📒 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 ≈250 μM (24)], complex I generates ≈40 nmol O2•− min−1 mg−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 [−0.335 V, pH 7.5 (32)]. E_FMN has been measured by EPR (−0.375V, pH 7.5)
- Predicted values of E1/2 for superoxide production (by using Eq. 3) are −0.44 V (33) and −0.38 V (34), close to the observed value of −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 O2•− from O2 [E0′ = −0.33 V, 1 atm (1 atm = 101.3 kPa) O2 (24)] is less favorable thermodynamically than formation of H2O2 [E0′ = +0.28 V, pH 7 (24)]. why a fully reduced flavin should produce O2•−?
- O2 and O2•− 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 “competent” enzyme and O2.