Oxidative Phosphorylation - ATP Synthase

Question 1

Describe the Proton-Motive Force (pmf)

Answer 1

The potion gradient across the inner mitochondrial membrane results in two energetic gradients:
1. a chemical gradient or pH gradient due to different H+ concentrations on either side of the membrane
2. An electrical gradient due to the charge difference across the membrane (positive in the intermembrane space, negative in the matrix)
This electrochemical gradient is known as the proton-motive force (pmf).
Mitchell proposed that the energy of the pmf drives ATP synthesis

Question 2

describe the electrical gradient part of the pmf

Answer 2

in an aqueous environment, we don’t actually have lots of protons. instead they exist as ions.
- in the inter membrane space where there is an increased concentration of H+, there is H3O+
- and in the matrix where there is a decreased concentration of H+ ions, there is OH-
So the electrical gradient is formed due to the charge difference of the two sides.

Question 3

describe chemiosmotic coupling hypothesis: experimental evidence with detergent

Answer 3

• Isolate mitochondria from cells
• treat with mild detergent, only removed outer membrane, ETC still works
• but ATP is NOT made, ATP is not synthesised in the ETC as an outer membrane is required for the gradient to form

Question 4

describe chemiosmotic coupling hypothesis: experimental evidence the artificial liposome

Answer 4

• artificial liposome (replicating the mitochondria)
• bacteriorhodopsin is a light inducible proton pump
• in light will pump protons to make a proton gradient
• ATP synthase: makes ATP when light switched on
• ETC is not present
• The proton gradient is sufficient for ATP synthesis

Question 5

describe chemiosmotic coupling hypothesis: experimental evidence with DNP

Answer 5

• 2,4-Dinitrophenol (DNP) is an uncoupler: shuttles H+ from the inter membrane space to the matrix, thus dissipating the proton gradient
• can do this as if can reversibly pick up or drop off a proton attached to its -O- group
• in the presence of DNP: ETC functions, no ATP is made, therefore pmf is necessary for ATP synthesis

Question 6

describe how DNP is a poison

Answer 6

banned for human consumption in NZ
If you take DNP: ETC functions but no ATP is made
- because no ATP is made, the fuel molecules keep going into the CAC, beta oxidation and PDH to make more reduced coenzymes.
- the coenzymes keep going through the ETC and releasing energy but because there is no proton gradient, ATP synthase can’t work, so energy from the electrons moving through the ETC is released as heat, you get mass organ failure and then die
- you do lose weight because you are using fuel molecules at a faster rate (but then you die)

Question 7

describe the F1Fo-ATP Synthase

Answer 7

Has a F1 region which is in the matrix
Has a Fo region which is in the inner mitochondrial membrane

• The ‘rotor subunits’ turn (rotor subunits: gamma stalk and red circular bit inside the membrane that the H+ ions go into)
• The ‘stator subunits’ do not turn (stator subunits: alpha and beta units in the IMM, stalk down the side, and the second circular bit that has the channels for H+ ions to get into the other circular bit in the membrane)
• proton flow drives rotor movement (protons come into the rotor in the IMM and this turns into kinetic energy, making the rotor move)
• rotor movement causes conformational changes in the stator that drives ATP synthesis

Question 8

describe how we know that F1Fo-ATP synthase works as a molecule motor

Answer 8

An experiment where we tip ATP synthase upside-down and stick it in a bead. The attach a fluorescently labelled actin on the end.
Will see that the actin filament moves around and around (so there must be a motor bit)

Question 9

describe how the movement of the rotor causes conformational changes in the catalytic alpha and beta subunits of F1

Answer 9

O = open (ATP release/ADP and Pi binding)
L = loose (holds ADP and Pi in preparation for catalysis)
T = tight (catalysis - ATP formation)
Changes for each dimer when rotor (gamma stalk) turns - each dimer O->L->T->O->L etc

Question 10

describe the energy accounting for this process overall

Answer 10

4 protons per 1ATP
NADH = 10 protons pumped in ETC so 2.5 ATP made
FADH2 = 6 protons pumped in ETC so 1.5 ATP made

oxidative phosphorylation is where you make most of the ATP