Background: TrxR1 is the only known reductant of thioredoxin. Results: The glutaredoxin system showed the capacity of reducing thioredoxin 1. Depletion of both TrxR1 activity and glutathione in cells induced Trx1 oxidation and cell death. Conclusion:The glutathione/glutaredoxin system is a backup of TrxR1 for reducing thioredoxin 1. Significance: We demonstrated the critical role of the thioredoxin redox state in cell survival and a new role of glutathione.

We have shown recently that halogenated quinones could enhance the decomposition of hydroperoxides and formation of alkoxyl/ hydroxyl radicals through a metal-independent mechanism. However, neither the proposed quinone enoxy radical intermediate, nor the major reaction products were unambiguously identified. In the present study, one of the major reaction products between 2,5-dichloro-1,4-benzoquinone (DCBQ) and t-butylhydroperoxide (t-BuOOH) was isolated and purified by semipreparative HPLC, and identified as 2-hydroxy-3-t-butoxy-5-chloro-1,4-benzoquinone [CBQ(OH)-O-t-Bu], which is the rearranged isomer of the postulated quinone-peroxide reaction intermediate. The formation of CBQ(OH)-O-t-Bu was found to be inhibited by the spin trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and concurrently, a new DMPO adduct with 1-chlorine isotope peak clusters at m/z 268 was observed. Further electron spin resonance (ESR) spintrapping, 1 H-NMR and HPLC/Fourier transform ion cyclotron resonance (FTICR) mass spectrometric studies with oxygen-17-labeled and unlabeled hydrogen peroxide strongly suggest that the radical trapped by DMPO is a carbon-centered quinone ketoxy radical, which is the spin isomer of the proposed oxygen-centered quinone enoxy radical. Analogous results were observed when DCBQ was substituted by other halogenated quinones. This study represents the first detection and identification of an unusual carbon-centered quinone ketoxy radical, which provides direct experimental evidence to further support and expand our previously proposed mechanism for metal-independent decomposition of hydroperoxides by halogenated quinones.ESR spin-trapping ͉ spin isomerization ͉ carbon-centered quinone ketoxy radical ͉ oxygen-centered quinone enoxy radical ͉ keto-enol tautomerization W e have shown recently that alkoxyl radicals can be produced by organic hydroperoxides and halogenated quinones independent of transition metal ions, and a reaction mechanism was proposed (1): A nucleophilic reaction may take place between 2,5-dichloro-1,4-benzoquinone (DCBQ) and t-butylhydroperoxide (t-BuOOH), forming a quinone-peroxide reaction intermediate 2-chloro-5-t-butylperoxyl-1,4-benzoquinone (CBQ-OO-t-Bu), which can decompose homolytically to produce t-butoxyl radical (t-BuO • ) and 2-chloro-5-hydroxy-1,4-benzoquinone enoxy radical (CBQ-O • ). CBQ-O• then disproportionate to form the ionic form of 2-chloro-5-hydroxy-1,4-benzoquinone (CBQ-OH) (see scheme 1 in ref. 1). We also found that hydroxyl radicals could be produced metal-independently by hydrogen peroxide and halogenated quinones (2-4). However, neither the major reaction products, nor the proposed quinone-peroxide reaction intermediate CBQ-OO-t-Bu and quinone enoxy radical CBQ-O • were unambiguously identified in the previous studies.Therefore, in the present study, we addressed the following questions: (i) was it possible to isolate and purify the proposed quinone-peroxide reaction intermediate; (ii) could CBQ-OH or any other major reaction products be isolated, purifi...

Background:The Cys-62/Cys-69 dithiol of Trx1 is predicted to have a profound effect on cell signaling. Results: The Cys-62/Cys-69 dithiol of Trx1 was oxidized by Prx1, and this disulfide was reduced by the GSH/glutaredoxin system. Conclusion: Trx1 is involved in redox regulation via reversible oxidation of the Cys-62/Cys-69 dithiol of Trx1. Significance: We demonstrated the critical role of Trx1 oxidation in cell signaling.

Aims: Mitochondrial thioredoxin (Trx) is critical for defense against oxidative stress-induced cell apoptosis. To date, mitochondrial thioredoxin reductase (TrxR) is the only known enzyme catalyzing Trx2 reduction in mitochondria. However, TrxR is sensitive to inactivation by exo/endogenous electrophiles, for example, 4-hydroxynonenal (HNE). In this study, we characterized the mitochondrial glutaredoxin 2 (Grx2) system as a backup for the mitochondrial TrxR. Meanwhile, as Grx2 is also present in the cytosol/nucleus of certain cancer cell lines, the reducing activity of Grx2 on Trx1 was also tested. Results: Glutathione alone could reduce oxidized Trx2, and the presence of physiological concentrations of Grx2 markedly increased the reaction rate. HeLa cells with Grx2 overexpression (particularly in the mitochondria) exhibited higher viabilities than the wild-type cells after treatment with TrxR inhibitors (Auranofin or HNE), whereas knockdown of Grx2 sensitized the cells to TrxR inhibitors. Accordingly, Grx2 overexpression in the mitochondria had protected Trx2 from oxidation by HNE treatment, whereas Grx2 knockdown had sensitized Trx2 to oxidation. On the other hand, Grx2 reduced Trx1 with similar activities as that of Trx2. Overexpression of Grx2 in the cytosol had protected Trx1 from oxidation, indicating a supportive role of Grx2 in the cytosolic redox balance of cancer cells. Innovation: This work explores the reductase activity of Grx2 on Trx2/1, and demonstrates the physiological importance of the activity by using in vivo redox western blot assays. Conclusion: Grx2 system could help to keep Trx2/1 reduced during an oxidative stress, thereby contributing to the anti-apoptotic signaling. Antioxid. Redox Signal. 21, 669-681.