Russian National Research Medical University
The respiratory chain: a strategy to recover
energy
The mitochondrial electron transport chain
functioning and control
Oxidative phosphorylation
Maxim A. Abakumov
Moscow, 2014
Energy flow in cell
Glicolysis
Electron transfer chain
Oxidative phosphorilation
TCA cycle
Outer membrane
Inner membrane
Intermembrane space
Matrix
Cristae
TCA total energy outcome
• Acetyl-CoA + 3 NAD+ + Q(FAD) + GDP + Pi +2 H20 
HS-CoA + 3NADH + QH2(FADH2)+ GTP + 2 CO2 + 2 H+
•
•
•
•
•
Isocitrate Dehydrogenase
α-ketoglutarate Dehydrogenase
Succinyl-CoA Synthetase
Sunccinate Dehydrogenase
Malate Dehydrogenase
1 NADH=2.5 ATP
1 NADH=2.5 ATP
1 GTP=1 ATP
1 QH2=1.5 ATP
1 NADH=2.5 ATP
• NADH and FADH2is not an ATP (no cash energy) – further
conversion is needed
NADH and NAD+
•
•
•
•
•
NAD – Nicotine Adenine Dinucleotide
Produced from niacine (vitamine B3)
NADH – reduced form
NADH – high energy (HE) electron carrier
NAD+ – oxidized form
• NAD+ + H+ + 2e- → NADH + H+
NADH and NAD+
O
H
NH2
O
HO P O
O N
HO P O
Reduction
+ H+ + 2eOH OH
NH2
N
HO P O
N
O
NH2
O
+
O
H
H
Oxidation
O N
O
OH OH
NH2
N
HO P O
N
O
O
N
O
OH OH
N
N
O
OH OH
N
FADH2 and FAD
•
•
•
•
•
FAD – Flavine Adenine Dinucleotide
Techically not a dinucleotide
FADH2 – reduced form (oxidizer)
FADH2 – high energy (HE) electron carrier
FAD – oxidized form (reductor)
• FAD + 2H+ + 2e- → FADH2
FADH2 and FAD
O
H3C
N
H3C
N
NH
Flavin
Ribitol
N
H
H
HO
H
HO
H
HO
H
O
NH2
N
O
O
N
O P O P O
O
N
-
O
OH
OH OH
ADP
N
FADH2 and FAD
FAD
FADH2
O
H3C
N
H3C
N
H3C
NH
N
H
H
HO
H
HO
H
HO
H
H
N
H3C
O
N
H
NH2
N
O
O
O P O P O
-
O
N
N
O
OH
OH OH
N
O
NH
N
H
O
Electron transfer chain
• Electron transfer chain (ETC) – group of enzymes
located on inner membrane of mitochondria
• Convert energy from NADH/FADH2 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 + Pi→ 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
QH2
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+
NAD+
FMN
FMNH2
Fe2+S
Fe3+S
CoQ
CoQH2
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
FADH2 and Fe-S clusters to CoQ
• Not a proton pump
Complex II
Complex III
CoQ-Cytochrome c Reductase
• 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 bL and bH
• Cytochromes, like Fe in Fe-S clusters, are oneelectron transfer agents
Complex III
• UQH2 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 O2 to produce 2H2O
• 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 FADH2 gives gives energy for 6 H+ transport
into intramembrane space
• How proton gradient energy is converted into
ATP energy?
ETC summary
• http://www.learnerstv.com/animation/animat
ion.php?ani=177&cat=biology
How is H+ gradient energy used for
ATP synthesis?
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-synthase
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
ATP-ADP Translocase
P/O ratio
• How many ATP is made per 2e-
• 1 ATP – 4H+
• NADH – 10H+
• P/ONADH = 10/4 = 2,5
• FADH2 = 6H+
• P/OFAD = 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 (Fe3+)
• 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 ATPsynthase (Fo 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
http://sbxsupplements.com/blog/Complementary-Treatment-ofSchizophrenia-with-Antioxidants
Antioxidants
Enzymatic
 Superoxide dismutase
 Catalase
 Glutathione peroxidase
 Glutathione reductase
Non-enzymatic
 Vitamins
 C and E
 Carotenoids
 Flavonoids
 Minerals
Manganese
 Copper
 Zinc
 Selenium
Antioxidant enzymes
Superoxide Dismutase
2O2- + 2H+
O2 +H2O2 + 2H2O
Catalase
2H2O2
O2 + 2H2O
Antioxidant enzymes
• 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
CH3
α-Tocopherol (vitamin E)
HO
CH3
H3C
O
CH3
CH3
CH3
CH3
CH3
Free radicals
CH3
O
CH3
H3C
O
CH3
CH3
CH3
CH3
CH3
Tocopheroxyl free radical
(Oxidized vitamin E)
Cell oxidative status