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25 Cards in this Set

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How much energy is available to a cell from electron transfer?

-220 kJ/mol

How does the cell use energy generated from electron transfer?

The cell uses this energy to pump protons in complex IV from the cytoplasm into the periplasm thus turning ATP synthase to activate the phosphorylation of ADP into ATP.

1. How much energy is expended pumping a mole of protons into the periplasm?


2. How many moles of protons does a single oxidation of NADH accomodate?

1. 18.92 kJ/mol

2. For each NADH oxidised, 10H+ are pumped across the membrane



How energy efficient is the proton pumping system?

The system conserves around about 90% of the energy within the system.

1. What problems oppose the proton pumping mechanism?


2. How might these issues be overcome?



1. a) Protons cannot be pumped indefinitely if there is not a refresh of protons to pump


b) Proton motive force will oppose electron transfer




2. a) PMF is consumed by ATP synthase


b) Inner membrane is made permeable


c) small but significant leak of protons back across the inner membrane

What are the functional domains of ATP synthase?

F1 and F0 domains

How is ATP synthase represented as a cartoon structure?

Describe the F1 domain.

Contains 3 alpha-beta heterodimers attached to the stem of the F0 domain.

How could you study ATP binding to the F1 domain?


What is the common molecule used?

1. Use an ATP analogue with a non hydrolyzable beta-gamma bond thus preventing release from the subunit.




2. The analogue commonly used is APP(NH)P

1. What is rotational catalysis?


2. What is the role of the alpha beta heterodimers of the F1 subunit in rotational catalysis?


3. How does the process favour ATP production?

1. Rotational catalysis is the process which the F1 subunit of ATP synthase uses to phosphorylate ATP.


2. The F1 domains 3 alpha-beta heterodimers work in unison. At any one time there is an ATP molecule bound to one of the heterodimers. One ADP...





1. Rotational catalysis is the process which the F1 subunit of ATP synthase uses to phosphorylate ATP.




2. The F1 domains 3 alpha-beta heterodimers work in unison. At any one time there is an ATP molecule bound to one of the heterodimers. One ADP molecule bound to another. As well as an unbound domain.




3. Hydrolysis and synthesis occur simultaneously within the F1 domain. However ATP binds more strongly thus the equilibrium tends towards the ATP product (as it will remain bound to the subunit rather than become unbound and subsequently hydrolysed)

What is the role of the gamma central shaft of the F0 subunit in rotational catalysis?

The gamma shaft moves round in a counter clockwise motion which comes into contact with an ATP bound domain.


The contact of the gamma subunit and the alpha-beta heterodimer causes a conformational change to the O conformation which releases the ATP.




Simultaneously the gamma shaft loses contact with a previously contacted alpha beta subunit which changes the conformation either to the T conformation (which catalyses ADP+Pi --> ATP) or the L confirmation which attracts ADP + Pi.

What process must occur before ATP can be released?

Until ADP+Pi are bound at another alpha-beta heterodimer.

How is the gamma central shaft moved?

By an actin filament attached to a molecule of avidin.

How many protons are used in the production of one ATP molecule?

4.3 protons per ATP

What is the P/O ratio?

The ratio of ATP produced per 2e- donated from oxygen (ATP/O)

What is the P/O ratio


a) with NADH


b) with succinate


?




Can P/O ratios be fractions of a whole?

a) NADH causes 10 protons to be displaced and (4.3 protons = 1 ATP) therefore = 2.3


b) Succinate causes 6 protons to be displaced therefore = 1.4




As calculated above it can be seen that P/O ratios are non integral.

How is electron transfer and its effect studied?

Inhibition - by using molecules which stop the electron transport chain we can observe the effects on ATP production.

1. What are the main electron inhibitors?


2. What stage of electron transfer does each inhibit?

Rotenone - inhibits electron transfer to Ubiquinone




Antimycin A -inhibits electron transfer to cytochrome c




Cyanide and Carbon monoxide - inhibit the final electron transfer to the terminal electron carrier - oxygen



What is the terminal electron carrier?

Oxygen

How can electron transfer be 'uncoupled' from ATP synthesis?

Usually ATP synthesis and electron transfer are mutually reliant.


The molecule 2-4 dinitrophenol (2,4 DNP) uncouples the process by allowing respiration to continue without ATP synthesis

How can we observe the uncoupling reaction?

By measuring O2 consumption and ATP synthesis against time.


First add succinate and then ADP+Pi to show ATP synth can only occur when both are present.


Then add an ATP synthase inhibitor such as venturicidin or oligomycin to halt ATP synthesis and thus significantly lower oxygen consumption (as the reactions are coupled)


Upon the addition of 2,4 DNP one can observe that Oxygen consumption will now occur in absence of ATP synthesis.

How do electron uncouplers work?

Typically ATP synthase is required to maintain the proton gradient through pumping into the periplasm.


Electron Uncouplers are highly hydrophobic and will readily accept protons, cross into the periplasm and readily dissociate. In this way the uncouplers maintain or heighten the proton gradient without the pumping action of ATP synthase.

How are most reactive oxygen species formed the in the mitochondria?

98% of ROS come from using Ubiquinone as an electron carrier between complexes 1,2 and 3.




To form QH2 (Ubiquinol - the reduced form of ubiquinone and thus the electron carrier),*Q- (a ubiquinone radical) is an intermediate.




The ubiquinone radical can sometimes pass the radical electron on to Oxygen to form peroxide (*O2-)

How does the mitochondria deal with radical oxygen species?

An enzyme called superoxide dismutase catalyses the production of Hydrogen Peroxide (H2O2) which is rendered harmless by another enzyme called Glutathione peroxidase.

What are the models for mitochondria complex interaction?

1. Solid state - all complexes bound together on membrane


2. Random collision - all complexes are separate and products randomly collide


3. Plasticity - many varieties of supercomplex form spontaneously, allowing interactions.