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Rapid inactivation of plant aconitase by hydrogen peroxide.

ABSTRACT: Preincubation of potato (Solanum tuberosum) tuber mitochondria with 300 microM-H2O2 for 10 min nearly stopped the State 3 rate of citrate oxidation. Addition of isocitrate resulted in resumption of O2 uptake. The State 3 rates of succinate, external NADH and 2-oxoglutarate oxidation were unaffected by H2O2 over the dose range 50-500 microM. Preincubation of mitochondria with 300 microM-H2O2 for 5 min unmasked in the matrix space a paramagnetic signal with a peak at a g value of approx. 2.03. Aconitase was purified over 135-fold to a specific activity of 32 mumol/min per mg (with isocitrate as substrate) from the matrix of potato tuber mitochondria. The native enzyme was composed of a single polypeptide chain (molecular mass 90 kDa). Incubation of purified aconitase with small amounts of H2O2 caused the build up of a paramagnetic 3Fe cluster with a low-field maximum of g = 2.03 leading to a progressive inhibition of aconitase activity. The results show that aconitase present in the matrix space was the major intramitochondrial target for inactivation by H2O2.

SUBMITTER: Verniquet F

PROVIDER: S-EPMC1151053 | biostudies-other | 1991 Jun

REPOSITORIES: biostudies-other

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Project description:Under oxidative stress conditions, mitochondria are the major site for cellular production of reactive oxygen species (ROS) such as superoxide anion and H2O2 that can attack numerous mitochondrial proteins including dihydrolipoamide dehydrogenase (DLDH). While DLDH is known to be vulnerable to oxidative inactivation, the mechanisms have not been clearly elucidated. The present study was therefore designed to investigate the mechanisms of DLDH oxidative inactivation by mitochondrial reactive oxygen species (ROS). Mitochondria, isolated from rat brain, were incubated with mitochondrial respiratory substrates such as pyruvate/malate or succinate in the presence of electron transport chain inhibitors such as rotenone or antimycin A. This is followed by enzyme activity assay and gel-based proteomic analysis. The present study also examined whether ROS-induced DLDH oxidative inactivation could be reversed by reducing reagents such as DTT, cysteine, and glutathione. Results show that DLDH could only be inactivated by complex III- but not complex I-derived ROS; and the accompanying loss of activity due to the inactivation could be restored by cysteine and glutathione, indicating that DLDH oxidative inactivation by complex III-derived ROS was a reversible process. Further studies using catalase indicate that it was H2O2 instead of superoxide anion that was responsible for DLDH inactivation. Moreover, using sulfenic acid-specific labeling techniques in conjunction with two-dimensional Western blot analysis, we show that protein sulfenic acid formation (also known as sulfenation) was associated with the loss of DLDH enzymatic activity observed under our experimental conditions. Additionally, such oxidative modification was shown to be associated with preventing DLDH from further inactivation by the thiol-reactive reagent N-ethylmaleimide. Taken together, the present study provides insights into the mechanisms of DLDH oxidative inactivation by mitochondrial H2O2.

Project description:The SrrAB two-component regulatory system (TCRS) positively influences the transcription of genes involved in aerobic respiration in response to changes in respiratory flux. Hydrogen peroxide (H2O2) can arise as a byproduct of spontaneous interactions between dioxygen and components of respiratory pathways. H2O2 damages cellular factors including protein associated iron-sulfur cluster prosthetic groups. We found that a Staphylococcus aureus strain lacking the SrrAB two-component regulatory system (TCRS) is sensitive to H2O2 intoxication. We tested the hypothesis that SrrAB manages the mutually inclusive expression of genes required for aerobic respiration and H2O2 resistance. Consistent with our hypothesis, a ?srrAB strain had decreased transcription of genes encoding for H2O2 resistance factors (kat, ahpC, dps). SrrAB was not required for the inducing the transcription of these genes in cells challenged with H2O2. Purified SrrA bound to the promoter region for dps suggesting that SrrA directly influences dps transcription. The H2O2 sensitivity of the ?srrAB strain was alleviated by iron chelation or deletion of the gene encoding for the peroxide regulon repressor (PerR). The positive influence of SrrAB upon H2O2 metabolism bestowed protection upon the solvent accessible iron-sulfur (FeS) cluster of aconitase from H2O2 poisoning. SrrAB also positively influenced transcription of scdA (ytfE), which encodes for a FeS cluster repair protein. Finally, we found that SrrAB positively influences H2O2 resistance only during periods of high dioxygen-dependent respiratory activity. SrrAB did not influence H2O2 resistance when cellular respiration was diminished as a result of decreased dioxygen availability, and negatively influenced it in the absence of respiration (fermentative growth). We propose a model whereby SrrAB-dependent regulatory patterns facilitate the adaptation of cells to changes in dioxygen concentrations, and thereby aids in the prevention of H2O2 intoxication during respiratory growth upon dixoygen.

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Rapid inactivation of plant aconitase by hydrogen peroxide.

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