Cellular Respiration III. Oxidative Phosphorylation
October 5, 2005
Chapter 9
Lecture Outline 1. What do we do with NADH + H+ and FADH2 reducing equivalents? 2. Electron Transport – the oxidation phase of Oxidative Phosphorylation 3. ATP synthesis – the Phosphorylation Phase of Oxidative Phosphorylation 5. Why believe the Chemiosmotic hypothesis? 6. Indirect Active Transport into the mitochondrion 7. Comparison of yield from Fatty Acid Oxidation and Monosaccharide Oxidation 8. Life without oxygen - Fermentation
1
Mitochondrial Functions
Electrons Given to Electron Transport chain
2
2 H+ + 2 e–
Controlled release of energy for synthesis of ATP ATP ATP
ATP
Energy Released Used To Make ATP
2 e– 1/
Electrons Eventually Given to Oxygen To form Water
O2
(from food via NADH)
Electron transport – oxidizes NADH and FADH2 back to NAD+ and FAD
1/
+
chain port trans
Generate >90% of Typical Cell’s ATP Oxidative Phosphorylation “electron transport” ATP synthesis Oxidize reduced carriers to produce ATP or equiv 3
2H
tron Elec
Oxidize compounds to CO2 + H2O Fatty acid Oxidation Produce Oxidation of Pyruvate reduced carriers TCA Kreb’s Cycle NADH & FADH2
2
Free energy, G
Lecture 15
2 H+
H2O
2
O2
How? 4
Figure 9.5 B
(b) Cellular respiration
Electron transport Oxidative part
Electron Transport Chain
3 discrete Proton pumps Powered by Passage of electrons
Oxidize NADH + H+ to NAD+ 2e- and 2H+ given to a multiprotein complex (complex I) 2e- get passed on 2H+ orphaned as electrons transferred
energy of oxidation pumps H+ to intermembrane space
Need O2 as final acceptor of reducing equivalents
2H2 + O2 = H2O + BOOM!
Complex I close up
5
6
Electron Transport Chain
pH 6
Oxidize NADH + H+ to NAD+ 2e- and 2H+ given to multiprotein complex (complex I) 2e- get passed on 2H+ orphaned as electrons transferred
2H+
energy of oxidation pumps H+ to intermembrane space
2 Fe++ 2
Fe+++
(2e-)
complex III) 2e- and 2H+ (where from?) get passed to another complex (complex orphaning of 2H+ repeated as 2e- passed on (2e-)
2e-
2H+
2H+
NADH +
H+
NAD+
CoQ
energy of oxidation pumps H+ to intermembrane space
(2e-)
(oxidized)
pH 8
CoQ H
H
(reduced) 7
complex IV) 2e- and 2H+ (where from?) get passed to a third complex (complex IV orphaning of 2H+ repeated as 2e- passed on
finally to oxygen energy of oxidation pumps H+ to intermembrane space
need to pick up 2H+ (where from?)to form water
8
2
H+
NADH 50
What happened to complex II?
pH 6
FADH2 40
I
FMN
Three pumping Steps from NADH + H+ oxidation
2
pH 8
Only TWO pumping Steps from FADH2 oxidation 2H+
OH-
Net Result of Electron Transport: an electrochemical H+ gradient
Free energy (G) relative to O2 (kcl/mol)
Fe•S
2H+
Multiprotein complexes
FAD Fe•S
II
O
III
Cyt b
30
Fe•S Cyt c1
IV
Cyt c Cyt a
Cyt a3
20
10
0
2 H + + 1⁄2
9
O2
10 Figure 9.13
H2O
What good is the H+ Electrochemical Gradient ?
Electron transport – oxidizes NADH and FADH2 back to
NAD+
H+
and FAD
H2O
Electron transport – electrons passed in series
from one carrier to another eventually give e- and H+ to oxygen to form water
OH-
pH 6
BUT PUMP H+ in the process across
the inner mitochondrial membrane into intermembrane space
FORMS BIG H+ gradient
11
pH 8 12
Mitochondrial H+ ATPase ATP synthase
H+ movement drives conformational change
pH 6
INTERMEMBRANE SPACE
H+ H+
A rotor within the membrane spins clockwise when H+ flows past it down the H+ gradient.
H+ H+
H+
H+
H+
A stator anchored in the membrane holds the knob stationary.
A rod (for “stalk”) extending into the knob also spins, activating catalytic sites in the knob. H+
ADP + ATP
Pi
pH 8
Three catalytic sites in the stationary knob join inorganic Phosphate to ADP to make ATP.
13
Figure 9.14
14
Reducing equivalents power the pump
ATP
ADP + Pi
Electron Transport Chain is a Proton Pump
H+ H
movt
Open
pH 8
MITOCHONDRIAL MATRIX
Mitochondrial H+ ATPase
H+
pH 6
H+
Open
ATP ADP + Pi ATP expelled 15
Gradient powers ATP Synthesis
16
Roughly 3 ATP for each NADH + H+ 2 ATP for each FADH2
Uncoupling Oxidation and Phosphorylation •Must have intact membranes to make ATP
Inner Mitochondrial membrane
Oxidative phosphorylation. electron transport and chemiosmosis
Glycolysis
ATP
ATP
ATP
H+
H+
• Chemiosmosis and the electron transport chain •Agents such as cyanide poison electron transport chain so no H+ gradient produced - but if supply H+ gradient can still make ATP
H+
Intermembrane space
Q III
17
FADH2 NADH+
FAD+
ADP +
Can Use
H
Electron transport chain Electron transport and pumping of protons (H+), which create an H+ gradient across the membrane
pH 8 -OH
H+
pyruvate-
How you get pyruvate (an acid) across the mitochondrion membrane? (not magic!)
Chemiosmosis ATP synthesis powered by the flow Of H+ back across the membrane
instead of making ATP + 2 ATP by substrate-level phosphorylation
Maximum per glucose:
+ 2 ATP
2 FADH2
Oxidative phosphorylation: electron transport and chemiosmosis
+ about 32 or 34 ATP
by substrate-level by oxidative phosphorylation, depending on which shuttle transports electrons phosphorylation from NADH in cytosol About 36 or 38 ATP
21
22 Figure 9.16
Compare to Fatty Acid Oxidation 18 carbon fatty acid (9 two carbon units) Each 2 carbon unit 1 X 2 NADH + 1 X 2 FADH2 Each acetyl CoA gives in TCA 3 NADH + H+ 1 FADH2 1 GTP Each 2 carbons
H+
In Metabolism:
3 X 6 ATP 2 X 4 ATP 9 2 1
Highly reduced CH3-CH2-CH2-(CH2)x-CH2-C-O + O2 Fatty acid O
ATP ATP ATP
Partially reduced
X ATP 17 22
Oxidation of 18 carbon fatty acid yields 198 X153ATP
8.5 X 11 ATP per FA carbon 6 ATP per glucose carbon
fully oxidized
carbohydrate
H2O + CO2 + energy
(captured)
fully oxidized + O2
H2O + CO2+ energy
(captured)
Why? 23
24
Organic Oxidation Series
CH4 R-CH2 -CH 3
X X X X X
Oxidative phosphorylation. electron transport and chemiosmosis
Glycolysis
Fatty Acids Start Here
R-CH=CH2
Life Without Oxygen
ATP
ATP
ATP
H+
H+
• Chemiosmosis and the electron transport chain H+
R-CH2-CH2 -OH Monosaccharides Start Here
R-CH2-C=O H R-CH2-C=O OH
Mitochondrial matrix
Life Without Oxygen
Cytosol
ATP Substrate-level phosphorylation
IV
III
II FADH 2 FADH2
NADH NADH + H+
X
NAD+
X
FAD+
2 H+ + 1/2 O2
H2O
ADP +
ATP synthase
ATP
Pi
+
H
Electron transport chain Electron transport and pumping of protons (H+), which create an H+ gradient across the membrane
Chemiosmosis ATP synthesis powered by the flow Of H+ back across the membrane
Oxidative phosphorylation
Figure 9.15
26
The Regeneration Energy Carriers
X
Glycolsis Pyruvate Glucose
Figure 9.6
Q
I
Inner mitochondrial membrane
H+
Cyt c
Protein complex of electron carners
(Carrying electrons from, food)
25
Electrons carried via NADH
Intermembrane space
+
O=C=O
Inner Mitochondrial membrane
X
Energy carriers (ATP, NAD+, FAD) present in only minute amounts
Electrons carried via NADH and FADH2
X X Citric acid cycle
Oxidative phosphorylation: electron transport and chemiosmosis
Mitochondrion
ATP Substrate-level phosphorylation
ATP Oxidative phosphorylation 27
X
Can’t
2e2H+ Captured in catabolism
Run out Of NAD+
X
Energy from catabolism (exergonic, energy yielding processes)
2e2H+ Cashed in
NADH + H+
X
Energy for cellular work (endergonic, energyconsuming processes)
NAD+
28
Life Without Oxygen - FERMENTATION Running Muscles
Glucose
CYTOSOL
Glucose
Pyruvate No O2 present Fermentation
Yeast
Wine/beer
Method To Recycle NADH + H+
X
2e2H+
MITOCHONDRION
Muscles
29 Figure 9.18
P1
Glucose
2 ATP
two pyruvate
O–
C
O
C
O
Glycolysis CH3
NAD+
H
H
C
2
CO2
H
OH
CH3
Figure 9.17
2 NADH
two ethanol 2 Ethanol
(a) Alcohol fermentation
C
O
CH3
H
C
O
C
O
2 NADH CH3
O
C
OH
CH3
two lactate 2 Lactate
(b) Lactic acid fermentation
Run in reverse When oxygen 30 returns
Fermentation and cellular respiration
– Differ in their final electron acceptor - oxygen - lactate - or ethanol
Cellular aerobic respiration
2 Pyruvate
Recycles NADH
alcohol
Keeps the Motor running
C
two pyruvate O–
O
Figure 9.17
2
Glycolysis
2 NAD+
Recycles NADH
Acetyl CoA
Citric acid cycle
2e2H+
2 ATP
P1
O2 present Cellular respiration
Ethanol or lactate
2 ADP + 2
2 ADP + 2
Gives the Fizz To Champagne Beer Hard cider
2 Acetaldehyde
31
– Produces tons more ATP
» 2 ATP in fermentation » 36 ATP in aerobic oxidation of glucose
Fermentation allows organisms to eek out a living in low or no O2
Ancient system – pre oxygen
32
Summary:
Where does oxygen come from?
1. Oxidative Phosphorylation is comprised of two distinct processes Electron Transport (oxidative) makes H+ gradient ATP synthesis (phosphorylation) uses H+ gradient coupled by H+ gradient
Next time:
photosynthesis
Light energy
2. Processes are separable: cyanide (poisons electron transport) uncouplers (DNP) (dissipate H+ gradient)
ECOSYSTEM
3. Electrochemical gradient powers mitochondrial H+ ATPase indirect active transport
Photosynthesis in chloroplasts Organic + O2 Cellular molecules respiration in mitochondria
CO2 + H2O
4. Oxidation of fatty acids yields more ATP than oxidation of glucose ATP powers most cellular work
Heat energy
Figure 9.2
33
• The catabolism of various molecules from food Proteins
Carbohydrates
Amino acids
Sugars
Fats
Glycerol
Fatty acids
Glycolysis
Glucose Glyceraldehyde-3- P
NH3
Pyruvate Acetyl CoA
Citric acid cycle
Figure 9.19
Oxidative phosphorylation
35
5. Can ferment sugars in absence of oxygen still produce ATP by substrate level phosphorylation but no reducing equivalents captured
