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

Mitochondrial superoxide (O-2(radical anion).) production is an important mediator of oxidative cellular injury. While NADH dehydrogenase (NDH) is a critical site of this O-2(radical anion) production; its mechanism of O-2(radical anion) generation is not known. Therefore, the catalytic function of NDH in the mediation of O-2(radical anion) generation was investigated by EPR spin-trapping. In the presence of NADH, O-2(radical anion) 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 O-2(radical anion) production. Destruction of the cysteine ligands of iron-sulfur clusters decreased O-2(radical anion) 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 O-2(radical anion) generation. O-2(radical anion) 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 O-2(radical anion)-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 (Cys(206)) and tyrosine (Tyr(177)) were specific sites of NDH-derived protein radical formation. Thus, two domains of the 51-kDa subunit, Gly(200-) Ala-Gly-Ala-Tyr-Ile-Cys(206)- Gly-Glu-Glu- Thr-Ala-Leu-Ile-Glu-Ser-Ile-Glu-Gly-Lys(219) and Ala(176)-Tyr(177)- Glu-Ala-Gly-Leu-Ile-Gly-Lys(184), were demonstrated to be susceptible to oxidative attack, and their oxidative modification results in decreased electron transfer activity.