Site-specific S-glutathiolation of mitochondrial NADH ubiquinone reductase

Chwen Lih Chen, Liwen Zhang, Alexander Yeh, Chun An Chen, Kari B. Green-Church, Jay L. Zweier, Yeong Renn Chen

Research output: Contribution to journalArticle

Abstract

The generation of reactive oxygen species in mitochondria acts as a redox signal in triggering cellular events such as apoptosis, proliferation, and senescence. Overproduction of superoxide (O2⊙-) and O2⊙--derived oxidants changes the redox status of the mitochondrial GSH pool. An electron transport protein, mitochondrial complex I, is the major host of reactive/regulatory protein thiols. An important response of protein thiols to oxidative stress is to reversibly form protein mixed disulfide via S-glutathiolation. Exposure of complex I to oxidized GSH, GSSG, resulted in specific S-glutathiolation at the 51 kDa and 75 kDa subunits (Beer et al. (2004) J. Biol. Chem. 279, 47939-47951). Here, to investigate the molecular mechanism of S-glutathiolation of complex I, we prepared isolated bovine complex I under nonreducing conditions and employed the techniques of mass spectrometry and EPR spin trapping for analysis. LC/MS/MS analysis of tryptic digests of the 51 kDa and 75 kDa polypeptides from glutathiolated complex I (GS-NQR) revealed that two specific cysteines (C206 and C187) of the 51 kDa subunit and one specific cysteine (C 367) of the 75 kDa subunit were involved in redox modifications with GS binding. The electron transfer activity (ETA) of GS-NQR in catalyzing NADH oxidation by Q1 was significantly enhanced. However, O 2⊙- generation activity (SGA) mediated by GS-NQR suffered a mild loss as measured by EPR spin trapping, suggesting the protective role of S-glutathiolation in the intact complex I. Exposure of NADH dehydrogenase (NDH), the flavin subcomplex of complex I, to GSSG resulted in specific S-glutathiolation on the 51 kDa subunit. Both ETA and SGA of S-glutathiolated NDH (GS-NDH) decreased in parallel as the dosage of GSSG increased. LC/MS/MS analysis of a tryptic digest of the 51 kDa subunit from GS-NDH revealed that C206, C187, and C425 were glutathiolated. C425 of the 51 kDa subunit is a ligand residue of the 4Fe-4S N3 center, suggesting that destruction of 4Fe-4S is the major mechanism involved in the inhibition of NDH. The result also implies that S-glutathiolation of the 75 kDa subunit may play a role in protecting the 4Fe-4S cluster of the 51 kDa subunit from redox modification when complex I is exposed to redox change in the GSH pool.

Original languageEnglish (US)
Pages (from-to)5754-5765
Number of pages12
JournalBiochemistry®
Volume46
Issue number19
DOIs
StatePublished - May 15 2007
Externally publishedYes

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Electron Transport Complex I
NADH Dehydrogenase
Oxidation-Reduction
Glutathione Disulfide
Spin Trapping
Sulfhydryl Compounds
Cysteine
Paramagnetic resonance
Electrons
Proteins
Mitochondria
Oxidative stress
Electron Transport
Oxidants
Superoxides
Disulfides
NAD
Mass spectrometry
Reactive Oxygen Species
Mass Spectrometry

ASJC Scopus subject areas

  • Biochemistry

Cite this

Chen, C. L., Zhang, L., Yeh, A., Chen, C. A., Green-Church, K. B., Zweier, J. L., & Chen, Y. R. (2007). Site-specific S-glutathiolation of mitochondrial NADH ubiquinone reductase. Biochemistry®, 46(19), 5754-5765. https://doi.org/10.1021/bi602580c

Site-specific S-glutathiolation of mitochondrial NADH ubiquinone reductase. / Chen, Chwen Lih; Zhang, Liwen; Yeh, Alexander; Chen, Chun An; Green-Church, Kari B.; Zweier, Jay L.; Chen, Yeong Renn.

In: Biochemistry®, Vol. 46, No. 19, 15.05.2007, p. 5754-5765.

Research output: Contribution to journalArticle

Chen, CL, Zhang, L, Yeh, A, Chen, CA, Green-Church, KB, Zweier, JL & Chen, YR 2007, 'Site-specific S-glutathiolation of mitochondrial NADH ubiquinone reductase', Biochemistry®, vol. 46, no. 19, pp. 5754-5765. https://doi.org/10.1021/bi602580c
Chen CL, Zhang L, Yeh A, Chen CA, Green-Church KB, Zweier JL et al. Site-specific S-glutathiolation of mitochondrial NADH ubiquinone reductase. Biochemistry®. 2007 May 15;46(19):5754-5765. https://doi.org/10.1021/bi602580c
Chen, Chwen Lih ; Zhang, Liwen ; Yeh, Alexander ; Chen, Chun An ; Green-Church, Kari B. ; Zweier, Jay L. ; Chen, Yeong Renn. / Site-specific S-glutathiolation of mitochondrial NADH ubiquinone reductase. In: Biochemistry®. 2007 ; Vol. 46, No. 19. pp. 5754-5765.
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