Persistent S-Nitrosation of Complex I and Other Mitochondrial Membrane Proteins by S-Nitrosothiols but Not Nitric Oxide or Peroxynitrite

Abstract

S-Nitrosation of mitochondrial proteins has been proposed to contribute to the pathophysiological interactions of nitric oxide (NO) and its derivatives with mitochondria but has not been shown directly. Furthermore, little is known about the mechanism of formation or the fate of these putative S-nitrosothiols. Here we have determined whether mitochondrial membrane protein thiols can be S-nitrosated on exposure to free NO from 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (DETA-NONOate) by interaction with S-nitrosoglutathione or S-nitroso-N-acetylpenicillamine (SNAP) and by the NO derivative peroxynitrite. S-Nitrosation of protein thiols was measured directly by chemiluminescence detection. S-Nitrosoglutathione and S-nitroso-N-acetylpenicillamine led to extensive protein thiol oxidation, with about 30% of the modified protein thiols persistently S-nitrosated. In contrast, there was no protein thiol oxidation or S-nitrosation on exposure to 3,3-bis (aminoethyl)-1-hydroxy-2-oxo-1-triazene. Peroxynitrite extensively oxidized protein thiols but produced negligible amounts of S-nitrosothiols. Therefore, mitochondrial membrane protein thiols are S-nitrosated by preformed S-nitrosothiols but not by NO or by peroxynitrite. These S-nitrosated protein thiols were readily reduced by glutathione, so S-nitrosation will only persist when the mitochondrial glutathione pool is oxidized. Respiratory chain complex I was S-nitrosated by S-nitrosothiols, consistent with it being an important target for S-nitrosation during nitrosative stress. The S-nitrosation of complex I correlated with a significant loss of activity that was reversed by thiol reductants. S-Nitrosation was also associated with increased superoxide production from complex I. These findings point to a significant role for complex I S-nitrosation and consequent dysfunction during nitrosative stress in disorders such as Parkinson disease and sepsis.

Nitric oxide inhibits insulin-degrading enzyme activity and function through S-nitrosylation

“…SNAP can directly release NO into solution by spontaneous cleavage of its NO moiety or can nitrosylate protein thiols by transfer of NO + [39,40]. The rate of NO release from this nitrosothiol compound is thought to be unaffected by ascorbate.…”

Regulation of Plant Glycine Decarboxylase byS-Nitrosylation and Glutathionylation

“…Therefore, the inhibition of GDC activity after GSNO treatment could have been an indirect effect of ROS induced by inhibition of complex I. It is well known that the activity of complex I of the mitochondrial electron transport chain is inhibited by nitrosothiols and peroxinitrite (Carreras et al, 2004;Dahm et al, 2006). This inhibition results in increased production of ROS, which can cause oxidative damage of GDC (Taylor et al, 2002).…”

“…Furthermore, NO activates mitogen-activated protein kinases in different plant species during stress signaling (Nakagami et al, 2005). However, direct biological activity of NO arises from chemical reactions between proteins and NO itself (Foster and Stamler, 2004;Dahm et al, 2006). S-Nitrosylation is a labile posttranslational modification with a half-life of seconds to a few minutes and represents a very sensitive mechanism for regulating cellular processes (Hess et al, 2005).…”

Novel interactions of mitochondria and reactive oxygen/nitrogen species in alcohol mediated liver disease

“…Because alterations in the redox state of protein thiols are important in regulating mitochondrial function such as respiration and oxidant production [57,58] , the identification of proteins with oxidized and/or modified thiol groups is critical for elucidating the mitochondrial defects that contribute to alcoholic liver disease.…”