Type of Document Dissertation Author Bitoun, Jacob Philip Author's Email Address email@example.com URN etd-07092009-144958 Title Biogenesis of Iron-Sulfur Clusters and Intracellular Iron Metabolism Degree Doctor of Philosophy (Ph.D.) Department Biochemistry (Biological Sciences) Advisory Committee
Advisor Name Title Huangen Ding Committee Chair John Battista Committee Member Joomyeong Kim Committee Member Yong-Hwan Lee Committee Member Megan Macnaughtan Dean's Representative Keywords
Date of Defense 2009-06-26 Availability unrestricted AbstractIron-sulfur ([Fe-S]) clusters represent one of natures most diverse and ubiquitous protein prosthetic groups. [Fe-S] proteins are integral for diverse biological processes. Reconstitution of apo-proteins can occur by exogenously adding excess iron and sulfide. However, due to the toxicity of iron and sulfide, it is most likely that proteins coordinate [Fe-S] cluster assembly in cells.
The first part of this dissertation addressed the iron donor for [Fe-S] cluster assembly. Recently, it has been shown that IscA is capable of binding iron with an association constant of 3.0 x 1019M-1. In addition, iron-loaded IscA is capable of donating iron to the proposed scaffold IscU for nascent [Fe-S] cluster assembly. We show that hIscA, the human homolog of E. coli IscA, functions as an iron chaperone for the assembly of [Fe-S] clusters in E. coli IscU. hIscA’s iron binding ability is similar to E. coli IscA. Moreover, hIscA is able to donate iron to IscU in the presence of 100-fold excess citrate, a metabolite capable of binding iron. This comparison signifies that [Fe-S] cluster assembly is conserved from bacteria to humans.
The second part of this dissertation determined the participation of Ferritin A (FtnA) in [Fe-S] cluster assembly. FtnA, the major iron storage protein in E. coli, could serve as an iron reservoir when intracellular iron is depleted. We have shown that FtnA is capable of buffering iron when oxidative stress disrupts nascent [Fe-S] clusters and alleviates the production of hydroxyl radicals. Moreover, when physiological conditions return, IscA is able to retrieve iron from FtnA for [Fe-S] cluster assembly.
The final part of this dissertation corroborated the interrelatedness of the oxidative and nitrosative stress response pathways. NsrR, a nitric oxide (NO) sensitive transcriptional repressor, is shown to coordinate a redox active [2Fe-2S] cluster with a midpoint redox potential of -346 ± 7 mV. The NsrR [2Fe-2S] cluster reacts with NO more quickly than other [Fe-S] proteins, signifying its role as a NO sensor. Finally, modification of the NsrR [2Fe-2S] cluster by NO results in the formation of a protein-bound dinitrosyl iron complex, relieving its DNA binding ability as a repressor.
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