📒 Vinogradov 2016
Oxidation of NADH and ROS production by respiratory complex I1
- Significant thermodynamic gap between the standard redox potentials of NADH/NAD+ (−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 → FMN → N3(N1a) → N1b → N4 → N5 → N6a → N6b → N2 → Q
- Time scale: μs
- Rate-limiting step: NAD dissociation / CoQ reduction
- 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 °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 (2ē)
- 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+/2ē
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 μmolar 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
- @ -50μM 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 “optimal” 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 (10μM) due to consumption. (Saturated air oxygen: 200μM)
- 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
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. ↩︎