Superoxide Radical Formation by Pure Complex I (NADH:Ubiquinone Oxidoreductase) from Yarrowia lipolytica

Abstract

Generation of reactive oxygen species (ROS) is increasingly recognized as an important cellular process involved in numerous physiological and pathophysiological processes. Complex I (NADH:ubiquinone oxidoreductase) is considered as one of the major sources of ROS within mitochondria. Yet, the exact site and mechanism of superoxide production by this large membrane-bound multiprotein complex has remained controversial. Here we show that isolated complex I from Yarrowia lipolytica forms superoxide at a rate of 0.15% of the rate measured for catalytic turnover. Superoxide production is not inhibited by ubiquinone analogous inhibitors. Because mutant complex I lacking a detectable iron-sulfur cluster N2 exhibited the same rate of ROS production, this terminal redox center could be excluded as a source of electrons. From the effect of different ubiquinone derivatives and pH on this side reaction of complex I we concluded that oxygen accepts electrons from FMNH 2 or FMN semiquinone either directly or via more hydrophilic ubiquinone derivatives.Over the last decade the processes leading to the production of superoxide and other reactive oxygen species (ROS) 1 have gained much attention. ROS seem to be involved in apoptosis, the development of various pathological states, aging, and the regulation of cell metabolism. It is generally accepted that production of reactive oxygen species is an inherent property of the mitochondrial respiratory chain of eucaryotic cells. Oxidation of certain redox centers in complex I and III by molecular oxygen results in the production of superoxide anion radical O 2. (see Ref. 1 for a review). Superoxide can then convert into hydrogen peroxide, the highly active hydroxyl radical (OH ⅐ ), and other ROS. It has been shown that O 2 . production by complex I occurs in the mitochondrial matrix, whereas the cytochrome bc 1 complex reduces oxygen primarily on the intermembrane side (2, 3) (see, however, Ref. 4). It has been demonstrated for the cytochrome bc 1 complex that oxygen reduction occurs at the Q P site and is increased markedly under conditions of "oxidant-induced reduction" (5, 6). However, much less is known about the site and mechanism of O 2 . generation in complex I. Thermodynamically, any of the complex I redox centers in the reduced state is capable of donating an electron to molecular oxygen to form a superoxide anion. Eucaryotic NADH:ubiquinone oxidoreductase (complex I or type I NADH dehydrogenase) is the largest and most complex enzyme of the respiratory chain, residing in the inner membrane of mitochondria. In mammals, the enzyme is composed of 46 different subunits (7) and contains non-covalently bound FMN and up to eight iron-sulfur clusters as redox cofactors. Two complex I-associated, electron paramagnetic resonancedetectable semiquinone species with different spin relaxation times have been characterized (8, 9). Complex I catalyzes the transfer of electrons from matrix NADH to membrane ubiquinone coupled to the translocation of four protons across the membrane (10, 11...

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