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Author ORCID Identifier
Open Access Dissertation
Doctor of Philosophy (PhD)
Year Degree Awarded
Month Degree Awarded
Michael J. Maroney
Biochemistry | Structural Biology
Ni(II) is one of the important cofactors involved in various enzyme functions. For organisms utilizing Ni(II), a regulation system is required to maintain Ni(II) homeostasis and prevent toxicity. The focus of this dissertation is on investigating the relationship between the Ni(II) site structure and the function of proteins, a Ni(II) sensor and a Ni(II) enzyme.
RcnR, a Ni(II)/Co(II) sensor in E. coli, controls the expression of the Ni(II)/Co(II) exporter proteins, RcnAB. Due to the lack of structural information, the mechanism of metal induced allosteric regulation and metal selection is not fully elucidated. Results presented here show that binding of cognate metals induces N-terminus ordering to the metal binding pocket, decrease helix 1 flexibility, and triggers conformational changes that restrict DNA interactions with the positively charged residues Arg14 and Lys17, leading to rcnAB expression. The critical role of His64 in tight metal binding and allostery, and the role of the loop1 region in cognate metal differentiation were also revealed. Results obtained from the loop1 region ligand deletion variant further point out that ligand selection rather than coordination number/geometry of the metal center is the key in allosteric regulation.
Ni-dependent superoxide dismutase (NiSOD), involved in the detoxification system of the superoxide, utilizes a redox-active Ni(II) alternating between oxidized and reduced forms to catalyze the disproportionation of superoxide, and this oscillation of reduced/oxidized metal center requires a unique protein ligation environment, a mixed amine/amide ligation, and the ligation of the two Cys residues. It was suggested that the mix amine/amide N-donor are involved in preventing the Cys residues from oxidation. A variant of additional Ala residue insertion to the N-terminus (Ala0-NiSOD) was made to test this hypothesis. This variant alters the mix amine/amide ligation environment to bis-amide ligation environment, and becomes air sensitive. The mass spectral analysis shows a mixture of several oxidation products in the air-exposed Ala0-NiSOD, while air-exposed WT-NiSOD remains intact. The oxidation products were found located at two Cys residues. Combined with the result from spectroscopy and computational data, the requirement of mix amine/amide ligation environment for preventing Cys oxidation is revealed.
Huang, Hsin-Ting, "Ni Site Structure and Function in Biological Sensing and Enzyme Activity" (2019). Doctoral Dissertations. 1552.