Superoxide generation from mitochondrial NADH dehydrogenase induces self-inactivation with specific protein radical formation

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

Research output: Contribution to journalArticlepeer-review

Abstract

Mitochondrial superoxide (O2.-) production is an important mediator of oxidative cellular injury. While NADH dehydrogenase (NDH) is a critical site of this O2.- production; its mechanism of O2.- generation is not known. Therefore, the catalytic function of NDH in the mediation of O2.- generation was investigated by EPR spin-trapping. In the presence of NADH, O2 .- generation from NDH was observed and was inhibited by diphenyleneiodinium chloride (DPI), indicating involvement of the FMN-binding site of NDH. Addition of FMN increased O2.- production. Destruction of the cysteine ligands of iron-sulfur clusters decreased O 2.- generation, suggesting a secondary role of this site. This inhibitory effect was reversed by addition of FMN. However, FMN addition could not reverse the inhibition of NDH by either DPI or heat denaturation, demonstrating involvement of both FMN and its FMN-binding protein moiety in the catalysis of O2.- generation. O2.- production by NDH also induced self-inactivation. Immunospin-trapping with anti-DMPO antibody and subsequent mass spectrometry was used to define the sites of oxidative damage of NDH. A DMPO adduct was detected on the 51-kDa subunit and was O2.--dependent. Alkylation of the cysteine residues of NDH significantly inhibited NDH-DMPO spin adduct formation, indicating involvement of protein thiyl radicals. LC/MS/MS analysis of a tryptic digest of the 51-kDa polypeptide revealed that cysteine (Cys206) and tyrosine (Tyr177) were specific sites of NDH-derived protein radical formation. Thus, two domains of the 51-kDa subunit, Gly200-Ala-Gly- Ala-Tyr-Ile-Cys206-Gly-Glu-Glu-Thr-Ala-Leu-Ile-Glu-Ser-Ile-Glu-Gly- Lys219 and Ala176-Tyr177-Glu-Ala-Gly-Leu-Ile- Gly-Lys184, were demonstrated to be susceptible to oxidative attack, and their oxidative modification results in decreased electron transfer activity.

Original languageEnglish (US)
Pages (from-to)37339-37348
Number of pages10
JournalJournal of Biological Chemistry
Volume280
Issue number45
DOIs
StatePublished - Nov 11 2005
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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