Oxidative Phosphorylation & Electron Transport

Question 1

What does the term oxidation mean?

Answer 1

Oxidation refers to the process of LOSING an electron.

Question 2

What does the term reduction refer to?

Answer 2

Reduction refers to the GAIN of electrons.

Question 3

What are monooxygenases?

Answer 3

Monooxygenases are enzymes that add ONE oxygen atom of O₂ to a molecule.

Question 4

What are dioxygenases?

Answer 4

Dioxygenases are enzymes that add both atoms of O₂ to a substrate.

This increases the solubility of nonpolar compounds.

Question 5

What are oxidases?

Answer 5

Oxidases are enzymes that catalyze REDOX reactions involving O₂ as the electron acceptor.

Oxygen will be reduced to either:

a) Superoxide (1 electron)
b) Hydrogen peroxide (2 electrons)
c) Water (4 electrons)

Question 6

What does reduction potential (E^o) refer to?

Answer 6

Reduction potential (E^o) is the measure of the ease with which a compound can be oxidized or reduced.

The more positive the value of E^o, the more readily it accepts electrons.

The more negative the value of E^o, the more readily it donates / loses electrons.

Question 7

What is the equation that relates delta G^o’ to the Nernst equation and what is it used for?

Answer 7

delta G^o’ = - nF delta E^o’

This equation is used to determine how much energy a molecule / system possesses based on its reduction potential.

n = # of electrons

F = 96.485 kj/V x mol

Question 8

Which direction do electrons flow?

Answer 8

Electrons will flow to any half reaction that has a higher or less negative reduction potential.

Question 9

True or False: Oxygen is an effective electron sink.

Answer 9

True: Oxygen has a very positive reduction potential and is therefore an effective electron acceptor.

Question 10

What does the generation of ATP via oxidative phosphorylation require?

Answer 10

To generate ATP via oxidative phosphorylation (the electron transport chain), an electron donor and an electron acceptor is needed.

Components of the electron transport chain.

ATP synthase.

An intact, inner mitochondrial membrane is needed as well.

NADH and/or FAD [2H] are electron donors and O₂ is the electron acceptor.

Question 11

How is the electron transport chain organized?

Answer 11

There are four protein complexes on the INNER mitochondrial membrane.

Lipid soluble coenzymes (UQ and CoQ) and a water soluble protein (cytochrome c) shuttle between the complexes.

Electrons FALL in energy as they are passed through the chain from Complex I and II to Complex IV.

Question 12

Picture of the Electron Transport Chain

Answer 12

Question 13

Energy Blance of Body

Answer 13

Question 14

What are the five(5) major protein components of the electron transport chain (oxidative phosphorylation)?

Answer 14

Complex I (NADH-CoQ Oxidoreductase)

Complex II (Succinate dehydrogenase)

Complex III (Cytochrome b-c₁ complex)

Complex IV (Cytochrome oxidase)

ATP Synthase

Question 15

What are the two coenzymes that are embeded in the inner mitochondrial membrane and what role do they play in the electron transport chain?

Answer 15

Coenzyme Q

Cytochrome C

Question 16

What is the function of CoQ₁₀ and what three states can it exist in?

Answer 16

CoQ₁₀ functions as an electron carrier from enzyme complex I and enzyme complex II to complex III.

CoQ₁₀ can exist in one of three oxidation states:

1) Fully oxidized to ubiquinone
2) Semiquinone
3) Fully reduced to ubiquinol

Question 17

What is the function of cytochrome C?

Answer 17

The heme group of cytochrome c accepts electrons from the b-c1 complex and transfers electrons to the cytochrome oxidase complex.

Cytochrome c is also involved in initiation of apoptosis. Upon release of cytochrome c to the cytoplasm, the protein binds apoptotic protease activating factor-1 (Apaf-1).

Question 18

What is the purpose of the electron transport chain?

Answer 18

The function of the electron transport chain is to produce a transmembrane mitochondrial proton electrochemical gradient as a result of the redox reactions.

If protons flow back through the mitochondrial membrane, they enable mechanical work.

Question 19

Why is the electron transport chain also called oxidative phosphorylation?

Answer 19

The entire process is called oxidative phosphorylation, since ADP is phosphorylated to ATP using the energy of hydrogen oxidation in many steps.

Question 20

Diagram of the overall pathway of the transfer of electrons within the electron transport chain.

Answer 20

**NADH** → ***Complex I*** → **Q** → ***Complex III*** → **cytochrome *c*** → ***Complex IV*** → **O<sub>2</sub>** ↑ ***Complex II*** ↑ ***FADH***

Question 21

What is the function of Complex I (NADH dehydrogenase)?

Answer 21

Complex I oxidizes NADH and reduces CoQ. It transfers two electrons from NADH to CoQ.

Ubiquinone is reduced to ubiquinol and difuses across the membrane and transfers four protons across the membrane to produce a proton gradient.

Complex I also transports protons from the mitochondrial matrix to the cytosolic side of the inner mitochondrial membrane.

The cytosolic side is more positive than the matrix side.

Question 22

How are electrons oxidized and what is their path at Complex I?

Answer 22

NADH is oxidized to NAD⁺, by reducing Flavin mononucleotide (FMN) to FMNH₂ in one two-electron step.

FMNH₂ is then oxidized in two one-electron steps, through a semiquinone intermediate. Each electron thus transfers from the FMNH₂ to an Fe-S cluster, from the Fe-S cluster to ubiquinone (Q).

Transfer of the first electron results in the free-radical (semiquinone) form of Q, and transfer of the second electron reduces the semiquinone form to ubiquinol.

During this process, four protons are translocated from the mitochondrial matrix to the intermembrane space.

Question 23

What is the purpose of succinate dehydrogenase (Complex II)?

Answer 23

Complex II oxidizes succinate and reduces CoQ.

When succinate is converted to fumarate in the TCA, there is a concomitant reduction of bound FAD to FADH₂ in succinate dehydrogenase.

The FADH₂ transfers its electrons to Fe-S centers which then pass them on to CoQ.

NOTE: It is the only TCA cycle enzyme that is an intergral membrane protein of the inner mitochondrial membrane.

NOTE II: The oxidation of one FADH₂ in the electron transport chain results in the synthesis of two ATPs. The oxidation of one NADH results in the production of three ATPs.

Question 24

What is unique about succinate dehydrogenase (Complex II)?

Answer 24

Succinate dehydrogenase is an enzyme complex, bound to the inner mitochondrial membrane.

It is the only enzyme that participates in both the citric acid cycle and the electron transport chain.

Question 25

What does the Complex III (Cytochrome c reductase / cytochrome _bc1) do?

Answer 25

Complex III mediates electron transport from CoQ to cytochrome c.

This cycle is known as the Q cycle.

Complex III also transports two protons across the inner mitochondrial membrane.

Question 26

What does Complex IV (cytochrome c oxidase) do?

Answer 26

Complex IV transfers electrons from cytochrome c to reduce oxygen in the mitochondrial matrix.

Cytochrom c oxidase and oxygen are the final destination for the electrons in the ETC.

The combined processes of oxygen reduction and proton transport require eight protons. Four are used to reduce two molecules of oxygen (2 per cycle) and four are transported from the matrix to the intermembrane space (2 per cycle)

Proton transport in Complex IV takes place via two channels that are lined with water molecules and polar amino acids.

Question 27

Picture of the electron transport chain.

Answer 27

Question 28

Picture showing relationship of electron transport chain and TCA.

Answer 28

Question 29

What does the term “coupling” refer to?

Answer 29

The electron transport chain and oxidative phosphorylation are coupled by a proton gradient across the inner mitochondrial membrane.

The efflux of protons from the mitochondrial matrix creates a proton gradient. This gradient is used by the ATP synthase complex to make ATP via oxidative phosphorylation.

The F_O component of ATP synthase acts as an ton channelthat provides for a proton flux back into the mitochondrial matrix. This reflux releases free energy produced during the generation of the oxidized forms of the electron carriers (NAD⁺ and Q). The free energy is used to drive ATP synthesis, catalyzed by the F₁ component of the complex.^<i></i>

Coupling with oxidative phosphorylation is a key step for ATP production. However, in specific cases, uncoupling the two processes may be biologically useful. The uncoupling protein, thermogenin—present in the inner mitochondrial membrane of brown fat—provides for an alternative flow of protons back to the inner mitochondrial matrix. This alternative flow results in thermogenesis rather than ATP production.^{<i> </i>}

^<i></i>Synthetic uncouplers (e.g., 2,4-dinitrophenol) also exist, and, at high doses, are lethal.^<i></i>

Question 30

What is the function of cytopchrome C?

Answer 30

Cytochrome C is a mobile electron carrier.

Electrons are passed from Complex III to cytochrome c.

It is associated with the inner mitochondrial membrane where it carries electrons in its reduced state to to cytochrome c oxidase.

Question 31

What is ATP synthase and what does it do?

Answer 31

ATP synthase is a combined enzyme, proton pump, and rotating molecular motor.

It is made up of the F₁ subunit and the F₀ subunit.

Question 32

What is the F₁ subunit of the ATP synthase and what does it do?

Answer 32

The F₁ subunit of the ATP synthase is the catalytic subunit of the ATP synthase; it catalyzes ATP production.

The F₁ subunit is made up of five modules: Alpha, beta, gamma, delta, and epsilon.

The F₁ portion of the ATP synthase is above the membrane, inside the matrix of the mitochondria.

The alpha and beta subunits are arranged in alternating patterns in the hexamer and are similar but not identical.

There are six ATP-binding sites on this subunit hexamer. Three of these, located on the beta subunit, ar ethe catalytic sites. The other three, located on the alpha subunit, are non-catalytic.

The release of newly synthesized ATP is what powers the subunits catalytic activity.

Question 33

What is the F₀ subunit and what is its funtion within the ATP synthase?

Answer 33

The F₀ subunit serves as the attachment point for the F₁ subunit.

It is made up three hydrophobic subunits (A, B, and C) and six additional subunits, d, e, f, g, F6, and 8.

The a and b subunits form part of the stator. This is a stalk-like structore that is anchored into the membrane and connects to the F₁ subunit. There is also a ring composed of 1-15 subunits; this makes up the rotor of the motor.

Protons flowing through the a-c complex cause the c-ring to rotate in the membrane.

The flow of protons through this subunit drives the conformational changes that result in the binding of substrate on the ATP synthase, ATP synthesis, and release of product (ATP).

Question 34

How exactly does the F_o subunit operate?

Answer 34

The proton-motive force across the inner mitochondrial membrane, generated by the electron transport chain, drives the passage of protons through the membrane via the F_O region of ATP synthase.

A portion of the F_O (the ring of c-subunits) rotates as the protons pass through the membrane. The c-ring is tightly attached to the asymmetric central stalk (consisting primarily of the gamma subunit), which rotates within the alpha₃beta₃ of F₁ causing the 3 catalytic nucleotide binding sites to go through a series of conformational changes that leads to ATP synthesis.

The binding change mechanism involves the active site of a β subunit’s cycling between three states.In the “open” state, ADP and phosphate enter the active site. The protein then closes up around the molecules and binds them loosely — the “loose” state. The enzyme then undergoes another change in shape and forces these molecules together, with the active site in the resulting “tight” state (shown in pink) binding the newly produced ATP molecule with very high affinity. Finally, the active site cycles back to the open state, releasing ATP and binding more ADP and phosphate, ready for the next cycle of ATP production.

Question 35

What would happen if compounds were partially reduced in the electron transport chain?

Answer 35

Hazardous compounds such as superoxides and free radicals would be produced.

Question 36

What are some inhibitors of oxidative phosphorylation and what steps do they block?

Answer 36

Inhibitors of Complex I, II, and III block electron transport:

a) Rotenone inhibits NADH-UQ oxidoreductase in Complex I; this prevents the reduction of CoQ and the oxidation of the Fe-S clusters of NADH-UQ oxidoreductase.
b) Blocking the activities of Q cytochrome c oxidoreductase prevents passage of protons on to cytochrome c
c) Cyanide, azide, and carbonmonoxide inhibit Complex IV (Cytochrome C oxidase): This inhibits passing protons on to O₂ to produce water.
d) ATP synthase inhibitors (Oligomycin and DCCD) block the movement of protons through the F_o subunit. No ATP synthesis occurs. This proves that electron transport and ATP synthysis is tightly coupled.

Question 37

What do uncouplers do?

Answer 37

Uncouplers disrupt the coupling of electron transport and ATP synthesis.

They dissapate the proton gradient across the inner mitochondrial membrane that is created by the ETC.

Examples include: 2,4-DNP

They are hydrophobic and have a dissociable proton.

They carry protons across the inner membrane to the matrix. In otherwords, protons leak back in via the uncouplers and so ATP synthasis does not occur.

Increased O₂ consumption and NADH oxidation.

The energy released in electron transport in this case is released as heat.

Question 38

What happens when no pH or charge gradient exists?

Answer 38

Instead of ATP being synthesized by ATP synthase, it is hydrolyzed.

Question 39

How does brown fat respond to cold temperatures?

Answer 39

Uncouple oxid. Phos. from ATP synthesis to generate heat.

In response to a temp drop, the release of hormones leads to the liberation of free fatty acids from triacylglycerols that in turn activate uncoupling protein 1 (UCP-1).

UCP-1 forms a pathway for the flow of H+ from the cytoplasm to the matrix.

The tissue with high UCP-1 called brown fat. (combination of greenish cytochromes and red hemoglobin in blood supply)

Question 40

How many protons are required by ATP synthase to generate a molecule of ATP?

Answer 40

2.7 protons are required to make each ATP.

Question 41

What is the ATP-ADP translocase?

Answer 41

ADP enters the mitochondrial matrix only if ATP exits.

ATP movement out of the matrix is favored due to the negative charge on the ATP; the cytosol is + relative to the matrix.

One ATP out requires the energy from a hydrogen.

Thus, making and transporting one ATP requires four hydrogens.

ATP-ADP translocase tightly couples the movement of of ATP out of the matrix with the movement of ADP into the matrix.

Question 42

How many protons are pumped out of the matrix per electron pair?

Answer 42

NADH-Q reductase - 4

Q-cytochrome C oxidoreductase - 2

Cytochrome c oxidase - 4

Thus 2.5 molecules of cytoplasmic ATP are generated as a result of the flow of a pair of electrons from NADH to O2 and those from the oxidation of succinate, the yield is about 1.5 molecules of ATP per electron pair.

Question 43

How are electrons of cytosolic NADH shuttled into the ETC?

Answer 43

a) Glycerophosphate shuttle: Two different glycerophosphate dehydrogenases (one in the cytosol and one on the outer face of the mitochondrial inner membrane) work together to carry electrons into the matrix of the mitochondrial matrix.

NADH produced in the cytosol transfers its electrons to dihydroxyacetone phosphate and it is reduced to glycerol-3-phosphate. It is reoxidized FADH₂ and its two protons are passed to UQ to form UQH.

This yields 1.5 molecules of ATP.

This mode is ireversible and operates when NADH levels are low.

b) Malate-Aspartate shuttle: Oxaloacetate is reduced in the cytosol and thus acquires the electrons from NADH (which is oxidized NAD⁺). Malate is transported across the inner membrane where it is reoxidized by malate dehydrogenase converting NAD⁺ to NADH in the matrix. This NADH enters the ETC. The oxaloacetate is transaminated to form aspartate and is transported back to the cytosolic face of the mitochondrial matrix.

Question 44

ATP synthase picture.

Answer 44

Question 45

ATP synthase picture II.

Answer 45

Question 46

ATP synthase picture III.

Answer 46

Question 47

ATP binding to F_o subunits.

Answer 47