πŸ“’ Kussmaul 2006

The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria1



  • mito-derived ROS from complex I and III
  • Most of these studies examined intact mitochondria or submitochondrial particles, in which it is difficult to correlate observations directly to complex I, or to define and control the conditions precisely
  • we propose a comprehensive kinetic and molecular mechanism for superoxide production by complex I


The Kinetic Mechanism of Superoxide Production

  • In air-saturated solution [O2 β‰ˆ250 ΞΌM (24)], complex I generates β‰ˆ40 nmol O2β€’βˆ’ minβˆ’1 mgβˆ’1
  • the reaction between complex I and NADH, common to all of the assays, is >2,000 times faster than the reaction of reduced complex I with O2. SOX generation vs NADH is saturated quickly
  • Superoxide generation by complex I is strongly inhibited by NAD+ => a preequilibrium is established between NADH, NAD+, and different states of complex I

The Molecular Mechanism of Superoxide Formation

  • In isolated complex I, superoxide is produced by the flavin, because it is not produced by any of the FeS clusters.
  • the pH dependence of E1/2 is further confirmation for the participation of the fully reduced flavin (FMNH-) in superoxide production. Only reduced flavin in an empty active site reacts with O2

  • NAD+ and NADH determine the equilibrium concentration of the free reduced flavin
  • E_NAD+ is well established [βˆ’0.335 V, pH 7.5 (32)]. E_FMN has been measured by EPR (βˆ’0.375V, pH 7.5)
  • Predicted values of E1/2 for superoxide production (by using Eq. 3) are βˆ’0.44 V (33) and βˆ’0.38 V (34), close to the observed value of βˆ’0.36 V, the strong disagreement between the two sets of values prevents any meaningful interpretation


  • Fully reduced flavin is a low potential electron donor capable of O2 reduction
  • formation of O2β€’βˆ’ from O2 [E0β€² = βˆ’0.33 V, 1 atm (1 atm = 101.3 kPa) O2 (24)] is less favorable thermodynamically than formation of H2O2 [E0β€² = +0.28 V, pH 7 (24)]. why a fully reduced flavin should produce O2β€’βˆ’?
    • O2 and O2β€’βˆ’ probably bind only weakly and nonspecifically in complex I. H2O2 generation is too slow.
  • In complex I, the flavin radical is thermodynamically unstable (30), supporting redistribution, but it is not possible to identify a single FeS cluster to oxidize (or re-reduce) it
  • the rate-limiting step in superoxide production by isolated complex I is a bimolecular reaction between β€œcompetent” enzyme and O2.


  1. Kussmaul L, Hirst J. The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria. Proc Natl Acad Sci U S A. 2006;103(20):7607-12. PMC1472492↩︎