Glutaredoxin 2 Catalyzes the Reversible Oxidation and Glutathionylation of Mitochondrial Membrane Thiol Proteins

Abstract

The redox poise of the mitochondrial glutathione pool is central in the response of mitochondria to oxidative damage and redox signaling, but the mechanisms are uncertain. One possibility is that the oxidation of glutathione (GSH) to glutathione disulfide (GSSG) and the consequent change in the GSH/GSSG ratio causes protein thiols to change their redox state, enabling protein function to respond reversibly to redox signals and oxidative damage. However, little is known about the interplay between the mitochondrial glutathione pool and protein thiols. Therefore we investigated how physiological GSH/GSSG ratios affected the redox state of mitochondrial membrane protein thiols. Exposure to oxidized GSH/GSSG ratios led to the reversible oxidation of reactive protein thiols by thiol-disulfide exchange, the extent of which was dependent on the GSH/GSSG ratio. There was an initial rapid phase of protein thiol oxidation, followed by gradual oxidation over 30 min. A large number of mitochondrial proteins contain reactive thiols and most of these formed intraprotein disulfides upon oxidation by GSSG; however, a small number formed persistent mixed disulfides with glutathione. Both protein disulfide formation and glutathionylation were catalyzed by the mitochondrial thiol transferase glutaredoxin 2 (Grx2), as were protein deglutathionylation and the reduction of protein disulfides by GSH. Complex I was the most prominent protein that was persistently glutathionylated by GSSG in the presence of Grx2. Maintenance of complex I with an oxidized GSH/GSSG ratio led to a dramatic loss of activity, suggesting that oxidation of the mitochondrial glutathione pool may contribute to the selective complex I inactivation seen in Parkinson's disease. Most significantly, Grx2 catalyzed reversible protein glutathionylation/deglutathionylation over a wide range of GSH/GSSG ratios, from the reduced levels accessible under redox signaling to oxidized ratios only found under severe oxidative stress. Our findings indicate that Grx2 plays a central role in the response of mitochondria to both redox signals and oxidative stress by facilitating the interplay between the mitochondrial glutathione pool and protein thiols.Oxidative damage and redox signaling can regulate protein thiol redox state (1-4). A major way in which this occurs is through the response of protein thiols to changes in the glutathione (GSH) to glutathione disulfide (GSSG) ratio (2, 5, 6). The intracellular GSH/GSSG ratio is usually kept high (Ͼ99% reduced) through reduction of GSSG to GSH by glutathione reductase, enabling GSH to act as an antioxidant (2, 6). However, during oxidative stress or redox signaling reactive oxygen species (ROS) 1 oxidize GSH to GSSG directly, or catalyzed by glutathione peroxidases. Protein thiols respond to the decreased GSH/GSSG ratio by forming mixed disulfides with glutathione through thiol-disulfide exchange between the thiolate anion and GSSG (protein thiols typically have pK a values of ϳ8 -9, but these can vary widely depending on the local e...

Site-Specific S-Glutathiolation of Mitochondrial NADH Ubiquinone Reductase

“…1A). This result is basically consistent with those reported in the literature (13,14). The 51 kDa subunit was specifically involved in the above redox modification when the Fp subcomplex was exposed to GSSG (Fig.…”

“…This result was further verified by immunoblotting as reported by Beer et. al (13). However, the molecular mechanism of the above redox event remains unclear and needs to be defined.…”

“…An important response of protein thiols (PrSH) to oxidative stress is to reversibly form protein mixed disulfides (PrSSG) via S-glutathiolation (11)(12)(13). This post-translational modification has been suggested as a common mechanism regulating protein functions related to pathological changes such as disruption of the electron transfer activity and induction of membrane permeable transition pores through the cross-linking of membrane protein thiol(s) (10).…”

“…It has been documented that Complex I is the major component of the ETC to host protein thiols, which comprise structural thiols involved in the ligands of iron sulfur clusters and the reactive/regulatory thiols which are thought to have biological functions of antioxidant defense and redox signaling (13,14). The physiological roles of Complex I-derived regulatory thiols have been implicated in the regulation of the respiration, nitric oxide utilization (15,16), and redox status of mitochondria (10)(11)(12)).…”

The chemistry of cell signaling by reactive oxygen and nitrogen species and 4-hydroxynonenal

“…Although glutathionylation of PTP1B can be achieved in vitro with the purified enzyme through oxidation by H 2 O 2 and addition of GSH (through the sulfenic acid and sulfenyl-amide as described above) (26) (27) or by addition of a very high concentration of GSSG (63), it is not known if glutathionylation occurs enzymatically or non-enzymatically in vivo and kinetic considerations clearly argue against the latter (87). While formation of glutathionylated PTP1B through disulfide exchange in the cytosol by reaction with GSSG enzymatically or non-enzymatically is unlikely due to the requirement for markedly increasing GSSG, a recent paper by Beer et al demonstrated glutaredoxin 2 dependent catalysis of glutathionylation/deglutationylation of mitochondrial proteins (88). Interestingly, a paper by Seth and Rudolph of recent publication extended the diversity of mechanisms utilized by PTPs to prevent irreversible oxidation of the active site.…”