8 Lecture Oxidative Phosphorylation (II)
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Important concepts
1. Memorize how many protons pumped out by each complex
Understand how O2 is reduced.
Understand the concept of proton-motive force
2. Understand how the ATP synthase works
Memorize the structure of ATP synthase
Understand the binding-change mechanism of F1
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Transfer of electrons from CoQ to Complex III
Pumping of 4 H+ through the Q cycle Transfer of electrons from CoQ (soluble in lipid) Through Iron-Sulfur Protein to cytochrome c1
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From Complex III to Complex IV
Electrons to soluble cytochrome c Apoptosis (programmed cell death) -- cytochrome c released out of mitochondrion to cytosol
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Complex IV: Cytochrome c Oxidase
Cytochromes Reduction of O2 to H2O Pumps 2 H+ Also uses up 2 H+ in reduction of O2 to add to gradient
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Cytochrome c Oxidase dimer
13 subunits -- cytochrome a, a3, CuA, CuB Reduction of O2 -- cytochrome a3-CuB complex
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Cytochrome Oxidase mechanism
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Cytochrome Oxidase mechanism
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A pair of electrons:
Pumps 4 H+ by Complex I Pumps 4 H+ by Complex III Pumps 2 H+ by Complex IV
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Reduction of O2 in oxidative phosphorylation -- Is there a downside?
If only partially reduced, create reactive oxygen species (ROS) Superoxide radical Or worse hydroxyperoxyl radical HO2--
Most potent ROS is hydroxyl radical
Superoxide + Hydrogen Peroxide
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Free Radicals
Extract e- from other molecules -- create other free radicals and thereby initiate chain reaction Oxidation of polyunsaturated lipids Damage to DNA Mitochondrion is probably most susceptible Leads to aging? Catastrophe Hypothesis -- damage leads to more damage, more damage leads to even more damage ...
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Antioxidants
Superoxide dismutase (SOD)
One cycle of superoxide dismutase uses two superoxide radicals and generates one hydrogen peroxide
Deficiency in amyotrophic lateral sclerosis (ALS) -- Lou Gehrig`s disease
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Electrons can be transferred between groups not in contact Complex II
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Important concepts
1. Memorize how many protons pumped out by each complex
Understand how O2 is reduced.
Understand the concept of proton-motive force
2. Understand how the ATP synthase works
Memorize the structure of ATP synthase
Understand the binding-change mechanism of F1
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Oxidative Phosphorylation: Converting the H+ gradient to ATP
ATP synthase -- a rotary machine converting energy in H+ gradient to ATP synthesis
H+ gradient called proton motive force`
~ 3-4 H+ drives synthesis of a single ATP
Proton-motive force = chemical gradient + charge gradient
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Chemiosmotic Theory
1963 to 1965, withdrew completely from scientific research Became architect and master of works for restoration of Glynn House, mansion a in Cornwall, England Furnished a major part of Glynn House for use as a research laboratory 1965 he started research on chemiosmotic reactions
Peter Mitchell (19201992) Nobel Prize for Chemistry 1978
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Testing the Chemiosmotic Theory
A light drive proton pump
When the vesicles exposed to light, ATP formed
The respiratory chain and ATP synthase are linked only by a 16 proton-motive force
How many protons required?
G=RTln(c2/c1) + ZFDV = 2.303RTlog10(c2/c1) + ZFDV =21.8 kJ/mol Z: electrical charge DV: potential across membrane
Then, DG = 21.8 kJ mol-1
Synthesis of ATP requires 40-50 kJ mol-1 So, at least 2, but more like 3 to 4 protons
NADH, 10 protons ~ 2.5 ATP
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FADH2, 6 protons ~ 1.5 ATP
FoF1 ATP Synthase
450 kDa multisubunit complex
Fo is transmembrane protein and the H+ pump F1 is watersoluble, peripheral membrane protein and the ATP synthase
Inside of mitochondrion (matrix)
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Micrograph of ATP FoF1-ATP synthase
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F1 (a3b3gde)
a 3b 3
ADP/ATP bind between a and b b synthesizes ATP g spins!
g
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How does F1 synthesize ATP?
Isotope exchange experiments
ATPs are synthesized but not released
About equal amounts of bound ATP and ADP at the equilibrium, even in the absence of a proton gradient Proton gradient releases ATP
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g binding makes b unequivalent
g rotation drives interconversion of the b forms
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Binding-change mechanism: ATP synthesis by ab subunits of F1
L is loose conformation, able to bind ADP + Pi T is tight conformation, promoting synthesis of ATP O is open conformation, unable to bind ADP/ATP
H+ gradient drives T to O conformational change
Transmitted from Fo by g subunit -- rotates
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How can we check that it actually rotates?
Attach something big (actin)
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Rotating g subunit
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Proton flow around the c ring powers ATP synthesis
Proton enters cytoplasmic half channel (Asp61 of c subunit protonated) but cannot move through Asp61 interacts with Arg210 on a subunit Asp61 of c subunit gives up proton (charged aspartate form) when in contact with a matrix half channel
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Proton motion from high to low concentration powers the c ring rotation Neutralized Asp favors hydrophobic environment -> rotate
Charged aspartate
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movie
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The number of c ring is significant
10 14 c rings
Rotation of 360 synthesize 3 ATP
1 ATP=10/3=3.3 protons
A resting human being require 85kg ATP = 3.3x1025 protons/day
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Summary
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Important concepts
1. Memorize how many protons pumped out by each complex
Understand how O2 is reduced.
Understand the concept of proton-motive force
2. Understand how the ATP synthase works
Memorize the structure of ATP synthase
Understand the binding-change mechanism of F1
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