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Title: Structure and function of human Mn(Fe)SOD containing iron in the active site
Authors: Sultana, Paul Michael (2020)
Keywords: Enzymes
Superoxide dismutase
Issue Date: 2020
Citation: Sultana, P. M. (2020). Structure and function of human Mn(Fe)SOD containing iron in the active site (Master’s dissertation).
Abstract: MnSOD is a mitochondrial protein that protects the cell from copious amounts of superoxide radicals that are generated by the electron transport chain in the healthy individual. It also plays an important role in free radical detoxification during pathologic state such as chronic inflammation and infection. As a metalloenzyme, MnSOD is only functional with manganese as the active site cofactor. Under certain cellular conditions, iron may replace manganese in the active site and the protein may serve as an iron reservoir in the mitochondria. Previous research carried out on the sub-cloned C. elegans MnSOD mutant, hMn(Fe)SOD [Q142H] has shown that a substitution of an active site residue was sufficient to change the metal specificity of the protein (Hunter et al., 2018). To the same end, the mutant hMnSOD Q143H was expressed in competent RosettaTM cells that were grown in minimal media supplemented with controlled quantities of selected metal salts. Expressed protein, induced by IPTG, were purified by MCAC and quantified using BCA and direct A280 measurements. CD structural probing revealed conservation of secondary structure integrity with increased stability in iron substituted mutants. The structure of hMnSOD Q143H, as refined through crystallographic diffraction data, was also modelled with electron density maps. Enzyme specific metal activity was reduced when compared to WT hMnSOD with activity for hMnSOD Q143H measured at 144 ±19 U mg−1 and 210 ±45 U mg−1 whereas for substituted mutants hMn(Fe)SOD Q143H activity was measured at 688 ±62 U mg−1. hMnSOD wild-type exhibited an activity of 9481 ±411 U mg−1. Metal occupancy for iron-substituted mutants was at 66% with Mn content for hMnSOD Q143H was measured at 70% and 99%, respectively. Therefore, hMn(Fe)SOD Q143H exhibited a shift in iron-supported activity but at the detriment of a 93% loss in catalytic function.
Appears in Collections:Dissertations - FacM&S - 2020
Dissertations - FacM&SPB - 2020

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