This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Although Mn plays a vital role for normal development and body functions excessive manganese exposure produces symptoms resembling those of idiopathic Parkinson?s disease. The mechanism by which Mn causes the neurodegenerative damage is not clearly understood. The key target protein for Mn induced neurotoxicity is aconitase. Its activity decreases dramatically in brain tissues of animals and in cell cultures subjected to elevated Mn levels. In vitro experiments demonstrated that aconitase is inhibited by Mn but did not providing much information about Mn binding site. The goal of this proposal is to detect the Mn redox state and coordination environment in the Mn-inhibited aconitase by XANES and EXAFS at Mn K-edge. Simultaneously, structural and redox effects at the Fe-S cluster of the Mn-inhibited aconitase will be analyzed by Fe K-edge XANES and EXAFS. We hypothesized that Mn replaces the fourth, weakly bound and lacking the protein ligand Fe atom in the 4Fe-4S cluster of aconitase. Additionally to its enzymatic activity in the conversion of citrate to isocitrate, cytosolic aconitase responds to and regulates Fe levels in cells. However, it remains unknown how inhibition by Mn can affect the ability of aconitase to convert to iron-responsive protein (IRP, aconitase becomes IRP when it loses the Fe-S cluster). Mn treated cells demonstrated increased levels of IRPs. Information about Mn binding site and changes in Fe-S cluster structure will help us to understand the influence of Mn on aconitase to IRP conversion. Overall, the results of the proposed experiments will help us to understand the damaging effects of cells exposure to elevated Mn concentration at the molecular level and its possible effects on the regulation of Fe uptake and storage.
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