Mitochondrial-Produced Reactive Oxygen Species Matthew Zimmerman, - PDF document

6/4/2013 Mitochondrial-Produced Reactive Oxygen Species Matthew Zimmerman, PhD Associate Professor Cellular & Integrative Physiology University of Nebraska Medical Center mczimmerman@unmc.edu Summer 2013 BIOC 998 ‐ 590 UNL Lecture Outline 1. Complex I and Complex III – Primary sources of ROS in mitochondria 2. Other sources of mitochondrial- produced reactive oxygen species (ROS)  NADPH oxidase (Nox4) 3. Mitochondrial-localized antioxidants 4. Methods to measure mitochondrial- produced ROS 5. Diseases associated with mitochondrial-produced ROS  Amyotrophic lateral sclerosis (ALS; aka Lou Gehrig’s Murphy MP. (2009) Biochem J. 417:1-13) disease) 1

6/4/2013 Sources of Reactive Oxygen Species • Mitochondria • NADPH oxidase • Xanthine oxidase • Lipoxygenase • Nitric oxide synthases Turrens JF. (2003) J Physiol. 552.2:335-344 NADPH oxidase Mitochondrial ‐ Produced ROS  Generally accepted that mitochondrial energy metabolism is the most quantitatively important source of ROS is most cells  ‐ ) is the primary (and  Superoxide (O 2 proximal) ROS generated by mitochondria  ≈ 0.2 ‐ 2 % of oxygen consumed by mitochondria is converted to superoxide  As electrons flow down chain they can “leak” o ff chain on to oxygen → superoxide  Presence of SOD in both matrix and intermembrane space indicates importance Zhang DX. (2006). Am J Physiol. 292:H2023-31)  ‐ from mitochondria of removing O 2  MnSOD knock ‐ out mice are perinatal lethal 2

6/4/2013 Mitochondrial ‐ Produced Superoxide  One-electron reduction of oxygen is thermodynamically favorable for many mitochondrial oxidoreductases due to the moderate redox potential of the superoxide/dioxygen couple Under what conditions can mitochondria electron transport chain (ETC) produced superoxide? 1. Mitochondria not making ATP and electron carriers are fully reduced 2. High NADH/NAD+ ratio in mitochondria matrix • Can be caused by damage to ETC, slow respiration, or ischemia Complex I: A Primary Source of Mitochondrial- Produced Superoxide Complex I (aka NADH-ubiquinone oxidoreductase; NADH dehydrogenase) • Major entry point for electrons into the electron transport chain (ETC) • Flavin mononucleotide (FMN) accepts electrons from NADH • FMN passes electrons to chain of FeS centers (n=7) and finally to CoQ  - from the reaction of oxygen • Produces O 2 with the fully reduced FMN (dependent on  - NADH/NAD + ratio) O 2 • Electrons may also leak off FeS centers • Inhibition of respiratory chain or increased levels of NADH increases NADH/NAD + ratio  - and, in turn produces O 2 3

6/4/2013 Complex I: A Primary Source of Mitochondrial- Produced Reactive Oxygen Species Reverse Electron Transfer (RET) production of superoxide RET: high  p and high CoQH 2 /CoQ Modified from Murphy MP. (2009) • Electrons are transferred against redox potential gradient (reduced CoQ NAD+) • Occurs during low ATP production resulting in a high protonmotive force (  p) and reduced CoQ (succinate or  -glycerophosphate supply electrons to reduce CoQ) • Rate of RET-dependent superoxide production may be the highest that can occur in mitochondria Complex I: A Primary Source of Mitochondrial- Produced Reactive Oxygen Species Increasing Complex I-produced superoxide experimentally: Rotenone- induced inhibition of Complex I • Rotenone binds to the CoQ-binding site • Electrons in Complex I “leak” from either FMN or FeS centers to oxygen producing superoxide Modified from Liu Y. et al. (2002). J Neurochem. 780-7. 4

6/4/2013 Complex III: A Primary Source of Mitochondrial- Produced Reactive Oxygen Species Complex III (aka ubiquinone:cytochrome c reductase) • Oxidizes CoQ using cytochrome c as  ‐ O 2 electron acceptor • Reduced CoQ (QH 2 ) transfers one electron to FeS protein (ISP, aka Rieske protein) and eventually cytochrome c • The resulting semiquinone (Q -  ) transfers electrons to cytochrome b, then to the Q i site which results in the reduction of another CoQ molecule (Q-cycle)  ‐ • The semiquinone (Q -  ) is unstable and can O 2 donate electron to oxygen forming Modified from Turrens JF, 2003 superoxide • In matrix and intermembrane space Complex III: A Primary Source of Mitochondrial- Produced Reactive Oxygen Species Increasing Complex III-produced superoxide experimentally: Antimycin- induced inhibition of Complex III • Antimycin blocks the transfer of electrons to the Q i -site, which results in the accumulation of the unstable semiquinone • The unstable semiquinone can transfer electrons to oxygen producing superoxide Andreyev A.U., et al. (2005). Biochemistry (Moscow). 70:200-14. 5

6/4/2013 Additional Sources of Mitochondrial- Produced Reactive Oxygen Species 1. Cytochrome b 5 reductase: • Outer mitochondrial membrane localization • Oxidizes cytoplasmic NAD(P)H • Reduces cytochrome b 5 in outer membrane  - (~ 300 nmol/min/mg protein) • May produce O 2 • Upregulated in schizophrenic patients 2. Monoamine oxidase (MAO): • Outer mitochondrial membrane localization • Critical in turnover of monoamine neurotransmitters • Catalyze the oxidative deamination of biogenic Bortolato M et al. Adv Drug Deliv Rev. amines aldehyde and release of H 2 O 2 2008 • May be involved in ischemia, aging, Parkinson’s disease Additional Sources of Mitochondrial- Produced Reactive Oxygen Species 3. Dihyroorotate dehydrogenase (DHOH): • Located at the outer surface of inner membrane • In the process of pyrimidine nucleotide synthesis, DHOH converts dihyroorotate to orotate • Electron receptor is coenzyme Q and in absence of coenzyme Q produces H 2 O 2 ( in vitro ) • Role in producing ROS in vivo remains unclear and controversial 4. Dehydrogenase of  -glycerophosphate: • Located at the outer surface of inner membrane • Uses coenzyme Q as electron receptor and catalyzes oxidation of glycerol-3-phosphate to dihydroxyacetone • Studies in mice and drosophila suggest it produces H 2 O 2 5. Aconitase: • Localized in matrix • Catalyzes conversion of citrate to isocitrate (tricarboxylic acid (TCA) cycle)  - and, in turn, produces OH  most likely via Fe 2+ release • Inactivated by O 2 6

6/4/2013 Additional Sources of Mitochondrial- Produced Reactive Oxygen Species 6.  -Ketoglutarate dehydrogenase complex: • Located on the matrix side of inner membrane Uses NAD+ as electron acceptor and catalyzes oxidation of  -ketoglutarate • to succinyl-CoA • Similar to other sources, limited supply of electron acceptor promotes production of ROS 7. Succinate dehydrogenase (SDH; aka Complex II): • Located at the inner surface of inner membrane • Flavoprotein that oxidizes succinate to furmarate using coenzyme Q as electron receptor • Isolated SDH can produce ROS (again in absence of electron Modified from Turrens JF, 2003 receptor) • Mutations in SDH subunits results in an increase in mitochondrial-localized ROS, particularly superoxide Link between NADPH oxidase and mitochondria? NADPH oxidase Modified from Wang G., et al. 2004 J Neurosci . 24(24):5516-24 • Gp91 phox (Nox2) is distributed in the cytoplasm of neurons and is “ particularly abundant near mitochondria”. 7

6/4/2013 NADPH Oxidase (NOX) ‐ Derived ROS  Multi ‐ subunit, membrane bound complex that passes electrons through the membrane from NADPH or NADH to oxygen → superoxide  100x more selective for NADPH over NADH  Phagocyte NADPH oxidase first example of a system that produces ROS as the primary function (not as a byproduct)  Responsible for phagocyte respiratory burst  Respiratory burst absent in Chronic Granulomatous Disease (CGD) – patients lacking cytochrome b 558 (gp91phox or Nox2 + p22phox)  NADPH oxidase subunits include:  7 Nox isoforms (Nox1, Nox2, Nox3, Nox4, Nox5, Duox1, Duox2)  Often referred to as the catalytic subunits  2 organizer subunits (p47phox, NOXO1)  2 activator subunits (p67phox, NOXA1)  2 Duox ‐ specific maturation subunits (DUOXA1, DUOXA2)  1 stabilizing subunit (p22phox)  p40phox  Active complexes made of a mixture of these subunits NADPH Oxidase Conserved Structural Properties Nox Enzymes  ‐ O 2 1. NADPH binding site in COOH terminus 2. Flavin adenine dinucleotide (FAD) binding region in COOH terminus 3. Six conserved transmembrane domains 4. Four conserved heme ‐ binding histidines  Electrons are passed from NADPH → FAD → 1 st heme → 2 nd heme → Oxygen = superoxide Bedard and Krause, Physiol Re v 2007 8

6/4/2013 Nox4, NADPH oxidase catalytic subunit, in mitochondria PNAS, 2009 106(34):14385-14390 0 Nox4 in neuron mitochondria Case AJ, et al. Mitochondrial-localized NADPH Oxidase 4 is a Source of Superoxide in Angiotensin II-stimulated Neurons. Am J Physiol Heart Circ Physiol. 2013 9

6/4/2013 Nox4 in neuron mitochondria MitoProt Score*: Nox4 – 0.977 ; MnSOD – 0.985; Prx3 – 0.993; LDH – 0.023; Actin – 0.016 * M.G. Claros, P. Vincens. Computational method to predict mitochondrially imported proteins and their targeting sequences . 1996 . Eur. J. Biochem. 241, 770-786. Case AJ, et al. Mitochondrial-localized NADPH Oxidase 4 is a Source of Superoxide in Angiotensin II-stimulated Neurons. Am J Physiol Heart Circ Physiol. 2013 Silencing Nox4 with siRNA attenuates AngII- induced increase in superoxide levels Case AJ, et al. Mitochondrial-localized NADPH Oxidase 4 is a Source of Superoxide in Angiotensin II-stimulated Neurons. Am J Physiol Heart Circ Physiol. 2013 10