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ETC & MITOCHONDRIAL OXIDATIVE PHOSPHORYLATION, Enzyme, Prosthetic…
ETC & MITOCHONDRIAL OXIDATIVE PHOSPHORYLATION
Energy Metabolism
Acetyl CoA
Common product of macromolecules
Recycle NADH/H+ & FADH2
Mitochondria
Heat Production / Proton leak / Mitochondrial uncoupling
Protons enter mitochondrial matrix w/o contributing to ATP synthesis
Unharnessed potential energy of proton electrochemical gradient = heat
Redox reaction = transfer of electron to O2
Cytosol to mitochondria
Respiratory Chain / Electron Transport Chain
Produces ATP
Oxidative Phosphorylation
Free energy trapped as high energy phosphate
Proton Gradient
Stored potential energy; basis of coupling in oxidative phosphorylation / chemiosmotic coupling
Reducing equivalents of ETC
NADH: cytosol to mitochondrial matrix
FADH2: mitochondrial matrix to inside mitochondria
Components and Enzymes in Mitochondrial Oxidative Phosphorylation
Complexes
II- Succinate Dehydrogenase
FAD, Fe-S Protein
Coenzyme Q
0
III- Q- Cyt C Oxidoreductase
Fe-S Protein, Cytochrome I
Cytochrome C
4
I - NADH-Q Oxidoreductase
Fe-S Centers, FMN
Coenzyme Q
4
IV- Cyt C Oxidase
Copper, Heme
Oxygen
2
1. Flavoproteins
2. Fe-S
3. Mobile Proteins
4. Enzymes
Hydroxyperoxidases
Hydrogen peroxide / Organic peroxide as substrate
Oxygenases
Direct transfer of O2 to substrate
Oxidases
Removes hydrogen using oxygen
Forms H2O or H2O2
Cytochrome Oxidase Complex 4, Flavoproteins
Dehydrogenases
Transfer 1 hydrogen from 1 substrate
Cannot use O2
NAD & FAD
Coenzyme C
Transfer 1 e- from Complex 3 to 4
Heme undergoes redox
Coenzyme Q
Transfer 2 e- from 1 to 2 or 2 to 3
3 Forms
Quinone / Ubiquinone
Oxidized Q
Hydroquinone / Ubiquinol
Reduced Q
Semiquinone
Free radical; intermediate between oxidized & reduced Q
Single electron transfer
Redox between Fe2+ & Fe3+
Accepts 1 electron = forms semiquinone intermediate
Oxidized FMN/FAD can be reduced
Complex 1
FMN --> FMNH2
Complex 2
FAD --> FADH2
Electron Transfer
Electron Transport Chain
Respiratory Chain Complexes
COMPLEX 2️⃣
FADH2 produced during conversion of succinate to fumarate in TCA
Succinate Q reductase
Steps
2 e- & 2 p+ transferred to complex 2 FAD = FADH2
FADH2 transfers e- to ubiquinone via Fe-S centers
Fe-S centers tranfer e- to CoQ = CoQH2
e- passed via Fe-S centers to Q
G3P & Acyl CoA pass e- to Q
G3P: generated in TAG breakdown / glycolysis
Succinate Dehydrogease / Succinate Ubiquinone Oxidoreductase
Succinate + Q --> Fumarate + QH2
Does not transport H+ in intermembrane
Produces 1.5 ATP per 1 mol FADH2
Bypass complex 1
COMPLEX 4️⃣
Oxidizes reduced cytochrome C
Cytochrome C Oxidase
4 Cyt C + O2 + 8 H --> 4 Cyt C + 2 H2O + 4H
2 e- used ; 1 H2O produced per cycle
Transports 2H in intermembrane
Cycle occurs 2x bec. of 2 Cyt C
Steps
Complex IV transfer e- from Cyt C to copper
e- passed to Cyt a, a3, CuB
CuB transfer e- to O2 = H2O
2 protons transferred
4 electrons needed to reduce 1 mole O2 to 2 mol H2O
COMPLEX 1️⃣
NADH binds
NADH + Q + 5H+ --> NAD + QH2 + 4H
NADH Dehydrogenase / NADH-Ubiquinone Oxidoreductase
Transports 4H in intermembrane space
Major source of ROS
Produces 2.5 ATP per 1 mol NADH
Steps
4 protons translocated
e- transferred via Fe-S centers
e- transferred from NADH to FMN = FMNH2
Source of NADH: fat oxidation, glycolysis, TCA
COMPLEX 3️⃣
Q cycle: e- passed from QH2 to cytochrome
Oxidation of 2 QH2 to Q: releases 4H
Reduction of Q to QH2: takes 2H
Cytochrome bc1 complex / Ubiquinol Cytochrome C Oxidoreductase
QH2 + 2 Cyt C + 2 H --> Q + 2 Cyt C + 4H
Transports 4H in intermembrane
3 Forms
Q: Quinone / Ubiquinone
QH2: Hydroquinone / Ubiquinol
QH: Semiquinone
Steps
CoQH2 donates e- to Fe-S centers
Fe-S centers transfer e- to cytochrome C
4 protons transferred
Oxidative Phosphorylation at Respiratory Chain
Any movement in F0 will lead to F1 movement
Y stalk
: attached F0 to F1
ATP Synthase
: generates ATP
Complex V Domains
F0 subunit
Hydrophobic; inner mitochondrial membrane
Movable subunit = main rotatory movement
F1 subunit
Alpha subunit
Binding site of substrates for ATP synthesis
For catalytic activity of enzyme
ATP synthesis
Beta subunit
each has opening
3 B-subunits in between a-subunits
Ball-shaped around an axis
NADH ➡️ FMN ➡️ Fe-S Centers ➡️ Q
Shuttle Systems
Malate Aspartate Shuttle
Mechanism
NADH enters via porins
Malate dehydrogenase: transfer reducing equivalent of NADH to oxaloacetate = yields malate
Malate enters mitochondria via malate-asparate shuttle
Malate a-KG antiporter
Malate dehydrogenases forms NADH & Oxaloacetate
Transaminase & Glutamate: transaminates oxaloacetate to aspartate
Aspartate cross inner mitochondria via glutamate-aspartate transporter
Aspartate aminotransferase:
Aspartate converted to oxaloacetate
Oxaloacetate regenerated in cytosol
1 more item...
Glutamate becomes a-KG
NADH pass directly to respiratory chain
Liver, Heart, Kidney
Enters through complex 1
Transfers reducing equivalent of NADH
Yields 2.5 ATP
Glycerol 3 Phosphate Shuttle
Muscles, Brain
Forms FADH2
Yields 1.5 ATP bec. complex 1 bypassed
Mechanism
G3P dehydrogenase with NADH: Reduce DHAP to G3P
G3P donates to FAD via mitochondrial G3P dehydrogenase
FADH2 reduces Q --> binds to Complex 2
DHAP crosses outer mitochondrial membrane
Phosphocreatine Shuttle
Creatine
Carrier of high energy phosphate group
Replenishes ATP
Creatine smaller than ADP = faster transport
Fast transfer system for new ATP
Transfers energy to active muscles (hearts/skeletal)
Inhibitory Toxins
ETC Inhibitors
Complex II
Carboxin, Thenoyltrifluoroacetone
Binds to ubiquinone binding site
Malonate
Succinate analog / Competitive inhibitor
Complex III
Antimycin
Binds to Qi site
Myxothiaxol, Stigmatellin
Binds to Qo site
Complex I
Rotenone
Binds to ubiquinone binding site
Piericidin, Amytal, Barbiturates
blocks e- transfer from Fe-S to ubiquinone
Complex IV
Cyanide, Axide, H2S
Binds to oxidized heme = Fe3+
CO
Binds to reduced heme = Fe2+
Uncouplers of Oxidative Phosphorylation
Inhibits phosphorylation of ADP w/o affecting ETC
O2 reduced to H2O, no ATP produced
Toxic = RR not controlled by ADP & Pi
Generates heat
Ionophores
2,4 dinitrophenol
Valinomycin
Thermogenin / Uncoupling protein
Generates body heat
For newborns during hibernation
Oxidative Phosphorylation Inhibitors
Atractyloside
Inhibits exchange of ATP/ADP
Carbonyl cyanide m-chlorophenyl hydrazone (CCPP) & 2,4 dinitrophenol
Used as poison / weightloss
Disrupts proton gradient, causes uncoupling of proton pump
Oligomycin
Prevents proton movement via ATP synthase
Clinical Correlation
Brown Adipose Tissue / Cold-induced thermogenesis
Uncoupling protein (UCP-1) carries protons and uncouples ATP synthesis
Leber Hereditary Optic Neuropathy
Blindness due to optic nerve death
Due to single base mutation in Complex 1
MELAS: Mitochondrial Encephalopathy, Lactic Acidosis, Stroke
NADH-Q oxidoreductase (Complex 1) or Cytochrome oxidase (Complex 4) deficiency
Mutation in mitochondrial DNA
Diabetes mellitus & Alzheimers
Reactive Oxygen Species (ROS)
Oxygen accepts 1 or 2 e- = forms superoxide anions/peroxide
Antioxidants protect against radical damage
Glutathione reductase: maintains reduced form of glutathione
Fatal Infantile Mitochondrial Myopathy and Renal Dysfunction
Absence/ deficiency of oxidoreductase enzyme
Enzyme
Prosthetic Groups
Acceptor
No. of H+ transported