Ch 19 - Oxidative Phosphorylation

Question 1

Q. 1: In all eukaryotic life forms, mitochondria are the cell’s “power plant” responsible for the synthesis of large amounts of ATP from food molecules. Our current understanding of ATP synthesis operating mitochondria is based on a (initially controversial) hypothesis which was introduced by Nobelist ________________ in 1961. This hypothesis led to the single most important theory in all of cell biology. What is the name of this theory? What does it state?

Answer 1

In all eukaryotic life forms, mitochondria are the cell’s “power plant” responsible for the synthesis of large amounts of ATP from food molecules. Our current understanding of ATP synthesis operating mitochondria is based on a (initially controversial) hypothesis which was introduced by Nobelist Peter Mitchell in 1961. This hypothesis led to the single most important theory in all of cell biology. What is the name of this theory? What does it state?

Chemiosmotic theory: the transmembrane differences in proton concentration across the inner membrane of the mitochondria are the reservoir for the energy extracted from biological oxidation reactions. Specifically, the theory suggests that most ATP synthesis in respiring cells comes from the electrochemical gradient across the inner membranes of mitochondria by using the energy of NADH and FADH₂ formed from the catabolism of energy-rich molecules like glucose to pump protons from the mitochondrial matrix to the intermembrane space, forming a transmembrane electrochemical gradient. The protons move back across the inner membrane through the enzyme ATP synthase, providing energy for ADP to combine with P_i to form ATP.

Question 2

Q. 2: The directed flow of protons across the inner mitochondrial membrane happens through specific protein channels which are provided by a protein complex, called _______________, that couples this proton flow to the phosphorylation of _____ and phosphate to ______.

Answer 2

The directed flow of protons across the inner mitochondrial membrane happens through specific protein channels which are provided by a protein complex, called ­ATP synthase, that couples this proton flow to the phosphorylation of ADP and phosphate to ATP.

Question 3

Q. 3: List some of the unique properties and components of the inner mitochondrial membrane.

Answer 3

1. Impermeable to most small molecules and ions, including H⁺
2. The only species that cross this membrane do so through specific transporters
3. Convolutions of this membrane (cristae) create a very large surface area that drastically increases the number of electron-transfer systems (respiratory chains) and ATP synthase distributed over the membrane surface
4. Contains the components of the respiratory chain (Complexes I – IV)
5. Contains ATP synthase

Question 4

Q. 4: What is the generic name of the enzymes which catalyze reversible chemical reactions of the following general type:

Reduced substrate + NAD⁺ à oxidized substrate + NADH + H⁺

Answer 4

Nicotinamide nucleotide-linked dehydrogenases

Question 5

Q. 5: One important concept observed in cell metabolism is that cells not only maintain separate pools of the reduction equivalents NADPH and NADH, but also have different uses for both. Which ones?

Answer 5

One of the main functions of NADH is to carry electrons from catabolic reactions to their point of entry into the respiratory chain (NADH dehydrogenase complex). In general, NADH is involved in catabolic reactions.

NADPH, on the other hand, generally supplies electrons to anabolic reactions (biosynthesis).

Question 6

Q. 6: Can NAD+/NADH or NADP+/NADPH molecules freely cross the inner mitochondrial membrane? Why?

Answer 6

No. NADH is generated in the cytosol (by glycolytic pathways, for example), but NADH cannot simply pass into mitochondria for oxidation by the respiratory chain because the inner mitochondrial membrane is impermeable to NADH and NAD⁺. The inner membrane must be impermeable to most molecules in order to maintain the electrochemical gradient necessary for cellular respiration. The solution is that electrons from NADH, rather than NADH itself, are carried across the mitochondrial membrane. One of several means of introducing electrons from NADH into the electron transport chain is the glycerol 3-phosphate shuttle, for example. In the heart and liver, electrons from cytosolic NADH are brought into mitochondria by the malate-aspartate shuttle.

Question 7

Q. 7: Both, NAD+ and FAD are very important cellular electron and proton shuttle molecules. But they have significant structural and functional differences. Name those. Which (dietary important) vitamins are needed for the synthesis of both?

Answer 7

NAD⁺ consists of two nucleotides joined through their phosphate groups. The nucleotides contain an adenine nucleobase and the other is nicotinamide. NAD exists in two forms: oxidized and reduced (NAD⁺ and NADH, respectively). Nicotinamide adenine dinucleotide is involved in redox reaction, carrying electrons from one reaction to another. For example, NADH carries electrons from catabolic reactions to their point of entry into the respiratory chain. NADH carries two electrons. Niacin (vitamin B₃) is needed for synthesis of NAD. Flavin adenine dinucleotide (FAD) is also a redox coenzyme, but it is usually very tightly associated with or covalently bonded to proteins. These proteins containing a flavin group (FMN or FAD) are known as flavoproteins. FAD consists of two main portions: AMP and FMN (flavin mononucleotide) bridged together through their phosphate groups. Riboflavin (vitamin B₂) is an important vitamin needed for the synthesis of FAD. Riboflavin is formed by a C-N bond between an isoalloxazine and a ribitol. FAD can accept either one electron (yielding the semiquinone form), or two electrons (yielding FADH₂).

Question 8

Q. 8: The mitochondrial respiratory chain, or often referred to as _____________ chain, consists of ___ major, membrane-intercalated protein complexes which act as a series of sequentially acting ____________ carriers.

Answer 8

Q. 8: The mitochondrial respiratory chain, or often referred to as electron transport chain, consists of four major, membrane-intercalated protein complexes which act as a series of sequentially acting electron carriers.

Question 9

Q. 9: Which of the following is/are (a) electron transfer(s) happening in the mitochondrial respiratory chain? Circle all of them.

A) reversible reduction of Fe3+ to Fe2+

B) transfer of methyl groups

C) transfer of a hydride ion (:H-)

D) transfer of hydrogen atoms; i.e. H⁺ + e-

E) transfer of hydroxyl ions

Answer 9

A) reversible reduction of Fe3+ to Fe2+

B) transfer of methyl groups

C) transfer of a hydride ion (:H-)

D) transfer of hydrogen atoms; i.e. H⁺ + e-

E) transfer of hydroxyl ions

Question 10

Q. 10: There are several types of electron-carrying molecules functioning within the mitochondrial respiratory chain which undergo reduction-oxidation (Redox) reactions. Which of the following are those? (Multiple answers possible)

A) ubiquinone

B) cytochromes

C) cobalt containing enzymes

D) iron-sulfur proteins

E) cysteine bound copper

Answer 10

A) ubiquinone

B) cytochromes

C) cobalt containing enzymes

D) iron-sulfur proteins

E) cysteine bound copper

Question 11

Q. 11: Coenzyme Q is a lipid-soluble benzoquinone with a long, lipophilic _______________ side chain. It is often referred to as a mitochondrial “redox cycler”. Explain this term with respect to its role in carrying electrons and protons in the inner mitochondrial membrane.

Answer 11

Coenzyme Q is a lipid-soluble benzoquinone with a long, lipophilic isoprenoid side chain. It is often referred to as a mitochondrial “redox cycler”. Explain this term with respect to its role in carrying electrons and protons in the inner mitochondrial membrane.

Question 12

Q. 13: The mitochondrial electron transport chain contains 3 classes of redox proteins called __________. They are designated a, b, and c. How are can they be distinguished? What doe they have in common?

Answer 12

The mitochondrial electron transport chain contains 3 classes of redox proteins called cytochromes. They are designated a, b, and c. How are can they be distinguished? What doe they have in common?

Cytochromes are distinguished by differences in their light-absorption spectra. Each type of cytochrome in its reduced form (Fe²⁺) state has three absorption bands in the visible range. Cytochromes have in a common an iron-containing heme prosthetic group.

Question 13

Q. 14: With respect to their heme groups, how are the three cytochromes different?

Answer 13

The heme groups of cytochromes a and b are tightly (not covalently) bound to their associated proteins; however, the heme group of cytochrome c is covalently attached to its associated protein through two Cys residues. Heme a of cytochrome a has a long isoprenoid tail attached to one of the five-membered rings. Heme b does not contain an isoprenoid tail nor covalent bonds to Cys residues, but it has two methylene groups attached to two five-membered rings.

Question 14

Q. 12: The Figure below shows the chemical structure of ubiquinone. Is it the oxidized or the reduced state? Label the functional group(s) which are involved in the alternating reduction-oxidation (redox) reaction of this important mitochondrial molecule. What is the name of the fully reduced form? What is the name of this molecule?

Answer 14

The above figure is ubiquinone, which is in the fully oxidized state. The functional groups involved in the alternating redox reactions are the ketone functional groups in the above figure. In the fully reduced ubiquinol, these functional groups are hydroxyl groups. This molecule is called coenzyme Q, also known as ubiquinone.

Question 15

Q. 15: At least ___\_ Fe-S proteins are found in mitochondrial electron transport chain. What is their function there? How is the iron organized? What is the ionic state (valence) of the iron in its reduced form? In its oxidized form?

Answer 15

At least eight Fe-S proteins are found in mitochondrial electron transport chain. What is their function there? How is the iron organized? What is the ionic state (valence) of the iron in its reduced form? In its oxidized form?

All iron-sulfur proteins participate in one-electron transfers where one iron atom of the iron-sulfur cluster is oxidized or reduced. The iron is present in association with inorganic sulfur atoms or with the sulfur atoms of Cys residues in the protein, or both. Iron-sulfur centers vary from simple structures with a single Fe atom coordinated to four Cys –SH groups to more complex iron-sulfur centers with two or four Fe atoms. The ionic state of iron in its reduced form is Fe²⁺ and Fe³⁺ in its oxidized form.

Question 16

Q. 16: During biological oxidation of NADH + H⁺ at the mitochondrial electron transport chain, __\_electrons are gives off, and all mitochondrial iron-sulfur proteins participate in __\_-electron transfers.

Answer 16

During biological oxidation of NADH + H⁺ at the mitochondrial electron transport chain, two electrons are gives off, and all mitochondrial iron-sulfur proteins participate in one-electron transfers.

Question 17

Q. 17: In the Fe-S centers of “simple” iron-sulfur proteins a single Fe ion is surrounded and held in place by the ______ atoms of ____ cysteine (Cys) residues.

Answer 17

In the Fe-S centers of “simple” iron-sulfur proteins a single Fe ion is surrounded and held in place by the sulfur atoms of four cysteine (Cys) residues.

Question 18

Q. 18: The direction of the flow of electrons within the components of the mitochondrial electron transport chain is determined by a measurable number, called ________, which is measured for each of the individual electron carriers. Write down the correct order of the mitochondrial redox carriers based on their measured values.

Answer 18

The direction of the flow of electrons within the components of the mitochondrial electron transport chain is determined by a measurable number, called reduction potential, which is measured for each of the individual electron carriers. Write down the correct order of the mitochondrial redox carriers based on their measured values.

NADH –> Q –> Cyt b –> Cyt c₁ –> Cyt c –> Cyt a –> Cyt a₃ –> O₂

Question 19

Q. 20: Complex I of the mitochondrial electron transport chain is called NADH:ubiquinone oxidoreductase or ___________. It is the largest enzyme complex composed of __ different polypeptide chains, including an ___-containing flavoprotein and at least ___ iron-sulfur centers.

Answer 19

Complex I of the mitochondrial electron transport chain is called NADH:ubiquinone oxidoreductase or NADH dehydrogenase. It is the largest enzyme complex composed of 42 different polypeptide chains, including an FMN-containing flavoprotein and at least six iron-sulfur centers.

Question 20

Q. 21: The mitochondrial electron transport chain is inhibited by a series of “respiratory toxins/poisons. Which of the following inhibit the flow of electron from the Fe-S centers

of Complex I to ubiquinone? (Multiple answers possible)

A) Amytal (a barbiturate drug)

B) Oligomycin

C) Rotenone (a plant-derived insecticide)

D) Cyanide

E) Piericidin A (an antibiotic)

Answer 20

A) Amytal (a barbiturate drug)

B) Oligomycin

C) Rotenone (a plant-derived insecticide)

D) Cyanide

E) Piericidin A (an antibiotic)

Question 21

Q. 19: In the mitochondrial electron transport chain, electrons are not only funneled into the chain via dehydrogenation of NADH + H⁺, but also through other substrates. Name those. At which level are they fed into the chain?

Answer 21

• Succinate at Complex II (and then to Q)
• Glycerol 3-phosphate donates electrons to a flavoprotein at glycerol 3-phosphate dehydrogenase on the outer face of the inner mitochondrial membrane (and then to Q)
• Fatty acyl-CoA at acyl-CoA dehydrogenase passes electrons to electron-transferring flavoprotein (ETF) and then on to Q via ETF:ubiquinone oxidoreductase
• Ubiquinone at Complex III
• Cytochrome c at Complex IV

Question 22

Q. 22: Which component of the mitochondrial electron transport chain (delivers the electrons (and protons) derived from the oxidation of succinate over to ubiquinone? What is the name of the enzyme complex? What is so unique about this component? Which coenzyme/cofactors does it require? Which vitamin supply does this coenzyme/cofactor require?

Answer 22

Complex II is where ubiquinone is the final electron acceptor from succinate. The name of the enzyme complex is succinate dehydrogenase, and it is unique because it is the only membrane-bound enzyme in the citric acid cycle. Complex II requires the following coenzymes/cofactors: heme b, coenzyme Q (ubiquinone), three 2Fe-2S centers, and FAD. Since Complex II contains FAD, it requires vitamin B₂ (riboflavin).

Question 23

Q. 23: What do we know about the directional flow of electrons in complex II? Which sequence?

Answer 23

The path of electron transfer in complex II: succinate –> FAD –> through the three 2Fe-2S centers –> Q. Once Q accepts the two electrons, it leaves the Q-binding site of complex II as QH₂ (ubiquinol).

Question 24

Q. 24: Complex II also contains a heme b group. What is its function? What are the potential consequences for the cell if this component is dysfunctional? Which human disease(s) have been brought in connection with a malfunction of this complex II component?

Answer 24

The heme b of Complex II is not in the direct path of electron transfer, but it most likely serves to reduce the occurrence of electrons leaking out of the system. If electrons leak out of the system, they can move from succinate to O₂, producing reactive oxygen species (ROS) like hydrogen peroxide and the superoxide radical. Humans with mutations in Complex II subunits near the heme b and/or Q-binding site are afflicted with hereditary paraganglioma (PGL). PGL is characterized by the development of benign, vascularized tumors in the head and neck, usually in the carotid body (CB), a chemoreceptive organ that sense oxygen levels in the blood.

Question 25

Q. 25: At which level of the mitochondrial electron transport chain are the electrons and protons derived from beta-oxidation of fatty acids funneled into the chain?

Answer 25

At the level of ubiquinone, but not through Complex II. Acyl-CoA dehydrogenase catalyzes the transfer of electrons from fatty acyl-CoA to the FAD of the dehydrogenase, then to electron-transferring flavoprotein (ETF). ETF then passes its electrons to ETF:ubiquinone oxidoreductase, which in turn transfers electrons into the respiratory chain by reducing ubiquinone. Reduced ubiquinone then shuttles to Complex III.

Fatty acyl-CoA → Acyl-CoA dehydrogenase → ETF → ETF:ubiquinone oxidoreductase → Q → Complex III.

Question 26

Q. 26: The cytochrome bc1 complex of the mitochondrial ETC is often referred to as _________________ and also called _____________________. What is the biological function of this enzyme complex? What is unique about it?

Answer 26

The cytochrome bc1 complex of the mitochondrial ETC is often referred to as Complex III and also called ubiquinon:cytochrome c oxidoreductase. What is the biological function of this enzyme complex? What is unique about it?

Complex III couples the transfer of electrons from ubiquinol (QH₂) to cytochrome c. It also accomplishes vectorial transport of protons from the matrix to the intermembrane space. One unique aspect of Complex III is that it is where QH₂ is oxidized back to Q via the Q cycle. Also, Complex III contains a Rieske iron-sulfur protein with a 2Fe-2S center, which is unique because one of the two Fe atoms is coordinated by two His residues rather than two Cys residues. Furthermore, Complex III is where the switch between the two-electron carrier (ubiquinone) and the one-electron carriers (cyt b, c₁, and c) takes place via the Q cycle.

Question 27

Q. 27: Explain the meaning and role of the mitochondrial “Q cycle”.

Answer 27

QH₂ + 2 cyt c₁(oxidized) + 2H_N⁺ –> Q + 2 cyt c₁(reduced) + 4H_P⁺

The meaning of the Q cycle is the cyclical reduction and oxidation of coenzyme Q during the electron transport chain that results in the transport of four protons from the mitochondrial matrix into the intermembrane space. Q is reduced at Complex I, Complex II, or at other electron entry points prior to Complex III (ETF:Q oxidoreductase or glycerol 3-phosphate dehydrogenase, for example), and then the reduced form (ubiquinol, QH₂) travels to Complex III where it is re-oxidized and transfers its electrons to cytochrome c. Thus, Q passes its electrons on to a carrier with a higher reduction potential and the cyclic nature of Q’s reduction then oxidation allows the electron transfer chain to continue because Q is recycled and reused. The Q cycle accommodates the transition between the two-electron carrier (ubiquinone) and the one-electron carriers (the cytochromes).

Question 28

Q. 28: Name the important “redox” components of complex III and write down the directional flow of electrons through the complex III of the mitochondrial ETC.

Answer 28

The important redox components of Complex III are cytochromes b₅₆₂, b₅₆₆, c₁, and the Rieske iron-sulfur protein. Cytochrome c is another important redox component, but it is not fixed to Complex III because it accepts an electron and moves to Complex IV. Ubiquinone is another important redox component in the Q cycle associated with Complex III, but it also is not fixed to Complex III. The flow of electrons for the first and second oxidation of QH₂ is depicted below.

Question 29

Q. 29: The mitochondrial electron transport chain also contains cytochrome c. Compared with the other ETC cytochromes it is quite different with respect to size, location and function. Explain.

Answer 29

The cytochromes of type a and b are integral proteins of the inner mitochondrial membrane. However, cytochrome c is an exception. It is a highly water-soluble protein that associates through electrostatic interactions with the outer surface of the inner mitochondrial membrane. Cyt c is relatively small (~12 kDa, primary structure of ~100 amino acids). Cytochrome b for example is ~400 amino acids, so it is much larger than cyt c. Cyt c binds its heme via two Cys residues in a characteristic -CXXCH- amino acid motif. Cyt c undergoes oxidation and reduction at its iron atom, but it does not bind oxygen – it transfers electrons (one electron) between Complex III and IV of the ETC.

Question 30

Q. 30: The biological function of complex IV of the mitochondrial ETC, also called ______________, molecularly explains the high dependency of aerobic life forms to molecular oxygen (O₂). Explain. Which ETC component delivers the electrons to this complex? What unique cofactor is found in this ETC component not found in other cytochromes?

Answer 30

The biological function of complex IV of the mitochondrial ETC, also called cytochrome oxidase, molecularly explains the high dependency of aerobic life forms to molecular oxygen (O₂). Explain. Which ETC component delivers the electrons to this complex? What unique cofactor is found in this ETC component not found in other cytochromes?

Complex IV explains the dependence of aerobic life forms on O₂ because Complex IV carries electrons from cyt c to O₂, reduing O₂ to H₂O. O₂ is the final acceptor of electrons in the ETC and has the highest reduction potential (+0.8166 V). Cytochrome c delivers the electrons to Complex IV. Complex IV contains two copper ion cofactors (Cu_A and Cu_B). Cu_A contains two Cu ions that are complexed with the –SH groups of two Cys residues in a binuclear center. Complex IV also contains two hemes, cytochrome a and a₃. Cu_B and cyt a₃ form a binuclear center that is the site of oxygen reduction.

Question 31

Q. 31: Map out the flow of electron through Complex IV starting from cytochrome c below. What about its function regarding the flow of protons (H⁺)?

Answer 31

1. Two reduced cyt c molecules give off a total of 2 electrons to the binuclear center Cu_A, and the two electrons pass through heme a. One electron stops at the Cu_B group (Cu²⁺ à Cu⁺) , while the other stops at heme a₃ (Fe³⁺ à Fe²⁺).
2. O₂ molecule binds the heme a₃—Cu_B (Fe-Cu center) and abstracts the two electrons to form a peroxide bridge.
3. Two more reduced cyt c are oxidized to transfer 2 electrons. Two H⁺ ions are obtained from the matrix to break the peroxide bridge to form Cu_B-OH and Heme a₃-OH.
4. The abstraction of two more H⁺ from the matrix oxidizes the heme a₃ and Cu_B back into their original state and releases two molecules of water. At the same time, four more protons are pumped from the matrix into the intermembrane space. The exact mechanism of this proton pump that transfers four protons across Complex IV is still unknown.

Question 32

Q. 32: In the mitochondrial electron transport chain, much of the energy of the flow of electrons along the reduction potential is used to do what? At how many sites does this take place? Which complexes are involved? How many protons per electron pair?

Answer 32

Much of the energy from the flow of electrons along the reduction potential is used to pump protons out of the matrix and into the intermembrane space, thereby establishing an electrochemical gradient. This takes place at three sites – Complex I, III, and IV. The transfer of two electrons from NADH to O₂ is accompanied by the outward pumping of 10 H⁺.

Question 33

Q. 33: The electrochemical energy generated by a functional electron transport chain is established in the form of a _________ gradient and also through the separation of charges across the ________ mitochondrial membrane. The energy stored in the gradient is also referred to as ________________________ or abbreviated “_____”.

Answer 33

Q. 33: The electrochemical energy generated by a functional electron transport chain is established in the form of a proton concentration gradient and also through the separation of charges across the inner mitochondrial membrane. The energy stored in the gradient is also referred to as proton-motive force or abbreviated “PMF”.