ATP Production 1

Question 1

Where in the mitochondria does oxidative phosphorylation occur?

Answer 1

Inner membrane (cristae)

Question 2

Equations for the re-oxidation of NADH and FADH2:

Answer 2

NADH + H+ + 1/2O2 –> NAD+ + H2O (-223 kJ/mol)

FADH2 + 1/2O2 –> FAD+ + H2O (-170 kJ/mol)

Question 3

Evidence to support mitochondria descended from prokaryotes that established an endosymbyotic relationship with eukaryotes

Answer 3

1. New mitochondria rise from pre-existing mitochondria
2. Possess their own genome - circular DNA, no associated histones
3. Own protein synthesising machinery resembling prokaryotic structures
4. First transcribed amino acid = fMet (resembling bacterial proteins) rather than Met (of eukaryotes)
5. Some antibiotics that block bacterial protein synthesis pathway, also block mitochondrial, although these have no effects on eukaryotes

Question 4

What does the ETC consist of?

Answer 4

Three enzymes (Membrane complexes – used electron energy to move protons across) :
NADH Dehydrogenase complex
Cytochrome b-c1 complex
Cytochrome oxidase complex

Two carriers (Mobile carriers – move electrons along chain):
Ubiquinone (a.k.a. co-enzyme Q)
Cytochrome C.

Question 5

What Krebs’ Cycle enzyme is located in the mitochondrial membrane and why is it positioned there?

Answer 5

Succinate Dehydrgenase – allows communication with coenzyme Q

Question 6

What are the 2 main steps to oxidative phosphorylation (Chemiosmotic Theory) ?

Answer 6

1. Movement of protons from mitochondrial matrix to inter membrane space
2. Pumped protons are allowed back into the matrix through ATP synthase

Question 7

How does the ETC work?

Answer 7

1. NADH donates electrons to NADH Dehydrogenase complex (which has a higher affinity for electrons)
2. Co-enzyme Q shuttles the electrons to b-c1 complex.
3. Cytochrome C shuttles the electrons to the cytochrome oxidase complex
4. Each consequent electron carrier protein has a a higher affinity for electrons, so energy released at each stage is used to pump H+ ions from the mitochondrial matrix across to the intermembrane space, via the cristae
5. When the cytochrome oxidase complex has 4 electrons, they combine with O2 and 4 H+ ions (that has diffused down the chemiosmotic gradient via ATP synthase), forming 2 H2O molecules

Question 8

What is a redox couple?

Answer 8

A substrate that can exist in both oxidised and reduced forms

Question 9

Does the free energy along the ETC increase or decrease?

Answer 9

Decrease

Question 10

Why does the redox potential become more positive along the ETC?

Answer 10

More likely to accept electrons, makes transfer of electrons along the chain energetically favourable

Question 11

Why do protons want to move back to the matrix (via the ATP synthase)?

Answer 11

Concentration gradient

Transmembrane electrical potential (intermembrane space more positively charged than matrix)

Question 12

What can go through ATP Synthase?

Answer 12

Only H+ ions

Question 13

What is ATP Synthase made of?

Answer 13

2 units:
f0 - consists of subunits a, b and c (transmembrane)
f1 - consists of subunits alpha, beta and gamma (protruding into the matrix)

Question 14

Which part of ATP Synthase are static, and which parts move/rotate?

Answer 14

Rotate: C, gamma
Static: Alpha, beta, attached to B and A subunits

Question 15

How does ATP Synthase work?

Answer 15

Protons are enter the ATP Synthase static shaft and embed onto the rotor. The gamma subunit rotates forcing the beta subunits to undergo conformational changes which alters their affinities for ADP and ATP. Torsional (rotational) energy flows from the catalytic subunit to the F1-ATPase, which catalyses ADP and Pi to promote formation of ATP

Question 16

What does the direction of flow of protons determine?

Answer 16

ATP synthesis or ATP hydrolysis
If protons move from the matrix to the intermembrane space, ATP Synthase reverses in direction and breaks down ATP instead.

Question 17

What does the Oxygen Electrode do?

Answer 17

It measures oxygen concentration in a solution, it contains two electrodes and the current is proportional to the concentration of O2.

Question 18

What is the Oxygen Electrode made of?

Answer 18

Oxygen permeable teflon membrane, silver anode, platinum cathode

Question 19

Equations and type of reactions at each electrode?

Answer 19

Reduction : platinum cathode -
O2 + 4H+ + 4e- —-> 2H2O
Oxidation : silver anode -
4Ag+ + 4Cl- —-> AgCl + 4e-

Question 20

Where can this Oxygen Electrode be used practically?

Answer 20

To measure oxygen concentrations in mitochondria, and the effect of substrates/ inhibitors in the ETC. This can be done by first preparing a suspension of mitochondria from a tissue, using the first minute to measure the baseline O2 concentration, then to measure the effects of inhibitors/substrates.

Question 21

How to measure the efficiency of the Mitochondrial Phosphorylation System?

Answer 21

Adding a known quantity of ADP to the mitochondria suspension.
Then the ratio of the amount of ADP phosphorylated by the mitochondria to the amount of oxygen consumed can be calculated.
This is the ADP:oxygen index (used to measure efficiency)

Question 22

What is the most common cause of oxidative phosphorylation failure? And how quick is the death?

Answer 22

Lack of oxygen e.g. hypoxia (diminished), anoxia (total)

Minutes = neurons, hours = muscles

Question 23

What is respiratory control? (A graph to demonstrate respiratory control)

Answer 23

The uptake of O2 which is dependent on/ controlled by ADP and Pi. On a graph, with O2 consumption on the y-axis and time along the x-axis, when ADP is added, O2 consumption increases, and when that supply of ADP is exhausted, O2 consumption levels off

Question 24

What are metabolic poisons?

Answer 24

Highly toxic, interrupt ATP synthesis by interfering with the flow of electrons through the ETC, or flow of H+ through ATP Synthase

Question 25

How does cyanide (CN) and azide (N3) act as a metabolic poison?

Answer 25

Cyanide binds to the Fe3+ haem group in the cytochrome oxidase complex with high affinity, blocking the flow of electrons through the ETC and consequently, the production of ATP

Question 26

How does malonate act as a metabolic poison?

Answer 26

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 27

How does Rotenone act as a metabolic poison?

Answer 27

Inhibits the transfer of electrons from complex I to ubiquinone

Question 28

How does Oligomycin act as a metabolic poison?

Answer 28

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. Causes a backlog of protons in the intermembrane space so it eventually becomes saturated with protons meaning that protons can no longer be pumped into the space by the ETC components. Therefore, flowing electrons in stopped, so respiration is stopped

Question 29

How does Dinitrophenol (DNP) act as a metabolic poison?

Answer 29

Disconnects/ uncouples oxidative phosphorylation from ATP production by transporting protons across the inner mitochondrial membrane

Question 30

How can DNP induce weight loss?

Answer 30

By uncoupling oxidative phosphorylation, it can increase metabolic rate, fat metabolism and body temperature as it reduces the efficiency of mitochondria to produce ATP causing the mitochondria to use more energy to create ATP

Question 31

Why was DNP discontinued in slimming/anti-fat products?

Answer 31

Caused deaths, several patients died before it was abandoned in 1937