Nitric Oxide Cytotoxicity and Iron-Sulfur Proteins

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DESCRIPTION (provided by applicant): Excessive production of NO has been implicated in causing neurodegenerative diseases, cardiovascular oxidative injuries and cancers. This proposal is aimed to test a hypothesis that modification of iron-sulfur clusters in proteins by NO represents an acute cellular oxidative damage of NO cytotoxicity. Unlike the reversible NO binding in heme, NO disrupts iron-sulfur clusters forming the stable protein-bound dinitrosyl iron complex (DNIC). As iron-sulfur proteins are involved in diverse biological processes, primarily in energy conversion, DNA repair, amino acid metabolism, heme and biotin biosynthesis and iron homeostasis, modification of iron-sulfur clusters by NO could lead to failure of multiple cellular functions and eventually contribute to development of human diseases such as cancer. The overall goals of the proposal are to investigate 1) the redox reaction underlying the biological modification of iron-sulfur clusters by NO, and 2) the cellular mechanism by which the NO-modified iron-sulfur clusters are repaired. Aim 1 is to quantitatively analyze the iron and sulfide released from iron-sulfur clusters and formation of the protein-bound DNIC by NO using the radioactive labeled iron-sulfur clusters. Aim 2 is to determine the relative reactivity of iron sulfur clusters and small molecular thiols with NO in both E. coli and cultured human fibroblast cells and to identify specific NO-modified iron-sulfur proteins in E. coli cells using the proteomic approaches. Aim 3 is to nvestigate the redox reactions of the L-cysteine-mediated decomposition of the protein-bound DNIC, based on preliminary studies showing that L-cysteine can decompose the protein-bound DNIC and facilitate reassembly of new iron-sulfur clusters in the proteins. Aim 4 is to explore the physiological role of L-cysteine in repairing the protein-bound DNIC in the E. coli cells in which the intracellular L-cysteine content will be modulated. The proposed research, if successful, will provide fundamental knowledge for developing therapeutic approaches to prevent or alleviate cellular oxidative damages inflicted by NO cytotoxicity.