oxidative phosphorylation

Question 1

what is the basis of oxidative phosphorylation?

Answer 1

use free energy provided by redox reactions to pump H+ from the matrix into the IMS so that H+ can travel back into the matrix via ATP synthase, driving the production of ATP in the process

Question 2

what are redox potentials and how are they used in the electron transport chain?

Answer 2

how well a couple can transfer electrons from one to the other. works like electrode potentials - the more -ve = loses/donates electrons/is oxidised/acts as a reductant.
like a battery, energy is released H+ across a membrane.

in the electron transport chain, electrons move from the -ve redox potential of NADH/NAD+ -320 mV to the +ve redox potential of O2/H2O +820 mV

Question 3

how do you measure the standard redox potential?

Answer 3

use a standard hydrogen cell - 10^-7 M solution of H+ (ox) and one atm of H2 gas (re)
your test substance A needs to be present in equimolar amounts of its reduced and oxidised form as you dont know which way it’ll go.
if electrons flow from H cell to A cell, A’s redox potential is more +ve than hydrogen’s etc…

Question 4

what are the two steps in calculating free energy change of a redox reaction?

Answer 4

first - calculate the redox potential (right or +ve - left or -ve)
the standard free energy change = -number of electrons transferred x Faradays constant (96485) x redox potential from step 1

Question 5

what does complex I do and how?

Answer 5

oxidises NADH to NAD+, transferring the 2e- to ubiquinone(UQ), forming ubiquinol (UQH2). free energy released is used to pump 4H+ to the IMS.

-NADH and UQ bind to the complex, conformational change promotes uptake of H+ into membrane arm of the complex.
-UQ is reduced causing a conformational change that causes bound H+ to be exposed to the IMS side of the membrane,
-NAD+ and UQH2 are release, affinity for H+ drops, H+ is released into the intermembrane space and the conformation is reset

Question 6

what is the overall reaction for complex I?

Answer 6

NADH + 5H+ (matrix) + UQ = UQH2 +NAD+ + 4H+ (IMS)

Question 7

what does complex II do?

Answer 7

succinate dehydrogenase reduces FAD in the krebs cycle, the FADH2 is used to provide electrons to reduce UQ to UQH2
(so doesn’t directly pump any protons to IMS)

Question 8

why isn’t FADH2 used at complex I?

Answer 8

its electrons are at a lower energy level, so are not as good at being donated

Question 9

what does complex III (Q-cytochrome C reductase) do?

Answer 9

it oxidises the UQH2 made at complexes I and II back to UQ, transferring electrons to and reducing cytochrome C (hence the complex name).
the free energy released is used to translocate 4H+ from the matrix to the IMS

Question 10

what is cytochrome C?

Answer 10

a soluble electron carrier, each molecule takes 1 electron, reducing its Fe3+ to Fe2+ in its haem group

Question 11

what is UQ and what does it do?

Answer 11

a lipid soluble electron carrier that takes electrons from complexes I and II to complex III.
Takes up protons from the matrix when reduced and releases them into the IMS when oxidised by complex III

Question 12

explain how the Q cycle works

Answer 12

its how UQ is reduced at complexes I and II, then oxidised at complex III. each UQ reduced takes up 2H+ in the process.
these 2H+ are released into the IMS at complex III when oxidised, so for every UQ reduced, 2H+ from the molecule and 2 from the matrix are transported into the IMS

Question 13

what does complex IV (cytochromeC oxidase) do?

Answer 13

carries out the final electron transfer to oxygen.
2e- from (2cytC) and 2H+ (from matrix) are used to reduce 1/2 O2 to H2O.

using the free energy released, 2H+ are pumped from the matrix into the IMS

Question 14

how does complex V (ATP synthase) work?

Answer 14

energy stored in the PMF (proton membrane force) is used to drive the energetically unfavourable formation of ATP via ATP synthase.
you’ve got the rotor ring in the membrane, protons move through binding to sections of the ring, allowing it to turn - this provides mechanical energy that moves the central stalk around in the F1ATPase head, driving the synthesis of ATP from ADP and Pi

Question 15

what is PMF?

Answer 15

the driver of ATP synthesis, it has 2 components:
membrane potential/difference in charge between two sides of a membrane
proton concentration gradient - causes a difference in pH

Question 16

how do you calculate PMF?

Answer 16

PMF is measured in mV so to combine tis two aspects, the pH difference needs to be converted to voltage

so PMF = membrane potential -2.3RT/F x pH change

F = 0.0965
R =0.00831
T = 298

Question 17

how do you calculate free energy change from PMF?

Answer 17

-n x F x PMF (perhaps look at the practice calculations from Jim Reid’s lecture)

Question 18

one full turn of the motor ring moves 8H+ across to form 3ATP.
why is 8H+/3ATP only a theoretical ratio?

Answer 18

there are import and export costs.
each pyruvate costs an H+
each ADP costs the equivalent of 1H+
each ATP requires a Pi, which costs an H+
each of the 2 NADH from glycolysis costs 4H+ to be imported
this mathematically changes the ratio to 14H+/3ATP

Question 19

how much ATP is produced for an NADH, and an FADH?

Answer 19

3.75
2.25

Question 20

what is the theoretical yield vs actual yield of ATP per glucose?

Answer 20

42 vs 26

Question 21

what was the most common belief to explain respiration and how did Peter Mitchell use electron microscopy to prove the electrochemical gradient theory?

Answer 21

most common belief = some unknown super reactive intermediate phosphorylated ADP to ATP
he showed mitochondria changed ratio of matrix to IMS, suggesting electron transport wad coupled to a change in osmotic potential

Question 22

how did Peter show that a proton gradient was needed?

Answer 22

added dinitrophenol to mitochondria, which stopped ATP synthesis because the compound facilitates diffusion of H+ across usually impermeable membranes.