πŸ“’ Vinogradov 2016

Oxidation of NADH and ROS production by respiratory complex I1



  • Significant thermodynamic gap between the standard redox potentials of NADH/NAD+ (βˆ’320 mV) and QH2/Q (+60 mV)
  • Mitochondrial and bacterial complex I also catalyzes NADH oxidation by oxygen, yeilding superoxide or H2O2

Intramolecular electron transfer

  • NADH β†’ FMN β†’ N3(N1a) β†’ N1b β†’ N4 β†’ N5 β†’ N6a β†’ N6b β†’ N2 β†’ Q
  • Time scale: ΞΌs
  • Rate-limiting step: NAD dissociation / CoQ reduction

Steady-state activity

  • the concentration of ubiquinone in the lipid phase is as high as about 10 mM
  • Turnover number of complex I as high as 500 1/s (pH 7.4, 25 Β°C)
  • Linear, not hyperbolic, dependence of the NADH oxidase rate on molar fraction of oxidized ubiquinone (Qox/(Qox + Qred)) => Qred competes with Qox for binding at the reactive site with similar affinity.
  • respiratory control phenomenon (slow NADH oxidation in state 4, CoQ are reduced)

Artificial electron acceptors

  • Ferricyanide & hexaammineruthenium(III) (HAR)
  • Ferricyanide: bell-shaped dependence => ping-pong mechanism => caution!
  • HAR: ternary complex mechanism, no inhibition from high NADH => convenient!
  • Bump the turnover rate to 2500 1/s => the half time of NAD+ release should be less than 0.3 msec
  • ATP acts as a competitive (with NADH) activator, thus decreasing the apparent Km for NADH

Proton pumping activity

  • remains a black box. Some subunits are homologs of bacterial Na+/H+ antiporters as proton-conductive channels.
  • 4 protons are translocated per molecule of NADH oxidized and ubiquinone reduced (2Δ“)
  • The NADH oxidase activity of bovine heart SMP could be inhibited by rotenone and piericidin
    • Rotenone-inhibited particles catalyze the NADH:Q1 reductase reaction coupled with proton translocation with the same stoichiometry of 4 H+/2Δ“

Complex I-mediated ROS production

  • Measuring contribution of complex I to overall ROS production by intact mitochondria are extremely difficult (other sources, ROS scavenging)
  • About 0.2–0.3% of the total oxygen consumption
  • Highest: coupled succinate oxidation (reverse electron transport, RET)
  • The dependences of superoxide production: bell-shaped (ping-pong). Only reduced enzyme–NADH complexes reduce oxygen? two NADH/NAD+-binding sites?
  • Generate both hydrogen peroxide and superoxide (Beard was right). The partitioning between the products depends on NADH concentration
  • The very low KmNADH for superoxide production => perhaps a component immediately reacting with oxygen has a substantially higher redox potential than FMN (e.g. iron-sulfur cluster N2)
    • N2 close to the ubiquinone-binding site, 3nm from the membrane plane.
  • C1-catalyzed ROS production is inhibited by ΞΌmolar NAD+ concentrations. It is safe to assume that all redox components of the enzyme are in equilibrium with the NAD+/NADH couple during the steady-state reaction
  • @ -50ΞΌM NADH: E_NAD = -350mV, close to E_FMN = -370mV. Н2О2 production fits the Nernst equation.
  • superoxide production does not fit the Nernst equation, goes on even when the nucleotide pool is 90% oxidized.

Ischemic-reperfusion ROS production

  • might be relevant to the unusual hysteretic kinetics of complex I
    • active (A) state-to-deactivated state (D) when low CoQ; D is equivalent to inhibition by rotenone
    • D- to A-form is a slow process
    • Deactivated enzyme will be directly oxidized by oxygen, not by ubiquinone => increased ROS production
  • Oxidizing externally added succinate cannot be considered as a model of any physiologically conceivable situation. No other quantitatively significant cytoplasmic sources of succinate exists in aerobic metabolic pathways.

NAD+/NADH ratio and ROS production

  • Pool (NAD + NADH): 4–7 mM in heart mitchondria
  • nucleotide-binding sites of complex I are always saturated. But free ones are not known.
  • NAD+/NADH ratio : 8 in liver mitochondria. 1 in heart mitochondria perfused in 10mM glucose.
  • But in vivo complex I-mediated ROS production is many-fold lower than measured under experimentally β€œoptimal” conditions.

PMF-dependent ROS production ?

  • The production of ROS depends on the amount (concentration) of reactive sites accessible to oxygen
  • PMF only influences redox state of the oxygen reactive sites.

physiological and pathophysiological significance of complex I and other mitochondrial enzyme-mediated ROS production

  • About 15% of intracellular hydrogen peroxide is produced by mitochondria in the rat liver.
  • Mitochondria do not produce that much ROS as we think. Local oxygen concentration is low (10ΞΌM) due to consumption. (Saturated air oxygen: 200ΞΌM)
  • All flavo- and iron-sulfur proteins could react wiht oxygen to produce ROS => protection from O2 attack and ROS scavenging are needed.
  • Are ROS real signaling molecules? They nonenzymatically oxidize the target instead of simple binding.
  • ROS production linearly depends on O2 concentration in RET.
  • Function of ROS? unfavorable leakage reaction vs normal metabolic intermediates
    • reductive stress can be induced by the antioxidants: abnormal anabolic activity (FA synthesis), malignancy, activation of ROS generation


  1. Vinogradov AD, Grivennikova VG. Oxidation of NADH and ROS production by respiratory complex I. Biochim. Biophys. Acta 2016;1857(7):863-871. doi:10.1016/j.bbabio.2015.11.004. ↩︎