Abstract
S-Nitrosylation is a reversible post-translational
modification
on cysteinyl thiols that can modulate the function of redox-sensitive
proteins. The S-nitrosylation of mitochondrial proteins has been shown
to regulate various mitochondrial activities involved in energy-transducing
systems and mitochondrion-driven apoptosis. In isolated rat brain
mitochondria, we demonstrate that mitochondrial protein S-nitrosylation
is regulated by respiratory substrates (glutamate/malate) through
a thiol-dependent pathway. Mitochondrial proteins become susceptible
to
S
-nitrosoglutathione (GSNO)-induced S-nitrosylation
in mitochondria with an oxidized environment (low glutathione (GSH),
NADH, and NADPH, and high GSSG, NAD
+
, and NADP
+
) caused by isolation of mitochondria using a discontinuous Percoll
gradient. Activation of mitochondrial respiration by respiratory substrates
leads to increased NAD(P)H and GSH levels, which in turn reduces mitochondrial
S-nitrosylated proteins. 1-Chloro-2,4-dinitrobenzene (CDNB), which
depletes mitochondrial GSH and inhibits the thioredoxin–thioredoxin
reductase system, prevented the denitrosylation of mitochondrial proteins
caused by respiratory substrate treatment. Using biotin-switch coupled
with LC-MS/MS, several mitochondrial proteins were identified as targets
of S-nitrosylation including adenine nucleotide translocase (ANT)
and voltage-dependent anion channel (VDAC), important components of
the mitochondria permeability transition pore (MPTP), as well as ATP
synthase. The S-nitrosylation of ATP synthase by GSNO was found to
inhibit its activity. These findings emphasize the importance of respiratory
substrates in regulating S-nitrosylation through a thiol-dependent
(GSH and/or thioredoxin) pathway, with implications for mitochondrial
bioenergetics and mitochondrion-driven apoptosis.