The focus of this thesis is an investigation of the rat enzyme cysteine dioxygenase (CDO). CDO is a non-haem mononuclear iron(II) enzyme that incorporates molecular oxygen into cysteine to form cysteine sulfinic acid. CDO has been implicated with a range of neurological disorders as well as many forms of cancer. Chemically, CDO has two fascinating and unique features. Firstly, it has a neutral three-histidine coordination environment; the more common environment found in most related enzymes is the negatively charged two-histidine carboxylate environment. Secondly, a post- translational thioether modification occurs between two secondary coordination sphere residues in the active site, C93 and Y157.
Although CDO has been investigated for many years, the mechanism is still not fully elucidated. The aim of this thesis is to develop the current understanding of the mechanism of cysteine dioxygenase by probing both multiple-turnover and single-turnover kinetics.
Multiple-turnover investigations of the secondary and tertiary coordination sphere have elucidated the roles of the crosslink, C93, Y157 and H155. The crosslink increases the rate of reaction. Contrary to current belief, the rate is not increased by the presence of the crosslink itself. Instead, the rate is increased by the act of removing the thiolate of C93 from the active site. This was shown by both the theoretically derived 100 % crosslinked enzyme having almost identical pH independent parameters as the C93G variant. However, removal of Y157 with Y157F did show a substantial variation in reactivity. Further investigations showed that Y157 works in tandem with H155 to protect the active site from secondary cysteine attack. Substrate binding studies support this as the Mössbauer spectra showed variations in the five and six-coordinate cysteine bound iron complexes when Y157 was modified and variations in just the six-coordinate complex when H155 was mutated.
Single turnover studies utilising stopped-flow spectrophotometry showed that two intermediates are formed in the visible region at pH 9.1. Using time-dependent chemical-quench with mass spectrometry, the intermediates were correlated to the formation of cysteine sulfinic acid and cysteine sulfonic acid. This is the first evidence of a single domain enzyme acting as a trioxygenase. Altogether, a detailed mechanism of CDO has been developed.
