The respiratory chain: a strategy to recover energy The mitochondrial electron transport chain functioning and control Oxidative phosphorylation Russian National Research Medical University Maxim A. Abakumov Moscow, 2014

Energy flow in cell Glicolysis TCA cycle Electron transfer chain Oxidative phosphorilation

Outer membrane Inner membraneIntermembrane space Cristae Matrix

TCA total energy outcome Acetyl-CoA + 3 NAD + + Q(FAD) + GDP + P i +2 H 2 0  HS-CoA + 3NADH + QH 2 (FADH 2 )+ GTP + 2 CO H + Isocitrate Dehydrogenase 1 NADH=2.5 ATP α-ketoglutarate Dehydrogenase 1 NADH=2.5 ATP Succinyl-CoA Synthetase1 GTP=1 ATP Sunccinate Dehydrogenase1 QH 2 =1.5 ATP Malate Dehydrogenase1 NADH=2.5 ATP NADH and FADH 2 is not an ATP (no cash energy) – further conversion is needed

NADH and NAD + NAD – Nicotine Adenine Dinucleotide Produced from niacine (vitamine B 3 ) NADH – reduced form NADH – high energy (HE) electron carrier NAD + – oxidized form NAD + + H + + 2e - → NADH + H +

NADH and NAD + + H + + 2e - Reduction Oxidation

FADH 2 and FAD FAD – Flavine Adenine Dinucleotide Techically not a dinucleotide FADH 2 – reduced form (oxidizer) FADH 2 – high energy (HE) electron carrier FAD – oxidized form (reductor) FAD + 2H + + 2e - → FADH 2

FADH 2 and FAD Flavin Ribitol ADP

FADH 2 and FAD FADFADH 2

Electron transfer chain Electron transfer chain (ETC) – group of enzymes located on inner membrane of mitochondria Convert energy from NADH/FADH 2 electrons into energy of proton gradient on inner membrane Proton gradient is needed for further ADP to ATP conversion Consists of four complexes

Oxidative phosphorilation ADP + P i → ATP Reaction is catalized by ATP-synthase. ATP-synthase is intramembrane multisubunit enzyme (nanorotor) ATP-synthase uses energy of proton gradient

Electron transfer chain Consists of four complexes A lipid soluble coenzyme (UQ, CoQ) and a water soluble protein (cyt c) shuttle between protein complexes Fe-S, Hem, FMN, Cu atoms acts as a cofactors in different complexes Electrons generally fall in energy through the chain - from complexes I and II to complex IV

Electron transfer chain

Coenzyme Q10 (CoQ) Lipid soluble molecule Transfer e - from complex I and II to complex III through membrane

FMN FMN –Flavin Mono Nucleotide Accepts e - from NADH and transfers them to QH 2

Complex I NADH-CoQ Reductase. Electron transfer from NADH to CoQ More than 30 protein subunits - mass of 850 kD Transfers 4H +

Complex I NADH-CoQ Reductase NADH + H + FMN Fe 2+ S CoQ NAD + FMNH 2 Fe 3+ SCoQH 2

Complex I - NADH-CoQ Reductase.

Complex II Succinate-CoQ Reductase Succinate dehydrogenase (from TCA cycle!) Membrane bound enzyme Accepts e - from succinate and pass through FADH 2 and Fe-S clusters to CoQ Not a proton pump

Complex II

Complex III CoQ in membrane passes electrons to cyt c in a unique redox cycle known as the Q cycle 4 H + are released into intermembrane space The principal transmembrane protein in complex III is the b cytochrome - with hemes b L and b H Cytochromes, like Fe in Fe-S clusters, are one- electron transfer agents CoQ-Cytochrome c Reductase

Complex III UQH 2 is lipid soluble e - carrier Cyt c is water soluble e - carrier

Complex IV Cytochrome c oxydase Electrons from cyt c are used in a four-electron reduction of O 2 to produce 2H 2 O Oxygen is the terminal acceptor of electrons in the electron transport pathway Cytochrome c oxidase utilizes 2 hemes and 2 copper sites Complex IV also transports 2H +

Complex IV

ETC

ETC summary 1 NADH gives energy for 10 H + transport into intramembrane space 1 FADH 2 gives gives energy for 6 H + transport into intramembrane space How proton gradient energy is converted into ATP energy?

ETC summary ion.php?ani=177&cat=biology ion.php?ani=177&cat=biology

How is H + gradient energy used for ATP synthesis?

ATP synthase – nanoscale rotating motor H + movement through the channel in ATP synthase leads to ATP synthesis 3H + leads to one full circle 3H + reguired for 1 ATP synthesis

ATP-synthase

ATP is synthesized in mitochondria matrix It must be transported to cytosol 1H + is spend for each ATP transfer 3H + is spend for each ATP synthesis Total 4H + is spend for 1 ATP synthesis ATP-ADP Translocase

P/O ratio How many ATP is made per 2e - 1 ATP – 4H + NADH – 10H + P/O NADH = 10/4 = 2,5 FADH 2 = 6H + P/O FAD = 6/4 = 1,5

ETC and OP inhibitors Rotenone inhibits Complex I Cyanide, azide and CO inhibit Complex IV, binding tightly to the ferric form (Fe 3+ ) Oligomycin and DCCD (Dicyclohexyl carbodiimide) are ATP synthase inhibitors

ETC and OP inhibitors Rotenone -inhibits the transfer of electrons from iron-sulfur centers in complex I to ubiquinone

ETC and OP inhibitors Oligomycin –blocks proton channel on ATP- synthase (F o subunit)

e - /H + transfer uncoupling Uncoupling of e - /H + leads to no proton gradient No ATP synthesis Affected by lipid soluble H + acceptors, such as dinitrophenol

NADH mitochondria/cytosol transport Most NADH used in electron transport is cytosolic and NADH doesn't cross the inner mitochondrial membrane Malate-aspartate shuttle system for NADH transport without actuall NADH transfer

NADH mitochondria/cytosol transport

Free radicals Contain unpaired electron on valent level Highly reactive Reactive Oxygen Species (ROS) Reactive Nitrogen Species (RNS)

Reactive oxygen species

Reactive nitrogen species

Lipid peroxides

Phagosome NADPH oxidase

Antioxidants Schizophrenia-with-Antioxidants

Antioxidants Enzymatic  Superoxide dismutase  Catalase  Glutathione peroxidase  Glutathione reductase Non-enzymatic  Vitamins  C and E  Carotenoids  Flavonoids  Minerals  Manganese  Copper  Zinc  Selenium

2H 2 O 2 O 2 + 2H 2 O Antioxidant enzymes Superoxide Dismutase 2O H + O 2 +H 2 O 2 + 2H 2 O Catalase

Glutathione (GSH) – tripeptide 2x GSH-reduced form GSSG-oxidized form Antioxidant enzymes

Antioxidants Enzymatic  Superoxide dismutase  Catalase  Glutathione peroxidase  Glutathione reductase Non-enzymatic  Vitamins  C and E  Carotenoids  Flavonoids  Minerals  Manganese  Copper  Zinc  Selenium

Vitamin E α-Tocopherol (vitamin E) Tocopheroxyl free radical (Oxidized vitamin E) Free radicals

Cell oxidative status