1

Suggested problems from the end of chapter 20: 1 (standard reduction potential of the FAD half-reaction can be taken to be 0.02V), 3 ( this question deals with a half reaction, at pH=8 Ehalf reaction =0.153V),4,5,6,7,8 (CoQ is UQ in Tables 20.1, and 3.5; the calculation in part c is for 25°C)9 (the calculation in part c is for 25°C)10, 13 (calculation is for 25°C, ∆Goverall = ∆Gimport + ∆Gsynthesis), 20.

Electron transport and oxidative phosphorylation in the mitochondrion

The mitochondria contains: •  pyruvate dehydrogenase.•  the enzymes involved in fatty acid oxidation.•  the citric acid cycle enzymes.•  the redox proteins involved in electron transport and oxidative phosphorylation.

Thus the mitochondria can be viewed as a cellular “power plant” for ATP production.A eukaryotic cell can have 1000’s of mitochondria. By volume, the mitochondria can take up to20% of the cell.

The presence of mitochondria in cells can be viewed as an example of cellular “symbiosis” byan early bacterium with the eukaryotic cell.

2

Mitochondrion organization

Outer membrane

Inner membrane

Cristae ( = crests)

Matrix

mitos = thread, chondros = granule

Freeze-fracture and freeze-etch electron micrographs of the mitochondrial membranesThe inner membrane containsabout twice as many particlesas the outer membrane. Electrontransport and oxidativephosphorylation proteins are foundwithin the inner membrane.

There is an asymmetricdistribution of particles betweenthe inner and outer faces ofthe membrane.

The concentration of proteinand DNA in the matrix is veryhigh, probably on the orderof 100 mg/ml. The viscosity ishigh, as expected.

Mitochondrial DNA, RNA andribosomes reside in the matrix.

Porin proteins reside within the outermembrane.

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The inner mitochondrion membrane does not contain an NADH transporter

1. A hydride and a proton are transferred to DHAP, forming 3-phosphoglycerol.

2. FAD is reduced to FADH2.

3. FADH2 is oxidized, and the electrons are transported to the reducing centers of the electron transport chain within themitochondria inner membrane.

ATP generated in the mitochondrion must be transported into the cytosol• The ADP-ATP translocator has 2 conformations. Both conformations have equal affinitiesto ADP and ATP.Would you predict that AMP, adenosine, or adenine could bind the transporter?• Ligand binding is required for conformational switches (unlike the glucose transporter).There is an overall charge difference across the membrane, where the inside of the membrane is kept more negative than the outside.How does this affect ATP transport?

See redox worksheet

4

Electron transport sequenceFour electron transport centers with (except for complex III) increasingly greater electron affinities (greater reduction potential) participate in electron transfer in the mitochondrial inner membrane.

Complex I catalyzes oxidation of NADH by Coenzyme Q (CoQ, ubiquinone):

NADH + CoQ (oxidized) ↔ NAD+ + CoQ (reduced)∆E°’ = 0.360 V, ∆G°’ = -69.5 kJ/mol

Complex III catalyzes oxidation of CoQ by cytochrome c:

CoQ (reduced) + 2 cytochrome c (oxidized) ↔ CoQ (oxidized) + 2 cytochrome c (reduced) ∆E°’ = 0.190 V, ∆G°’ = -36.7 kJ/mol

Complex IV catalyzes oxidation of cytochrome c by O2:

2 cytochrome c (reduced) + 0.5 O2 ↔ 2 cytochrome c (oxidized) + H2O∆E°’ = 0.580 V, ∆G°’ = -112 kJ/mol

Complex II catalyzes oxidation of FADH2 by cytochrome c (ATP is NOT produced in this step):

FADH2 + CoQ (oxidized) ↔ FAD + CoQ (reduced)∆E°’ = 0.085 V, ∆G°’ = -16.4 kJ/mol

Electron transport sequence

5

Use of inhibitors to reveal the sequence of mitochondrial electron transport

Mitochondrial membrane preparations were made by purifying mitochondria from enrichedsources (bovine heart, insect flight muscle).The rate of NADH-dependent (or FADH2 - dependent) O2 consumption by this preparation can be measured. This measurement indicates the respiration rate.The effect of inhibitors on the O2consumption rate, then, can be measured.

Rotenone and amytal block electron transport in complex I

N

NO

O

O

CH2CH2CH(CH3)2

CH2CH3

Amytal

H3CO

OCH3

O

O

O

O CH2

CH3

CRotenone

Use of inhibitors to reveal the sequence of mitochondrial electron transport

Antimycin A inhibits complex III

-C N

Cyanide

Cyanide inhibits complex IV

In some cases one can “rescue” the inhibited respiratory chain by introducing compounds that would supply electrons downstream of the inhibited complex.

O

O

O

CH3

O

(CH2)5CH3O

H3C

HN

C

O

CH2CH(CH3)2

C

O

OH

HN CHO

Antimycin A

6

Arrangement of the electron transport chain within the inner mitochondrial membrane

These complexes are not arranged in higher-order structures. They seem to be able to diffusefreely within the membrane.

Proton translocation as a consequence of electron transfer

This is a hypothetical model for proton translocation across the mitochondrial inner membrane.Binding of protons and sunsequent reduction may induce a change in the protein transporter. The affinity for protons then is decreased, and a concomitant change in the overall structure of the protein makes proton release into the intermembrane space likely. Oxidation of the complex induces a switch to the starting conformation.

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Electron transfer in complex I

The free radical form of flavin mononulceotide (FMN) and the reduced (FMNH2) are stable.Thus FMN can transfer 2 electrons, whereas the iron-sulfurcenters can transfer only one electron at a time.

Because NADH can only transfer 2 electrons per reaction, FMN can serve as a conduit between the 2 - electron transfer reaction of NADH and the 1 - electron transferreaction of the iron-sulfur centers.

Complex I consists of 43 polypeptides and a total mass of 850 kDa. The groups that are active in electron transfer are 6 to 7 iron-sulfur clusters and one flavin mononucleotide.

Electron transfer in complex I

The iron-sulfur clusters can undergo a singleelectron transfer reaction. Overall the ironmoieties can have either a +2 or a +3 charge.These groups cannot transfer more than oneelectron at a time.The exact sequence of reducing equivalenttransfer is not known because the system“equilibrates” faster than it is possible tomeasure.

8

Electron transfer in complex I

CoQ can accept and transfer 1 or 2 electrons becausethe semiquinone form is a stable free radical.It seems that only some of the time 2 electrons aretransferred.

CoQ has a hydrophobic tail that makes it soluble in theinner membrane. In contrast to FMN, CoQ is not tightly bound to the protein moiety of complex I. In mammalian cells the number of C5 isoprenoid units is 10 and is designated Q10CoQ.In plants and bacteria, this tail may only be a Q6 or Q8.

Proton translocation as a consequence of electron transfer

For each pair of electrons donated by NADH, 4 protons are pumped from the matrix across the mitochondrial inner membrane to the intermembrane space.

The mechanism is not well understood, but almost certainly a conformational change within a trans-membrane protein is involved. Bacteriorhodopsin may have some structural similarities to the mitochondrial proton pump.

When light is received by the retinal group, the protein conformation changes. There are two main conformations, and protons are driven across the membrane as a resultof these conformational switches.In general, this system confirms that changes in one part of the protein can haveprofound consequences on the overall structure.

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Complex IIThe reduction potential of complex II is not sufficient to drive ATP synthesis. Nevertheless,complex II serves to insert the electrons contributed by FADH2 into the electron transport chain.Complex II contains succinate dehydrogenase, with a covalently bound FAD group, threeiron-sulfur clusters, and one cytochrome b560. Cytochromes are redox-active proteins containing heme groups. The electron transfer potentialof the heme groups is very high and the protein moiety must be situated properly to prevent non-specific electron transfer.

Complex IIIComplex III transfers electron from reduced CoQ to cytochrome c. Complex III contains twocytochrome b proteins, one cytochrome c, and one iron-sulfur center.

Complex III’s function is to catalyze the transfer of 2 electrons from CoQH2 to two molecules ofcytochrome c. This is done using the Q cycle (actually 2 cycles), which allows protons to be pumped across the mitochondrial inner membrane.

The ISP is the iron-sulfur protein,and cytochrome c is reducedby the ISP.The other electron eventually is transferred to cytochrome bH.CoQ dissociates from site Q0and rebinds to site Q1, where itaccepts the electron from cytochrome bH.

matrix

inter-membranespace

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Complex III

In cycle 2, another CoQH2repeats the steps of cycle 1.There is a free radical CoQ-bound at the Q1 site, andthis free radical acceptsthe electron fromcytochrome bH, as well astwo protons from themitochondrial matrix.

Overall:two CoQH2 were consumed and one was re-generated.2 cytochrome c1 were reduced.4 protons were pumpedacross the membrane.

This model depends on: 1. Stability of free-radical CoQ-. 2. The presence of 2 binding sites for CoQ, so-called Q0 and Q1.

Structural evidence for 2 CoQ binding sites within Complex III

• The crystal structure of Complex III is known, and different electron transport inhibitors weresoaked into the crystal to identify their site of inhibition.

• Actimycin A has been shown to block electron flow from heme bH to CoQ or CoQ-. Thisinhibitor binds near bH, and, most likely, at site Q1.

• Myxothiazol has been shown to inhibit electron flow from CoQH2 to the ISP and to heme bL.This inhibitor binds near the ISP and heme bL. Most likely, this is site Q0.

N

S

S

N

CH2N

O

CH3

OCH3 OCH3

CH3

CH3

CH3

Myxothiazol

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Complex IV (cytochrome c oxidase)The overall oxidation of 4 cytochrome c molecules yields:

4 cytochrome c (Fe2+) + 4 H+ + O2 → 4 cytochrome c (Fe3+) + 2 H2O

The complex is a homodimer of 400 kDa. There are 4 redox centers: cytochrome a, cytochrome a3, CuB (a copper atom), and CuA (a pair of copper atoms). Mg2+ and Zn2+ alsowere found bound to the complex.From the crystal structure the path of electron may be traced.

Cytochrome c binds near CuA and transfersits electron to the copper atom.Heme a, about 20 Å away, receives theelectron and transfers it to heme a3, a distanceof less than 5 Å. 4.5 Å from there, CuB receives the electron.

Reduction of O2 by cytochrome c oxidase

These reactions take place at the very end of the electron transfer reactions, and involve theelectron transfer between heme a3 and CuB. 4 electrons and 4 H+ are required to reduceone molecule of O2. This cycle is complete within 1 msec.

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Oxidative phosphorylation• ATP synthesis must be linked to the free energy released by electron transport.• The coupling between electron transport and ATP synthesis remained elusive for a long timebecause high energy intermediates were sought. No such intermediates have been found.• The current paradigm argues that the proton gradient generated during electron transfer isharnessed to generate ATP• Recent advances in structural and dynamic studies of the F0F1 ATPase enzyme have beeninstrumental in buttressing this argument.

Observations concerning the proton gradient-driven ATPase activity

1. An intact inner mitochondrial membrane is required for ATP synthesis. 2. The inner mitochondrial membrane is impermeable to H+, Cl-, K+, OH-, and Na+, whichserves to maintain a charge gradient. 3. Electron transport results in a proton gradient across the membrane. 4. Electron transport continues in the presence of compounds that increase the permeabilityof the membrane. Oxidative phosphorylation, however, is sensitive to the presence of suchagents. If the membrane electrochemical potential is discharged, ATP synthesis is “uncoupled”from electron transport.

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The thermodynamic “value” of a proton gradient

The membrane potential is a combination of the chemical and charge differences across themembrane. For a substance A, the electrochemical potential (for transport into the cell) is:

Where ZA is te overall charge of the particle, F is faraday’s constant, 96485 C/mol, �ΔΨ is the membrane charge potential (in volts, 1 V = 1 J/C), where the mitochondrial inside is negative. For liver mitochondria, ΔΨ = -0.1 V. The transport of protons into the mitochondrial matrix, therefore, yields a negative term for the charge potential, which is favorable.The chemical term also is favorable under these conditions because H+ is transported along its chemical gradient.

1. What is the electrochemical gradient if the pH on the outside of the membrane is 6.5 and the pH on the inside is 7.4?

2. Calculate the minimal pH gradient that is required for ATP synthesis in liver mitochondria under the following conditions: pH (matrix) = 7.4; [ATP]=1mM; [ADP]=10mM; [Pi]=2mM; ∆G0’ for ATP hydrolysis is 30.5 kJ/mol.

€

ΔG A = RTln([A]in

[A]out) + ZAFΔΨ

F1F0 ATPaseThe ATP synthase is a multisubunit transmembrane protein of 450 kDa.The F0 portion is a water-insoluble transmembrane proton channel containing at least 8 distinctpolypeptides.The F1 portion is water-soluble, peripheral membrane protein. Pure F1 contains an ATPasebut no ATP synthase activity.Recent crystallographic studies agree with this general picture:

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The F0 component

DCCD is a lipid-soluble molecules which can react with carboxyl groups. This moleculereacts with a single glutamic acid residue on the mammalian F0 component and inhibits proton transport. This can indicate that the glutamic acid residue is buried within a hydrophobic environment.Six DCCD binding proteins form a putative H+ transport channel within F0. It is possible,however, that 12 proteins are actually present there, and that each duplex reacts with one DCCD molecule.

N C N

Dicyclohexylcarbodiimide (DCCD)

The F1 component

20 Å

15

The F1 component

Positive potential is colored in blue, negative potential in red. Note the absence of charge on the inner part of this sleeve. There is a pseudo 3-fold and 6-fold symmetry due to the highsimilarity between the α and β subunits.

Boyer’s model of proton-driven ATP synthesisAny model must account for three activities: 1. Translocation of protons by F0. 2. Phosphoanhydride bond formation by F1. 3. Coupling of F1 to F0 activities.

The Boyer model proposes differential binding of ATP within the three ATP binding sites within F1. The affinities for ATP can be described as low (L), high (T), and undetectable (0). Interconversion of these three ATP binding modes depends on proton pumping by the F0 component. T is the only catalytically-active site.

1. ADP + Pi bind the L site. 2. Energy-driven conformational change converts the L site to a T site. 3. ATP bound at the 0 site is released, and ATP is synthesized at the T site. 4. Two more counter-clockwise turns of the stalk, with respect to the F1 component, bring the enzyme to the starting conformation.

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Boyer’s model of proton-driven ATP synthesis

• Boyer proposed that the energy of proton transfer is coupled to the conformational changevia mechanical 120° turns.This proposal is corroborated by the finding that attachment of the F1 via the stalk region to aglass plate results in an ATP-dependent clockwise spin, as assayed by labeled actin attachedto the stalk region.• At low ATP concentrations, one can observe sequential, stepwise, 120° turns.• At high ATP concentrations, one can observe fast clockwise turns that eventually stop as a result of twisting the stalk.• Taking into account the drag on the actin filament, one can calculate the amount of forceexerted by the motor.

If this model is correct, in which direction would you expect a similar ATP synthase motor to turn in the mitochondrial inner membrane?

Look up animations online (there are many and they turn over rapidly)

Synthesis of ATP in terms of the reduction potential of NADH and FADH2

Experiments with isolated mitochondria show:~3 ATP synthesized / NADH (contributes 2 electrons into Complex I)~2 ATP synthesized / FADH2 (contributes 2 electrons into Complex II)~1 ATP synthesized / Tetramethyl-p-phenylenediamine (this compound contributes an electronpair directly into Complex IV).

N+

N+

H3C

H3C

CH3

CH3

Tetramethyl-p-phenylenediamine

Starting the electron transport at Complex I, 10 protons are pumped out of the mitochondrial membrane per electron pair. This gives sufficient free energy to synthesize ~3 ATP molecules.Starting at Complex II, 6 protons are pumped across the membrane, enough for ~2 ATP.Transit of 2 electrons through Complex IV yields 2 protons, enough for ~1 ATP.

These ratios are almost always non-integral numbers. There is some leakage of protons acrossthe membrane, which dissipates the gradient. Also, the point of harnessing the proton potentialis at the end of the cycle, which means that the amount of protons translocated need not be amultiple of the amount of protons required for ATP synthesis.

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ATP yields from glycolysis, citric acid reactions, and oxidative phosphorylation

An examination of the stoichiometries of glycolysis products and their utilization shows that (1) the amount of ATP produced per NADH is 2.5-3, and (2) the amount of ATP produced per FADH2 is 1.5-2.

Overall, how much ATP is synthesized per glucose?

I. Glycolysis yields 2 ATP II. The citric acid cycle yields 2 ATP, 10 NADH, and 2 FADH2. III. Oxidative phosphorylation utilizes 10 NADH which yields 25 to 30 ATP, and 2 FADH2 which yields 3 to 4 ATP.

Overall, we get 32 to 38 ATP per glucose.