Site-Specific S-Glutathiolation of Mitochondrial NADH Ubiquinone Reductase

Chwen Chen

Liwen Zhang

Alexander Yeh

Chun Chen

Kari Green-Church

Jay Zweier

Yeong Chen

^{Davis Heart & Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal Medicine, The Ohio State University, Columbus, OH 43210}

^{Davis Heart & Lung Research Institute, Division of Cardiovascular Medicine, Department of Molecular and Cellular Biochemistry, College of Medicine, The Ohio State University, Columbus, OH 43210}

^{Campus Chemical Instrument Center, Proteomics and Mass Spectrometry Facility, The Ohio State University, Columbus, OH 43210}

^{Address correspondence to: Yeong-Renn Chen, 607 Davis Heart & Lung Research Institute, The Ohio State University, 473 W. 12}^{Ave. Columbus, OH 43210, Tel.: 614-688-4054; Fax: 614-292-8778; E-mail:}ude.cmuso@nehc.nner-gnoey

^{Authors contributed equally to this study.}

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 O}₂^{-derived oxidants change 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. Here, to investigate the molecular mechanism of S-glutathiolation of Complex I, we prepared isolated bovine Complex I under non-reducing 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 (C₃₆₇) 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, O₂^{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 C}₂₀₆, 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 inhibiton 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.

Abstract

Mitochondrial Complex I (EC 1.6.5.3. NADH:ubiquinone oxidoreductase) is the first energy-conserving segment of the electron transport chain (ETC) (1-3). The enzyme catalyzes electron transfer from NADH to ubiquinone coupled with the translocation of four protons across the membrane. In addition to its functions of electron transfer and energy transduction, the catalysis of Complex I provides the major source of oxygen free radical generation in mitochondria (4-6). Two regions of the enzyme complex are hypothesized to be responsible for generating the superoxide anion radical (O₂^{). One is located on the FMN cofactor and is modulated by its binding protein moiety (}4, 5, 7), while the other is likely located on the ubiquinone-binding site and probably acts in the mediation of ubiquinone reduction (8, 9).

The generation of O₂^{and the oxidants derived from it in mitochondria can act as a redox signal in triggering cellular events such as apoptosis, proliferation, and senescence. The redox pool in mitochondria is enriched in glutathione (GSH) with a physiological concentration of 5-10 mM (}10). Overproduction of O₂^{and O}₂^{-derived oxidants increases the ratio of GSSG to GSH in mitochondria.}

The proteins of mitochondrial ETC are rich in protein thiols (11, 12). 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 (10-12).

An important response of protein thiols (PrSH) to oxidative stress is to reversibly form protein mixed disulfides (PrSSG) via S-glutathiolation (11-13). 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).

With the use of chaotropic anions such as perchlorate, Complex I can be resolved into three fractions: a flavoprotein fraction (Fp), an iron-sulfur (Fe-S) protein fraction (Ip), and a hydrophobic protein fraction (Hp) (17). The Fp fraction contains the enzymatic activity of NADH dehydrogenase and can be isolated as a three-subunit subcomplex from submitochondrial particles (SMP) (7). As demonstrated by EPR spin trapping with DEPMPO, the mechanism of O₂^{generation by NDH is mainly controlled by FMN cofactor and its binding protein moiety at the 51 kDa subunit (}7).

In previous studies we demonstrated that the biological relevance of C₂₀₆ of the 51 kDa subunit in the oxidative damage of NADH dehydrogenase is to play the unique role involved in oxidative damage with protein radical formation based on the evidence of immunospin trapping with DMPO and mass spectrometry (7). Taylor et. al have employed a thiol-specific probe and proteomic approach to examine the redox biochemistry of mitochondria (14). Both the 51 kDa and 75 kDa subunits of Complex I have been implicated as hosts of the redox thiol(s) and are potentially involved in protein S-glutathiolation. 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.

The current study was undertaken to address the fundamental questions regarding the deep insights into the redox biochemistry of Complex I. Here we have identified the specific cysteine residues involved in the protein S-glutathiolation of Complex I. We have also functionally characterized NQR and NDH, including their electron transport and O₂^{generation activities resulting from site-specific S-glutathiolation.}

Abbreviations

NQR	NADH ubiquinone reductase, or mitochondrial Complex I
GS-NQR	glutathiolated NQR
NDH	NADH dehydrogenase or flavin protein subcomplex of Complex I
GS-NDH	glutathiolated NDH
O₂^·-	superoxide anion radical
ETC	electron transport chain
SMP	submitochondrial particles
DEPMPO	5-diethoxylphosphoryl-5-methyl-1-pyrroline N-oxide
FMN	flavin mononucleotide
GSH	glutathione
GSSG	oxidized glutathione
SOD	superoxide dismutase
SDS-PAGE	SDS polyacrylamide gel electrophoresis
EPR	electron paramagnetic resonance
MS	mass spectrometry
MS/MS	tandem mass spectrometry
PBS	phosphate buffered saline
β-ME	β-mercaptoethanol

Abbreviations

Footnotes

^{C.L. Chen, J.L. Zweier, and Y.R. Chen, unpublished results.}

Footnotes

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