New insights into protein S-nitrosylation. Mitochondria as a model system

Area of Science:

• Biochemistry
• Molecular Biology
• Cellular Signaling

Background:

• Nitric oxide (NO) mediates biological effects through S-nitrosylation of cysteine thiols.
• Previous studies suggested molecular oxygen (O(2)) is required for NO-mediated S-nitrosylation, but its role at low physiological oxygen levels was unclear.

Purpose of the Study:

• To investigate S-nitrosylation reactions in situ using a proteomic approach.
• To identify endogenously S-nitrosylated proteins and elucidate the mechanisms of S-nitrosylation under varying oxygen conditions.

Main Methods:

• Proteomic analysis to identify S-nitrosylated proteins in subcellular organelles.
• In situ studies under strictly anaerobic and aerobic conditions.
• Investigation of S-nitrosylation mechanisms, including those involving covalently bound flavin and transnitrosation.

Main Results:

• Identified endogenous S-nitrosylated proteins (dihydrolipoamide dehydrogenase, catalase, hydroxymethylglutaryl-CoA synthase, sarcosine dehydrogenase (SarDH)) in subcellular organelles.
• Demonstrated S-nitrosylation of these proteins by NO under strictly anaerobic conditions.
• Proposed a novel mechanism for SarDH S-nitrosylation involving its covalently bound flavin.
• Identified mitochondrial proteins susceptible to S-nitrosylation via multiple pathways (anaerobic/oxidative, NO/O(2), GSNO-mediated transnitrosation).
• Observed higher steady-state S-nitrosylation levels in mitochondrial extracts compared to intact organelles, indicating the role of denitrosylation.

Conclusions:

• S-nitrosylation by nitric oxide can occur under anaerobic conditions, challenging previous assumptions.
• Novel mechanisms of S-nitrosylation, including flavin-dependent pathways, are involved in cellular signaling.
• Denitrosylation reactions play a crucial role in regulating S-nitrosothiol levels within subcellular compartments.