the investigation of the metal binding site in Aβ. Fe³⁺ found to be in a six-coordinate environment with oxygen-rich ligands while Cu²⁺ is in a five-coordinate environment with histidine-rich ligands.

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