1 (Standard Reduction Potential of the FAD Half-Reaction Can Be Taken to Be 0.02V)

Home , Electron transport chain, Heme a, Intermembrane space, Q cycle, Reducing equivalent

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 to 20% of the cell.

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

1 Mitochondrion organization

mitos = thread, chondros = granule

Outer membrane Inner membrane

Cristae ( = crests)

Matrix

Freeze-fracture and freeze-etch electron micrographs of the mitochondrial membranes The inner membrane contains about twice as many particles as the outer membrane. Electron transport and oxidative phosphorylation proteins are found within the inner membrane.

There is an asymmetric distribution of particles between the inner and outer faces of the membrane.

The concentration of protein and DNA in the matrix is very high, probably on the order of 100 mg/ml. The viscosity is high, as expected.

Mitochondrial DNA, RNA and ribosomes reside in the matrix.

Porin proteins reside within the outer membrane.

2 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 the mitochondria inner membrane.

ATP generated in the mitochondrion must be transported into the cytosol •The ADP-ATP translocator has 2 conformations. Both conformations have equal afﬁnities to 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

3 Electron transport sequence Four 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

4 Use of inhibitors to reveal the sequence of mitochondrial electron transport

Mitochondrial membrane preparations were made by purifying mitochondria from enriched sources (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 OCH3

H3CO O O

CH2CH2CH(CH3)2 N

CH2CH3 O O O N O O CH2

Rotenone C Amytal

CH3

Use of inhibitors to reveal the sequence of mitochondrial electron transport

Antimycin A inhibits complex III Cyanide inhibits complex IV

O O

H O C N CH3

-C N O H3C Cyanide O C CH2CH(CH3)2 OH O HN CHO (CH2)5CH3 O

Antimycin A

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

5 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 diffuse freely 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.

6 Electron transfer in complex I

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.

The free radical form of ﬂavin mononulceotide (FMN) and the reduced (FMNH2) are stable. Thus FMN can transfer 2 electrons, whereas the iron-sulfur centers 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 transfer reaction of the iron-sulfur centers.

Electron transfer in complex I

The iron-sulfur clusters can undergo a single electron transfer reaction. Overall the iron moieties can have either a +2 or a +3 charge. These groups cannot transfer more than one electron at a time. The exact sequence of reducing equivalent transfer is not known because the system “equilibrates” faster than it is possible to measure.

7 Electron transfer in complex I

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

CoQ has a hydrophobic tail that makes it soluble in the inner 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 result of these conformational switches. In general, this system conﬁrms that changes in one part of the protein can have profound consequences on the overall structure.

8 Complex II The 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, three iron-sulfur clusters, and one cytochrome b560. Cytochromes are redox-active proteins containing heme groups. The electron transfer potential of the heme groups is very high and the protein moiety must be situated properly to prevent non-speciﬁc electron transfer.

Complex III Complex III transfers electron from reduced CoQ to cytochrome c. Complex III contains two cytochrome 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 of cytochrome 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 reduced by the ISP. The other electron eventually is transferred to cytochrome bH. CoQ dissociates from site Q0 and rebinds to site Q1, where it accepts the electron from cytochrome bH.

intermembrane space matrix

9 Complex III

In cycle 2, another CoQH2 repeats the steps of cycle 1. There is a free radical CoQ- bound at the Q1 site, and this free radical accepts the electron from cytochrome bH, as well as two protons from the mitochondrial matrix.

Overall: two CoQH2 were consumed and one was re-generated. 2 cytochrome c1 were reduced. 4 protons were pumped across 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 were soaked 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 . This inhibitor 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.

CH OCH3 OCH3 3

CH3

N N

S CH3 H2N C CH3 S O Myxothiazol

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

2+ + 3+ 4 cytochrome c (Fe ) + 4 H + O2 → 4 cytochrome c (Fe ) + 2 H2O The complex is a homodimer of 400 kDa. There are 4 redox centers: cytochrome a, 2+ 2+ cytochrome a3, CuB (a copper atom), and CuA (a pair of copper atoms). Mg and Zn also were found bound to the complex. From the crystal structure the path of electron may be traced.

Cytochrome c binds near CuA and transfers its electron to the copper atom. Heme a, about 20 Å away, receives the electron and transfers it to heme a3, a distance of 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 the + electron transfer between heme a3 and CuB. 4 electrons and 4 H are required to reduce one molecule of O2. This cycle is complete within 1 msec.

11 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 time because high energy intermediates were sought. No such intermediates have been found. •The current paradigm argues that the proton gradient generated during electron transfer is harnessed to generate ATP •Recent advances in structural and dynamic studies of the F0F1 ATPase enzyme have been instrumental 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+, which serves 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 permeability of the membrane. Oxidative phosphorylation, however, is sensitive to the presence of such agents. If the membrane electrochemical potential is discharged, ATP synthesis is “uncoupled” from electron transport.

12 The thermodynamic “value” of a proton gradient

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

[A] ΔG = RTln( in ) + Z FΔΨ A [A] A out

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; 0’ [ADP]=10mM; [Pi]=2mM; ∆G for ATP hydrolysis is 30.5 kJ/mol.

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

13 The F0 component

DCCD is a lipid-soluble molecules which can react with carboxyl groups. This molecule reacts 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

14 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 high similarity between the α and β subunits.

Boyer’s model of proton-driven ATP synthesis Any 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.

15 Boyer’s model of proton-driven ATP synthesis

•Boyer proposed that the energy of proton transfer is coupled to the conformational change via mechanical 120° turns.

This proposal is corroborated by the ﬁnding that attachment of the F1 via the stalk region to a glass plate results in an ATP-dependent clockwise spin, as assayed by labeled actin attached to 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 force exerted 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 electron pair directly into Complex IV).

H3C CH3

N+ N+

H3C 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 across the membrane, which dissipates the gradient. Also, the point of harnessing the proton potential is at the end of the cycle, which means that the amount of protons translocated need not be a multiple of the amount of protons required for ATP synthesis.

16 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.