The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria.

TitleThe mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria.
Publication TypeJournal Article
Year of Publication2006
AuthorsKussmaul, L, Hirst, J
JournalProc Natl Acad Sci U S A
Volume103
Issue20
Pagination7607-12
Date Published2006 May 16
ISSN0027-8424
KeywordsAnimals, Cattle, Electron Transport Complex I, Electrons, Flavins, Humans, Hydrogen Peroxide, Mitochondria, Heart, NAD, Oxidants, Oxidation-Reduction, Oxygen, Superoxides
Abstract

NADH:ubiquinone oxidoreductase (complex I) is a major source of reactive oxygen species in mitochondria and a significant contributor to cellular oxidative stress. Here, we describe the kinetic and molecular mechanism of superoxide production by complex I isolated from bovine heart mitochondria and confirm that it produces predominantly superoxide, not hydrogen peroxide. Redox titrations and electron paramagnetic resonance spectroscopy exclude the iron-sulfur clusters and flavin radical as the source of superoxide, and, in the absence of a proton motive force, superoxide formation is not enhanced during turnover. Therefore, superoxide is formed by the transfer of one electron from fully reduced flavin to O2. The resulting flavin radical is unstable, so the remaining electron is probably redistributed to the iron-sulfur centers. The rate of superoxide production is determined by a bimolecular reaction between O2 and reduced flavin in an empty active site. The proportion of the flavin that is thus competent for reaction is set by a preequilibrium, determined by the dissociation constants of NADH and NAD+, and the reduction potentials of the flavin and NAD+. Consequently, the ratio and concentrations of NADH and NAD+ determine the rate of superoxide formation. This result clearly links our mechanism for the isolated enzyme to studies on intact mitochondria, in which superoxide production is enhanced when the NAD+ pool is reduced. Therefore, our mechanism forms a foundation for formulating causative connections between complex I defects and pathological effects.

DOI10.1073/pnas.0510977103
Alternate JournalProc. Natl. Acad. Sci. U.S.A.
Citation Key10.1073/pnas.0510977103
PubMed ID16682634
PubMed Central IDPMC1472492
Grant ListMC_U105663141 / / Medical Research Council / United Kingdom