New insights into the superoxide generation sites in bovine heart NADH-ubiquinone oxidoreductase (Complex I) The significance of protein-associated ubiquinone and the dynamic shifting of generation sites between semiflavin and semiquinone radicals

作者：Ohnishi S Tsuyoshi^*; Shinzawa Itoh Kyoko; Ohta Kazuhiro; Yoshikawa Shinya; Ohnishi Tomoko

来源：Biochimica et Biophysica Acta-Bioenergetics, 2010, 1797(12): 1901-1909.

DOI：10.1016/j.bbabio.2010.05.012

• NADH ubiquinone oxidoreductase complex I
• Electron transport
• Superoxide generation
• Semiflavin radical
• Iron-sulfur cluster N2
• Semiquinone radical
• Quinone pocket inhibitors

Considerable disagreement still exists concerning the superoxide generation sites in the purified bovine heart NADH-ubiquinone oxidoreductase (complex I) Majority of investigators agree that superoxide is generated at the flavin site Here we present a new hypothesis that the generation of superoxide reflects a dynamic balance between the flavosemiquinone (semiflavin or SF) and the semiquinone (SQ) like a "tug-of war" through electrons All preparations of bovine heart complex I which have been isolated at Yoshikawa s laboratory have one protein-bound endogenous ubiquinone per complex I (Shinzawa-Itoh et al Biochemistry 49 (2010) 487-492) Using these preparations we measured (i) EPR signals of the SF the SQ and iron-sulfur cluster N2 simultaneously with cryogenic EPR and (ii) superoxide production with both the room temperature spin-trapping technique and the partially acetylated cytochrome c method Our experimental evidence was (1) without added decylubiquinoni (DBQ) no catalytic oxidation of NADH occurs The NADH addition produced mostly SF and It generated superoxide as reported by Kussmaul and Hirst (PNAS 103 (2006) 7607-7612) (2) During catalytic electron transfer from NADH to DBQ the superoxide generation site was mostly shifted to the SQ (3) A quinone-pocket binding inhibitor (rotenone or piericidin A) inhibits the catalytic formation of the SQ and it enhances the formation of SF and increases the overall

• 出版日期2010-12