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4. Cell energetics
 An overview of cell metabolism
 Glycolysis and the tricarboxylic acid cycle
(Krebs cycle)
 Fatty acid oxidation (beta-oxidation)
 Oxidative phosphorylation
 Electron transfer in proteins
1
General organization of cell metabolism
The human basal metabolic rate is about 2000 kcal/day
Digestive tract
endosomes-lysosomes
hydrolysis
glucose
2 ATP
24 kcal/mole
Cytosol
ANAEROBIC
glycolysis
2 pyruvate
+ 6 O2
28 ATP
336 kcal/mole
6 CO2 + 6
H2O
686 kcal/mole
Mitochondria
AEROBIC
Citric acid cycle
Oxidative phosphorylation
Excretions
2
Molecular intermediates in energy production
Glucose C6H12O6 + 6 O2  6 CO2 + 6 H2O
Palmitate (C16H32O2) + 23 O2  16 CO2 + 16 H2O
686 kcal/mole
2340 kcal/mole
Acetyl Coenzyme A is an acetyl group carrier
Nicotinamide adenine dinucleotide is an
intermediate electron carrier
E0 (pH 7) = - 0.32 V/SHE
3
Flavin adenine dinucleotide is an intermediate electron carrier
E0 (pH 7) = - 0.22 V/SHE
FAD : 2 e- 2 H+
NAD : 2 e- 1 H+
4
Overview of metabolic reactions
fatty acids
glucose
ATP
glycolysis
b oxidation
NADH
pyruvate
NAD
CO2
+
lactate
ethanol
NADH
Chemical intermediate :
 acetyl  CO2
Storage of redox chemical
energy (e-): NAD+  NADH
2. NADH oxidation
and O2 reduction
3. Energy conversion  ADP phosphorylation
Proton gradient
1. Carbon oxidation
glucose
ATP
NADH
pyruvate
net yield
+ 2 ATP
+ 2 NADH ~ 6 ATP
Glycolysis yield6
Pyruvate fates in anaerobic conditions
 NAD+ regeneration
Krebs cycle
In prokaryotes and
higher eukaryotes
In prokaryotes and
yeast
7
pyruvate
Pyruvate decarboxylation
CoA-SH
NAD+
CO2
NADH
CO2
NADH
H+
pyruvate
dehydrogenase
complex
E2
Components subunits
E1 pyruvate
24
dehydrogenase
prosthetic groups
thiamine
pyrophosphate
E2 dihydrolipoyl 24
transacetylase
lipoate
CoA
E3 dihydrolipoyl 12
dehydrogenase
FAD
NAD+
E3 binding protein
E1
AcetylCoA
E3
8
The tricarboxylic acid cycle (Krebs cycle, citric acid cycle)
ubiquinone (Q)
2eubiquinol (QH2)
9
Glucose oxidation yield
Complete glucose oxidation
Step
Metabolite
ATP equivalent
Total
+ 2 ATP
+ 2 NADH
2
1.5 (2.5)
2
3 (5)
2.5
2.5
Glycolysis
Pyruvate decarboxylation
+ 1 NADH
+ 1 CO2
TCA cycle
+ 1 GTP
+ 3 NADH
+ 1 FADH2
+ 2 CO2
Total
6 CO2
1
2.5
1.5
1
7.5
1.5
in the presence of O2
x2
30 (32)
10
Triacylglycerol is an energy storage molecule
adipocyte
G
L
Y
C
E
R
O
L
FATTY ACID
FATTY ACID
FATTY ACID
triacylglycerol + H2O
Palmitate (C16H32O2) + 23O2
glycerol + 3 fatty acids
16 CO2 + 16 H2O
2340 kcal/mole
ATP)
1272 kcal/mole
in mitochondria
+ 106 (ADP + Pi
11
Example of a palmitate :
1. activation
http://rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/fatcatab.htm
C16-COOH + ATP
Acyl-adenylate
Acyl-CoA
+ 2 Pi
12
The fatty acid cycle (beta oxydation)
1. Acyl CoA
dehydrogenase
1egc
4. Acetyl-CoA Cacyl transferase
2. Enoyl CoA
hydratase
1mj3
3. L-b hydroxyacyl CoA
dehydrogenase 1f17
13
Example of a palmitate :
2. b-oxidation
fatty acids
b oxidation
Acetyl CoA
C16
C14
C16
C14
C12
C10
C8
C6
C4
C2
14
Energy yield of fatty acid oxidation
Step
Metabolite
ATP equivalent
Initial fatty acid activation
ATP
-2
Fatty acid cycle
per acetyl group
FADH2
NADH
Acetyl CoA
Total
1.5
2.5
10
14
for a palmitate (C16)
10.5
17.5
70
98
Last acetyl CoA
10
Total
106
15
Mitochondria structure
Matrix: Krebs cycle,
lipid beta oxidation
Internal membrane:
oxidative phosphorylation
Inter-membrane space:
nucleotide
transphosphorylation
External membrane:
filter < 5000 Da
Cytosol: glycolysis
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Oxidative phosphorylation
Energy coupling through a proton gradient
- 53 kcal/mole
Proton gradient
+ 12 kcal/mole
10 protons per reduced O
3 protons per phosphorylated
ADP
respiratory
complexes
ATPsynthase
17
The three respiratory complexes
4 H+
complex I
4 H+
complex III
2 H+
complex IV
18
Proton flow through the ATPsynthase F1F0 is coupled to
ADP phosphorylation
ADP + Pi
F0
F1
Proton
flow
Mechanical
work
ATP
Turnover : about 100 ADP are phosphorylated per
second
ATP
Mechanical
work
Proton
flow
ADP + Pi
19
The proton motive force
Free energy to transport one mole of
H+ from the interspace to the matrix
(electrochemical gradient)
DG = NeDV - RT*ln(10) DpHmatrix-interspace
DG/F = DV - RT*ln(10)/Ne DpH
Proton motive force (in mV) = DY = DV - 60 DpH
 Some active transport processes
are driven by a H+ electrochemical
gradient
In the mitochondria : ADP-ATP
exchange, pyruvate and phosphate
import
In bacteria : lactose import
20
Complex I
DG = DG’0 + RTln(C2/C1) = - nFDE’0 +
RTln(C2/C1)
NADH + Q + 5H+matrix → NAD+ + QH2 + 4H+cytosol
Complex III
QH2 + 2Cytcox + 2H+matrix → Q + 2Cytcred + 4H+cytosol
Complex IV
2Cytcred + ½ O2 + 4H+matrix → 2Cytcox + H2O + 2H+cytosol
Net reaction
NADH + ½ O2 + 11H+matrix → NAD+ + H2O + 10H+cytosol
NAD+ + 2e- + H+ → NADH
E’0 = -0.32 V
½ O2 + 2H+ + 2e- → H2O
E’0 = +0.82 V
NAD+/NADH ratio is about 1
[O2] is about 2 µM
10 x {H+matrix → H+cytosol}
{ DpH = 1.4
DV = 0.14 V
DGADP phosphorylation= 12 kcal/mole
DG’0 = -52.6 kcal.mol-1
DG = -56.7 kcal.mol-1
DGproton gradient = 5.2 kcal.mol-1 }
3 protons per ATP molecule
3 ATP per NADH oxidized
– 0.5 ATP to transport phosphate
21
Electron transfer through ubiquinone (coenzyme Q)
Q
TCA cycle
oxidative
phosphorylation
+
2e- + 2 H+
Succinate oxidation
(via FADH2)
NADH oxidation
QH2
complex II
complex I
complex II
22
Electron transfer rate
… depends on the distance between
electron-carrying groups
… depends on the free-energy of the
transfer reaction
Environment coupled
quantum tunneling
temperature sensitive
quantum tunneling
temperature insensitive
Marcus’s theory :
log k = 13 -0.6(R -3.6) -3.1(DG + l)2/l
k is the rate in units of s-1
R is the edge-to-edge distance in Å
DG is the driving force in eV
l is the reorganization energy in eV
23
Functional staining of active mitochondria
Bovine pulmonary
endothelial cell
5 µm
bull spermatozoids
10 µm
24
Example : Mitotracker (Molecular Probes)
freely diffuses
through membranes
membrane
impermeant
complexed to
proteins
(fixable using
formaldehyde)
25
Inhibitors of oxidative phosphorylation
Inhibitor
Target
Effect
Cyanide
CN-
Cytochrome oxidase
(complex IV)
binds more strongly than O2 to the Fe-Cu
center, preventing O2 reduction
Oligomycins
ATP synthase
blocks the proton flow through the F0 subunit
CCCP
proton gradient
uncouples proton pumping from ATP synthesis
malonate,
oxaloacetate
succinate dehydrogenase
(complex II)
competitive inhibitors
Bongkrekic acid
ADP/ATP transporter
(carboxy)atractyloside
inhibitors of ADP import and ATP export through
the inner mitochondrial membrane
Carbonyl cyanide mchlorophenyl hydrazone
Oligomycin A
26
carboxyatractylate
27
Cell energetics
 An overview of cell metabolism
 Glycolysis and fatty acid oxidation
 Tricarboxylic acid cycle
 Oxidative phosphorylation
 Experimental evidence of mitochondrial proton
gradient
 Mitochondria labeling and inactivation
 Mitochondrial diseases
28
Mesure of mitochondrial activity
 O2 consumption : Clark electrode
O2 + 2 e− + 2 H2O → H2O2 + 2 OH−
 ATPase activity : released Pi
 Cytochrome redox state : difference spectra
 NADH is detected spectroscopically at 340
nm or by its fluorescence (emission 460 nm)
29
http://www.atpsynthase.info/Protocols.html#Sec1.4
In vitro reconstitution of proton gradient coupled ATP synthesis
 ATP hydrolysis: coupled assay
Pyruvate Kinase
phosphoenolpyruvate + ADP → pyruvate + ATP
 ATP synthesis: direct assay
luciferase
luciferin + ATP + O2 → oxyluciferin + AMP + PPi+ CO2
Lactate dehydrogenase
NADH + pyruvate → NAD+ + lactate
NADH is detected spectroscopically at 340 nm
or by its fluorescence (ex 340 nm, em 460 nm)
30
Mitochondrial diseases
Mitochondria contain a short (14000 bp in humans) circular chromosome,
coding for 13 genes. Other proteins are synthesized in the cytoplasm and
translocated across the
membrane(s).
Mitochondria are essentially
maternally inherited.
ND : NADH dehydrogenase (complex I)
CYB : cytochrome b (complex III)
CO : cytochrome c oxidase (complex IV)
ATP : ATP synthase
A given cell contains hundreds of different mitochondria, which may divide
and fuse. Mitochondrial diseases are therefore rather complex. Fluctuations in
the percentage of mutated mitochondria lead to large variations in the nature
and severity of the symptoms of the pathological condition as well as the time
of onset.
31
Mitochondrial disease and symptoms
Molecular origin
Aminoglycoside-induced deafness
sensitivity of mitochondrial
ribosomes to some antibiotics
Kearns-Sayre syndrome
progressive neuromuscular disorder
partial deletion of mitochondrial DNA
Leber hereditary optic neuropathy
progressive blindness
point mutation in complex I subunits
Leigh syndrome
progressive central nervous system
degeneration
various mutations resulting in
decreased ATP production
NARP (Neuropathy Ataxia
Retinitis Pigmentosa)
mutation in ATP6
→ decreased ATP production
MELAS (Mitochondrial Encephalomyopathy,
Lactic Acid accumulation, Stroke-like)
A-G point mutation in the
tRNALeu gene → brain ischemia
→ brain infarct
Pearson’s syndrome
deletion in tRNA genes
Bone marrow deficiency → low blood cell content
Progressive external ophtalmoplegia
Progressive muscle paralysis
deletions in mtDNA
32
Integration of the energetic pathways in the cell metabolism
glucose
glycogen synthesis
glycogen
glucose-6P
glycogen degradation
amino acid synthesis
(not essential amino acids)
pentose phosphate pathway :
sugars, ribose,
purine metabolism
pyruvate
fatty acids synthesis
acetyl-CoA
gluconeogenesis
fatty acids
b-oxidation
malate
amino acid degradation
and urea cycle
lipids
fumarate
ATP
pyrimidine metabolism
33
Arthur HARDEN
Phosphorylated sugars
1865-1940, NP1929
Peter MITCHELL
chemiosmotic theory
1920-92, NP1978
Hans KREBS
tricarboxylic acid cycle
1900-81
NP1953
NP1997 Paul BOYER
John WALKER
ATP synthase mechanisms
1918
1941
Fritz LIPMANN
CoenzymeA
1899-1986
Jens SKOU
Na+/K+-ATPase
1918
34
æ [ ADP]ic [ Pi]ic ö
÷÷
ATP carries chemical energy in cells DG = DG0 + RT ln çç
è [ ATP ]ic ø
ATP + H2O
ADP + Pi + H+
DG ≈ - 12 kcal/mole
ATP + H2O
AMP + PPi + H+
DG ≈ - 12 kcal/mole
PPi + H2O
2 Pi + H+
DG ≈ - 12 kcal/mole
 Many intracellular chemical reactions are coupled to ATP phosphorylationdephosphorylation
 The human basal metabolic rate is about 2000 kcal/day
 Cells maintain the ATP/ADP intracellular ratio of about 1000
35
Cell energetics
 An overview of cell metabolism
 Glycolysis and fatty acid oxidation
 Tricarboxylic acid cycle
 Oxidative phosphorylation
 Experimental evidence of mitochondrial proton
gradient
 Mitochondria labeling and inactivation
 Mitochondrial diseases
36
5 nm
Glycolysis
enzymes 37
citrate
synthase
1csc
complex II
aconitase 7acn
citrate
dehydrogenase
1lwd
succinate
dehydrogenase
2acz
fumarase
3e04
oxoglutarate
dehydrogenase
1l1f
succinyl CoA
synthetase
1cqj
malate
dehydrogenase
4mdh
38