Excess production of nitric oxide (NO) has been attributed to a number of human pathological conditions including cardiovascular diseases. Recent studies indicated that iron-sulfur proteins are the primary targets of NO in bacterial cells.  Upon exposure to NO, iron-sulfur clusters in proteins are quickly disrupted, forming the protein-bound dinitrosyl iron complex (DNIC).  However, much less is known on metabolism of iron-sulfur proteins in mammalian cells in response to NO.  The goal is to investigate specific modification and repair of iron-sulfur proteins in rat heart cells in response to NO stress.  When rat heart cells are exposed to NO, significant amounts of protein-bound DNICs are formed with concomitant inactivation of mitochondrial aconitase, a key iron-sulfur enzyme in energy metabolism.  Re-assembly of iron-sulfur clusters using cysteine desulfurase, L-cysteine and ferrous iron fully restores the enzyme activity of mitochondrial aconitase in the cell extracts prepared from the NO-treated rat heart cells, suggesting that NO only disrupts iron-sulfur clusters in proteins without significantly modifying protein polypeptides.  Western blotting analyses of total nitrotyrosine and S-nitrosylation in the cell extracts further show that iron-sulfur proteins in rat heart cells are much more sensitive to NO than the residue tyrosine or cysteine in proteins.  While the protein-bound DNICs are stable under aerobic or anaerobic conditions, they are able to exchange among iron-sulfur proteins or with small molecular thiols.  Furthermore, unlike the protein-bound DNICs, the L-cysteine-bound DNIC rapidly decomposes under aerobic conditions and releases ferrous iron and nitrite, suggesting that L-cysteine may have a vital role in repairing the NO-modified iron-sulfur proteins.  The results led us to propose that iron-sulfur proteins are the primary targets of NO in rat heart tissues, and that the protein-bound DNICs may exchange among proteins or with small molecular thiols for efficient re-activation of the NO-modified iron-sulfur proteins.

Dynamic metabolism of iron-sulfur proteins in rat heart cells in response to nitric oxide

248th National Meeting (2014)

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