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

Alexander Galkin,Ulrich Brandt

doi:10.1074/jbc.m504709200

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 FMNH2 or FMN semiquinone either directly or via more hydrophilic ubiquinone derivatives.

Highlights

Over the last decade the processes leading to the production of superoxide and other reactive oxygen species (ROS)1 have gained much attention
As was typically observed for the membrane preparation used that largely consists of mitochondrial fragments, rates of dNADH oxidation were 2–3 times lower than those of dNADH: ubiquinone oxidoreductase activities
As the effect was approximately the same no matter which complex was inhibited, it seems likely that a higher reduction level of the redox centers in complex I was responsible for the increase in superoxide production as has been shown previously for bovine heart submitochondrial particles [39]

Summary

Introduction

Over the last decade the processes leading to the production of superoxide and other reactive oxygen species (ROS) have gained much attention. Oxidation of certain redox centers in complex I and III by molecular oxygen results in the production of superoxide anion radical O2. Production by complex I occurs in the mitochondrial matrix, whereas the cytochrome bc complex reduces oxygen primarily on the intermembrane side [2, 3] (see, Ref. 4). 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. In recent studies the involvement of other cofactors has been discussed, i.e. the most negative iron-sulfur cluster, N1a of complex I [21], or tightly bound semiquinone molecules [22, 23]. Even an enzyme-bound NAD radical has been considered as a possible source of electrons by some authors [25]

Methods

Results

Conclusion