Regulation of mitochondrial glutathione redox status and protein glutathionylation by respiratory substrates

Abstract

Brain and liver mitochondria isolated by a discontinuous Percoll gradient show an oxidized redox environment, which is reflected by low GSH levels and high GSSG levels and significant glutathionylation of mitochondrial proteins as well as by low NAD(P)H/NAD(P) values. The redox potential of brain mitochondria isolated by a discontinuous Percoll gradient method was calculated to be -171 mV based on GSH and GSSG concentrations. Immunoblotting and LC/MS/MS analysis revealed that succinyl-CoA transferase and ATP synthase (F(1) complex, α-subunit) were extensively glutathionylated; S-glutathionylation of these proteins resulted in a substantial decrease of activity. Supplementation of mitochondria with complex I or complex II respiratory substrates (malate/glutamate or succinate, respectively) increased NADH and NADPH levels, resulting in the restoration of GSH levels through reduction of GSSG and deglutathionylation of mitochondrial proteins. Under these conditions, the redox potential of brain mitochondria was calculated to be -291 mV. Supplementation of mitochondria with respiratory substrates prevented GSSG formation and, consequently, ATP synthase glutathionylation in response to H(2)O(2) challenges. ATP synthase appears to be the major mitochondrial protein that becomes glutathionylated under oxidative stress conditions. Glutathionylation of mitochondrial proteins is a major consequence of oxidative stress, and respiratory substrates are key regulators of mitochondrial redox status (as reflected by thiol/disulfide exchange) by maintaining mitochondrial NADPH levels.

FIGURE 1.

Effect of DTT on GSH and GSSG levels in isolated brain and liver mitochondria. Liver mitochondria were isolated using differential centrifugation (DC) or discontinuous Percoll gradient (DPG) as described under “Experimental Procedures.” Brain mitochondria were isolated using discontinuous Percoll gradient. Following isolation, mitochondria were incubated at 37 °C for 5 min with or without DTT (1 mm). Mitochondria were subsequently pelleted, resuspended in 5% metaphosphoric acid, and centrifuged once more. GSH (A) and GSSG (B) levels were measured in the supernatant using HPLC with electrochemical detection. Shaded bar, no DTT added; black bar, plus 1 mm DTT.

FIGURE 2.

Effect of respiratory substrates on GSH and GSSG levels in brain mitochondria. Brain mitochondria were isolated using a discontinuous Percoll gradient. Following isolation, mitochondria were incubated with either glutamate/malate/ADP (○) or DTT (■). ●, no additions. At the indicated times, mitochondrial GSH and GSSG levels were measured using HPLC with electrochemical detection as described under “Experimental Procedures.” The assay conditions were: glutamate/malate, 2.4 mm; ADP, 0.07 mm; and DTT, 1 mm.

FIGURE 3.

Effect of respiratory substrates on GSH and GSSG levels in liver mitochondria. Liver mitochondria were isolated using a discontinuous Percoll gradient. Following isolation, mitochondria were incubated with either glutamate/malate/ADP (○) or DTT (■). ●, no additions. At the indicated times, mitochondrial GSH and GSSG levels were measured using HPLC with electrochemical detection. The assay conditions were as described in the legend to Fig. 2.

FIGURE 4.

Effect of respiratory substrates on GSH and GSSG levels in liver mitochondria isolated by differential centrifugation. Liver mitochondria were isolated using differential centrifugation. Following isolation, mitochondria were incubated with either glutamate/malate/ADP (●) or DTT (■). ○, no additions. At the indicated times, mitochondrial GSH and GSSG levels were measured using HPLC with electrochemical detection. The concentrations of reactants are as described in the legend to Fig. 2.

FIGURE 5.

Identification of glutathionylated mitochondrial proteins: regulation by respiratory substrates. A, immunoblotting of glutathionylated proteins in mitochondria isolated using a discontinuous Percoll gradient. Following isolation, brain mitochondria were incubated at 37 °C for 10 min in the presence or absence of either glutamate/malate (7.5 mm) plus ADP (50 μm) or DTT (1 mm). Mitochondria were lysed in a 2% CHAPS/nonreducing and reducing buffer and underwent a series (three times) of freezing and thawing to ensure maximal protein release. Then mitochondria were separated via SDS-PAGE, transferred to a nitrocellulose membrane, and probed using a Virogen GSH antibody (1:500). B, immunoprecipitation (IP) of glutathionylated mitochondrial proteins following mitochondria isolation using discontinuous Percoll gradient and identification using LC/MS/MS. Following isolation, brain mitochondria were treated with 2% CHAPS/nonreducing buffer and immunoprecipitated using GSH antibody (1:200). Immunoprecipitation proteins were then separated via SDS-PAGE. Protein bands stained by SYPRO® Ruby were excised and identified by LC/MS/MS.

FIGURE 6.

Effect of mitochondrial respiratory substrates and inhibitors on NADH/NAD⁺ and NADPH/NADP⁺ values. Brain mitochondria were isolated using a discontinuous Percoll gradient. Following isolation, mitochondria were incubated with mitochondrial respiratory substrates and/or inhibitors for 10 min at 37 °C. Pyridine nucleotides were measured by HPLC with fluorescence detection as described under “Experimental Procedures.” G/M, glutamate/malate. The assay conditions were: glutamate/malate, 2.4 mm; ADP, 0.7 mm; and rotenone, 5 μm.

FIGURE 7.

Regulation of brain mitochondria GSH and GSSG by respiratory substrates following H₂O₂ treatment. Brain mitochondria were isolated by a discontinuous Percoll gradient with a buffer containing DTT to conserve mitochondrial GSH. Isolated mitochondria were resuspended in a respiration buffer and incubated with various concentrations of H₂O₂ for 10 min at 37 °C. When added, glutamate/malate concentration was 2.4 mm. A, brain mitochondria GSH content. B, brain mitochondria GSSG content. C, immunoblotting of glutathionylated ATP synthase.