Cell integrity

Question 1

ATP equivalent

Answer 1

GTP

Question 2

Bulk of ATP produced in … in process called…

Answer 2

Mitochondria in oxidative phosphorylation

Question 3

Mitochondria structure

Answer 3

Outer membrane (which limits the size of the organelle).

Inner membrane (folds that project inward called cristae), reactions of oxidative phosphorylation take place here. Numerous folds within the cristae increase the surface area upon which o. p. can take place.

Krebs Cycle reactions occur in the matrix.

Question 4

Reoxidation of NADH and FADH₂ reaction

Answer 4

NADH + H+ + ½ O2 → NAD+ + H20

FADH2 + H+ + ½ O2 → FAD + H20

Occurs Within the mitochondria.

ΔG for ATP hydrolysis is -31 kJ/mol.

Energy released from the re-oxidation of NADH and FADH2 is enough to generate several phosphoanhydride bonds.

Much of this energy is recovered by the components of the electron transport chain and used to synthesise ATP.

Question 5

Electron transport chain 4 membrane proteins

Answer 5

Complex I (NADH-Q oxidoreductase or NADH dehydrogenase)

Complex II (Succinate-Q reductase or succinate dehydrogenase)

Complex III (Q-cytochrome C oxidoreductase)

Complex IV (cytochrome c oxidase)

Question 6

ETC mobile carriers

Answer 6

Co-enzyme Q (ubiquinone)

Cytochrome C

Question 7

WHich complexes accept e^-

Answer 7

Complexes I, III and IV accept electrons and in doing so, a proton (H+) from the aqueous solution. As electrons pass through each of these complexes, a proton is passed or ‘pumped’ to the intermembrane space as shown in pic

Question 8

What does succinate dehydrogenase ( complex II ) do

Answer 8

Enzyme of the TCA cycle and sits in the inner mitochondrial membrane. It uses FAD as a cofactor and can communicate directly with Coenzyme Qwhich is also in the membrane. As electrons are passed from FADH2 to Coenzyme Q it also picks up a pair of protons, thereby regenerating FAD and forming QH2.

Question 9

Why are fewer protons pumped to inner membrane space when FADH2 is reoxidised compared with NADH.

Answer 9

Complex I bypassed, because FAD cofactor for complex II so fewer protons are pumped to the inter membrane space.

Protons flowing back into the matrix via ATP synthase are used to generate ATP. Thus, fewer ATP molecules are generated from the reoxidation of FADH2 compared to NADH.

Electrons donated by other FADH2 molecules such as those generated in the glycerol phosphate shuttle and the β-oxidation pathway all bypass complex I in this fashion.

Question 10

Redox reaction meaning

Answer 10

E tranfer, reducing agent /reduced substrate (donate e) and oxidising agent (oxidised substrate) (accepts e).

A substrate that can exist in both oxidised and reduced forms is known as a redox couple.

e.g. NAD+/ NADH; FAD / FADH2; Fe3+/Fe2+; ½ O2/H2O

Question 11

Redox potential

Answer 11

The ability of a redox couple to accept or donate electrons, also known as the reduction potential.

Question 12

How can standard redox potentials (E₀) be measured experimentally

Answer 12

Using hydrogen electrode as reference

Question 13

-ve E₀ implies that redox couple has tendency to..

Answer 13

donate e^- and so has more reducing power than H. Opposite for +ve E₀

Question 14

Transfer of e^- from one complex to another is …

Answer 14

energetically favourable, as they progress along the chain, the electrons lose energy.

Question 15

ATP synthase

Answer 15

• Multimeric (multiple polypeptide chains) enzyme.
• Consists of a membrane bound part (F0) and a F1 part which projects into the matrix space.
• Rotation of the enzyme drives transitions states, with altering affinities for ATP and ADP. So conformational energy flows from the catalytic subunit into the bound ADP and Pi to promote formation of ATP (chemical energy).
• The direction of proton flow dictates ATP Synthesis v ATP Hydrolysis, i.e. depending on the direction of the flow of protons through the ATP synthase, the complex can either generate ATP or consume it.

Question 16

The oxygen electrode

Answer 16

• Device that measures the oxygen concentration in a solution in a small chamber.
• Base of the chamber formed by a teflon membrane permeable to oxygen.
• Underneath this membrane, is a compartment containing two electrodes, a Platinum (Pt) cathode and a silver (Ag) anode.
• Small voltage of 0.6 volts applied between anode (+) and cathode (-). Oxygen diffuses through teflon membrane and is reduced to water at the Pt cathode:

O2 + 4H+ + 4e- → 2 H2O

• The circuit is completed at the silver anode, which is slowly oxidised to AgCl by the KCl electrolyte.

4 Ag0 + 4Cl- → AgCl + 4e-

• The resulting current is therefore proportional to the O conc in the sample chamber.
• We can use the oxygen electrode to dissect various components of the electron transport chain.
• First step is to prep a suspension of mitochondria from a tissue and place them into the chamber of the oxygen electrode.
• If the oxygen consumption of the suspension is then monitored for a set time period, the effects of various substrates and inhibitors on the electron transport chain can be determined.

Question 17

Clark type oxygen electrode

Answer 17

• Suspension of mitochondria placed into chamber of O electrode and recording is started.
• The baseline respiration is measured.
• If quantity of ADP added to the suspension is known then the ratio of the amount of ADP phosphorylated by the mitochondria to the amount of O consumed can be calculated, this is known as the ADP oxygen index and measures the efficiency of the mitochondrial phosphorylation system.
• Eventually ADP will be consumed and it returns to the level of basal respiration.

Question 18

ATP consumption

Answer 18

Each ATP molecules has a lifespan of between 1-5 mins.

Humans possess only about 250 g of ATP at any given moment with each ATP molecule recycled approximately 300 times per day.

So any interruption to the process of oxidative phosphorylation /ATP synthesis, means that a cell rapidly becomes depleted of ATP and is likely to die.

The most common cause of a failure of oxidative phosphorylation is simply a lack of oxygen e.g. hypoxia (diminished), anoxia (total).

Depending on the cell type and their metabolic requirements, death will be within a few minutes (neurons) or a few hours (muscle).

Question 19

Respiratory control

Answer 19

Uptake of oxygen by mitochondria is controlled by the components of ATP production: Inorganic phosphate (Pi), and ADP.

This is known as respiratory control and allows the body to adapt oxygen consumption to actual energy requirements.

Question 20

What are metabolic poisons

Answer 20

M​olecules that interfere with either the flow of electrons along the ETC or the flow of protons through ATP synthase by definition, interrupt ATP synthesis. As such, they are highly toxic and are termed metabolic poisons.

Question 21

Name the metabolic poisons

Answer 21

Cyanide (CN-) and azide (N3-)

M​alonate

Rotenone

Oligomycin

Dinitrophenol (DNP)

Question 22

Cyanide (CN-) and azide (N3-)

Answer 22

Bind with high affinity to the ferric (Fe3+) form of the haem group in the cytochrome oxidase complexblocking the final step of the ETC.

Question 23

Malonate

Answer 23

Closely resembles succinate and acts as a competitive inhibitor of succinate dehydrogenase. It slows down the flow of electrons from succinate to ubiquinone by inhibiting the oxidation of succinate to fumarate.

Question 24

Rotenone

Answer 24

Isoflavone found in the roots and seeds of some plants. It inhibits the transfer of electrons from complex I to ubiquinone.

Question 25

Oligomycin

Answer 25

Antibiotic produced by Streptomyces that inhibits oxidative phosphorylation by binding to the ‘stalk’ of ATP synthase and blocking the flow of protons through the enzyme.

Question 26

Dinitrophenol (DNP)

Answer 26

• Proton ionophore which can shuttle protons across the inner mitochondrial membranes.
• Can induce weight loss by transporting protons across the mitochondrial membrane, thereby uncoupling o.p. from ATP production and markedly increasing the metabolic rate and body temperature.
• Subsequently found its way into “anti-fat” medicines for the treatment of obesity. However, the margin between the slimming dose and that required to poison or kill is slight - so slight that several patients died and many suffered permanent injury before use of the drug was abandoned