Mictochondra and oxidative phosphorylation

Question 1

What are mitochondria?

Answer 1

Powerhouse of the cell and generate the majority of the ATP
- Folds of the inner mitochondria (cristae) gives the inner membrane a large surface area. Cristae is the location of the ETC

Question 2

What is the evolutionary origin of mitochondria?

Answer 2

• Inherited maternally
• Evolutionary descendant of a prokaryote.
• Consumed by eukaryote and an endosymbiotic relationship was established.
• Occurred early in the history of life on earth and following this, many of the genes needed for mitochondrial function were moved to the nuclear genome.

Question 3

What is the evidence supporting endosymbiosis theory?

Answer 3

• Mitochondria can only arise from pre-existing mitochondria (or chloroplasts in the case of plants).
  Mitochondria possess their own genome, resembling that of prokaryotes, being a single circular molecule of DNA, with no associated histones.
• Mitochondria have their own protein-synthesizing machinery, which again resembles that of prokaryotes not that of eukaryotes.
• The first amino acid of mitochondrial transcripts is a formylated methionine residue (fMet) as is the case in bacteria and not methionine (Met) as in eukaryotic proteins.
• A number of antibiotics (e.g., streptomycin) that act by blocking bacterial protein synthesis also block protein synthesis within mitochondria. They do not interfere with protein synthesis in the cytoplasm of the eukaryotes.

Question 4

How does NADH donate electrons?

Answer 4

Protons go to the solvent surrounding the enzyme complex.

Electrons join the Electron Transport Chain.

Question 5

How are NADH and FADH2 re-oxidised?

Answer 5

• Re-oxidised using molecular oxygen.

NADH + H+ + 1/2 O2 —> NAD+ + H2O

FADH2 + 1/2 O2 —> FAD + H20

• Each reaction has DG of -220 and -167 kJ/mol respectively - much more free energy than ATP so there is lots of energy to be harnessed.
• The energy released from the re-oxidation of NADH and FADH2 is sufficient to generate several phosphoanhydride bonds.
• Part of this energy is recovered by parts of the electron transport chain and used to synthesise ATP.

Question 6

Steps involved in oxidative phosphorylation

Answer 6

Oxidative Phosphorylation occurs in two steps:

1. Movement of protons from within the matrix of the mitochondria into space in between mitochondrial membranes - controlled by the electron transport or respiratory chain.
2. Pumped protons are allowed back into the mitochondria through a specific channel which is coupled with an enzyme which can synthesise ATP called ATP synthase

• The flow of protons back into the matrix is coupled to ATP synthesis
• The pumping of protons into the intermembrane space establishes a gradient which can be looked at as a potential gradient or a pH gradient
• The proton motive force that drives H+ back into the matrix space consists of a pH gradient AND a transmembrane electrical potential

Question 7

Complexes and electron carriers involved in the ETC

Answer 7

Enzymes:

• NADH Dehydrogenase complex
• Cytochrome b-c1 complex
• Cytochrome oxidase complex

Carriers:

• Ubiquinone (a.k.a. co-enzyme Q)
• Cytochrome C.
• These proteins accept electrons and in doing so, a proton (H+) from the aqueous solution. As electrons pass through each of the complexes, a proton is passed or ‘pumped’ to the intermembrane space.
• Each unit in the chain has a HIGHER AFFINITY for electrons than the previous unit, allowing them to flow in a logical order.

Question 8

What is the function of ubiquinone (co-enzyme Q)?

Answer 8

• Ubiquinone (co-enzyme Q) is an electron carrier which transfers an electron from NADH Dehydrogenase Complex to Cytochrome b-c1.
• It can pick up one or two electrons (together with an H+ from solution)
• The hydrophobic tail of ubiquinone confines it to the lipid membrane

Question 9

What is the function of cytochrome oxidase?

Answer 9

• Cytochrome Oxidase is the last membrane complex in the electron transport chain. It initially receives 2 electrons from cytochrome C in the first cycle of the electron transport chain, then the cycle repeats so that cytochrome oxidase has 4 electrons in total.
• Cytochrome oxidase passes the electrons to Oxygen to generate water.
• Furthermore, 4 protons are pumped into the inter-membrane space, thus enhancing the proton gradient.

4e- + 4H+ + O2 —> 2H2O

-Oxygen is the ideal terminal electron acceptor because it has a high affinity for electrons, proving a driving force for oxidative phosphorylation.

Question 10

Importance of redox reactions in oxidative phosphorylation

Answer 10

• Electron transfer reactions involving a reduced substrate and an oxidised substrate.
• A substrate that can exist in both oxidised and reduced states is known as a redox couple e.g. NAD+/NADH, FAD/FADH2
• The ability of a redox couple to accept or donate electrons is known as the reduction potential or redox potential.
• Standard Redox Potentials
  NEGATIVE redox potential = tendency to DONATE
  POSITIVE redox potential = tendency to ACCEPt
• Each successive membrane complex or carrier has a more positive redox potential than the previous component of the ETC. This means that the transfer of electrons from one complex to the next is energetically favourable. As they progress along the chain, the electrons lose energy.

Question 11

Structure of ATP synthase

Answer 11

A multimeric enzyme consisting of a membrane bound part (F0) and a part which projects into the matrix space (F1).
F1 and F0 consist of three different subunits:
F0 = a, b and c
F1 = alpha, beta and gamma

• When H+ flows through the membrane via the pore, the disc of c subunits rotate.
  The gamma-subunit in the F1 unit is fixed to the disc and so rotates with it.
• HOWEVER, alpha and beta-subunits in the F1 unit CANNOT ROTATE because they are locked in position by the b subunit which is anchored to subunit ‘a’ in the membrane.
  To summarise:
• C and gamma subunits can rotate
  alpha and beta-subunits CANNOT rotate because they are fixed in position by a and b subunits
• As the gamma subunit functions as an asymmetrical axle, the beta-subunits are compelled to undergo structural changes. This rotation drives transitions of the catalytic portions of the beta-subunits, which alters their affinities for ATP and ADP.
• So, TORSIONAL ENERGY flows from the catalytic subunit into the bound ADP and Pi to promote the formation of ATP.

Question 12

What is the function of succinate dehydrogenase?

Answer 12

• Succinate Dehydrogenase is an integral membrane protein that is firmly attached to the inner surface of the inner mitochondrial membrane.
• This enzyme is responsible for catalysing the reaction which produces FADH2.
• Its location allows it to communicate directly with ubiquinone.

Succinate —> Fumarate

• using succinate dehydrogenase
• also FAD –> FADH2
• Because FADH2 passes electrons directly to ubiquinone (co-enzyme Q), fewer protons are pumped out so less ATP is produced.
• Ubiquinone is the entry point for electrons donated by FADH2.

Question 13

Explain the mechanism of action of metabolic poisons

Answer 13

• Cyanide - binds to Fe3+ in haem group in cytochrome oxidase, inhibiting it. This blocks the flow of electrons through the ETC so no ATP is produced.
• Carbon Monoxide - binds to Fe2+ in haem group and blocks flow of electrons
• Malonate - competitive inhibitor to succinate dehydrogenase - slows down the flow of electrons from succinate to ubiquinone by inhibiting the oxidation of succinate to fumarate
• Oligomycin - binds to stalk of ATP synthase - blocks flow of protons through the enzyme . Therefore build up of protons so no more can be pumped as conc gradient very high
• Dinitrophenol - uncouples oxidative phosphorylation from ATP production - provides an alternate pathway for protons to travel through the membrane. Promotes weight loss. Non-shivering thermogenesis
  increases metabolic rate and body temperature