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

Abstract

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 decylubiquinone (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 superoxide generation. This suggests that if electron transfer was inhibited under pathological conditions, superoxide generation from the SF would be increased.

Engineering the Respiratory Complex I to Energy-converting NADPH:Ubiquinone Oxidoreductase

“…Recently, it was proposed that the site where ROS are generated is variable and determined by the reduction state of the individual cofactors (47). Thus, the various sites of ROS production reported under different experimental conditions might derive from different states of the complex (47). Under our experimental conditions the ROS are produced at the flavin site because piericidin A did not inhibit ROS production ( Fig.…”

“…Most investigators agree on the flavin site, whereas the ubiquinone-binding site and the Fe/S clusters N1a and N2 have also been proposed to be responsible for the generation of ROS (46). Recently, it was proposed that the site where ROS are generated is variable and determined by the reduction state of the individual cofactors (47). Thus, the various sites of ROS production reported under different experimental conditions might derive from different states of the complex (47).…”

Mitochondrial Respiratory Supercomplex Association Limits Production of Reactive Oxygen Species from Complex I

“…This may take place in (23). The site of electron escape to oxygen is controversial and either FMNH 2 or ubisemiquinone or iron sulfur cluster N2 have been suggested as the possible sites (23,26,30,52,55).…”

Mammalian NADH:ubiquinone oxidoreductase (Complex I) and nicotinamide nucleotide transhydrogenase (Nnt) together regulate the mitochondrial production of H2O2—Implications for their role in disease, especially cancer

“…The reaction with O 2 has also been studied with Yoshikawa's Complex I (Ohnishi et al 2010). Based on EPR studies it was hypothesized that the semiquinone forms of both FMN (i.e.…”