<p class="first" id="d4194891e72">Our group has previously observed that protein S-glutathionylation
serves as an integral
feedback inhibitor for the production of superoxide (O2●-)/hydrogen peroxide (H2O2)
by α-ketoglutarate dehydrogenase (KGDH), pyruvate dehydrogenase (PDH), and complex
I in muscle and liver mitochondria, respectively. In the present study, we hypothesized
that glutathionylation would fulfill a similar role for the O2●-/H2O2 sources sn-glycerol-3-phosphate
dehydrogenase (G3PDH), proline dehydrogenase (PRODH), and branched chain keto acid
dehydrogenase (BCKDH). Surprisingly, we found that inducing glutathionylation with
disulfiram increased the production of O2●-/H2O2 by mitochondria oxidizing glycerol-3-phosphate
(G3P), proline (Pro), or α-keto-β-methylvaleric acid (KMV). Treatment of mitochondria
oxidizing G3P or Pro with rotenone or myxothiazol increased the rate of ROS production
after incubating in 1000 nM disulfiram. Incubating mitochondria treated with disulfiram
in both rotenone and myxothiazol prevented this increase in O2●-/H2O2 production.
In addition, when adminstered together, ROS production decreased below control levels.
Disulfiram-treated mitochondria displayed higher rates of ROS production when oxidizing
succinate, which was inhibited by rotenone, myxothiazol, and malonate, respectively.
Disulfiram also increased ROS production by mitocondria oxidizing KMV. Treatment of
mitochondria oxidizing KMV with disulfiram and rotenone or myxothiazol did not alter
the rate O2●-/H2O2 production further when compared to mitochondria treated with disulfiram
only. Analysis of BCKDH activity following disulfiram treatment revealed that glutathionylation
does not inhibit the enzyme complex, indicating this α-keto acid dehydrogenase is
not a target for glutathione modification. However, treatment of mitochondria with
rotenone and myxothiazol without disulfiram also augmented ROS production. Overall,
we were able to demonstrate for the first time that glutathionylation augments ROS
production by the respiratory chain during forward electron transfer (FET) and reverse
electron transfer (RET) from the UQ pool. Additionally, we were able to show that
BCKDH is not a target for glutathione modification and that glutathionylation can
also increase ROS production in mitochondria oxidizing branched chain amino acids
following the modification of enzymes upstream of BCKDH.
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