Title page for ETD etd-07072009-151947


Type of Document Dissertation
Author Ren, Binbin
URN etd-07072009-151947
Title Nitric Oxide Cytotoxicity and Functions of Iron-sulfur Enzymes in Escherichia coli
Degree Doctor of Philosophy (Ph.D.)
Department Biochemistry (Biological Sciences)
Advisory Committee
Advisor Name Title
Ding, Huangen Committee Chair
Battista, John R. Committee Member
Lee, Yong-Hwan Committee Member
Moroney, James V. Committee Member
Yu, Tin-Wein Committee Member
Janes, Marlene E. Dean's Representative
Keywords
  • Nitric Oxide
  • Iron-Sulfur Enzymes
  • Escherichia coli
Date of Defense 2009-06-19
Availability unrestricted
Abstract
Iron-sulfur proteins are ubiquitous in biological processes. Here we report that NO, a physiological free radical, can effectively inhibit cell growth of Escherichia coli in minimal medium under anaerobic growth conditions. Fractionation of the cell extracts obtained from NO-exposed cells shows a broad distribution of the protein-bound dinitrosyl-iron complexes (DNICs) formed by NO. On the other hand, the cell growth of E. coli can be restored when NO-exposed cells are either supplemented with the branched-chain amino acids (BCAAs) anaerobically or returned to aerobic growth conditions. It turns out that dihydroxyacid dehydratase (IlvD), an iron-sulfur enzyme essential for the BCAAs biosynthesis, is completely inactivated by NO along with formation of the IlvD-bound DNICs. Nevertheless, the IlvD-DNICs, together with other protein-bound DNICs, are sufficiently repaired under aerobic growth conditions without new proteins synthesis. It is proposed that cellular deficiency to repair the NO-modified iron-sulfur proteins may directly contribute to the NO-induced bacteriostasis under anaerobic conditions. We further identify a new iron-sulfur enzyme as a potential target of NO, a DNA damage-inducible helicase DinG, which is a member of the DNA helicase superfamily II involved in DNA replication and repair. We find that E. coli DinG contains a redox-active [4Fe-4S] cluster with a midpoint redox potential (Em) of -390 23 mV (pH 8.0). An oxidized [4Fe-4S] cluster in DinG is required for its helicase activity and reduction of the [4Fe-4S] cluster can reversibly switch off its helicase activity. While DinG is resistant to 100-fold excess of hydrogen peroxide, it can be readily inactivated by NO, implying that inactivation of DinG by NO could impact the DNA repair and result in genomic instability. The NO-modified DinG can be fully reactivated by reassembly of a new [4Fe-4S] cluster, indicating that DinG is the primary target of NO exposure. The results from this study suggest that NO can effectively modify the iron-sulfur cluster in proteins, thus contributing to the NO-mediated bacteriostasis and genomic instability.
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