ATP Synthase and Completing Photosynthesis

Question 1

Phosphoanhydride Bond

Answer 1

• bonds between phostphate groups
• formed during ATP synthesis and broken in hydrolysis
• sometimes called ‘high energy’ phosphate bond

Question 2

ATP Hydrolysis Reaction

Answer 2

ATP (-4) + H20
->
ADP (-3) + HPO4(-2) + H+
-where numbers in bracket indicate charge

Question 3

What is the standard free energy change for ATP hydrolysis?

Answer 3

-the STANDARD free energy change (at 1M concentrations, pH7, 298K, 1atm) is:
ΔGo’ = -30.5 kJ / mol
-this means the standard reaction is spontaneous

Question 4

What is ATP hydrolysis used for?

Answer 4

• in biology, the exergonic process of ATP breakdown is coupled to other energy-requiring biochemical reactions
• thus the energy which would have been released by hydrolysis is used to drive an endergonic reaction

Question 5

Actual Free Energy Change of ATP Hydrolysis

Answer 5

ΔG = ΔGo’ + RT ln( [ADP][Pi]/[ATP] )

-where [X] indicates concetration of X in M

Question 6

How do you find the free energy change of the backwards reaction from the forward reaction?

Answer 6

-the free energy of the reverse reaction is just the negative of the free energy of the forward reaction

Question 7

Chemiosmosis

Definition

Answer 7

-the movement of ions across a semipermeable membrane, down their electrochemical gradient (i.e. from high to low concentration)

Question 8

Postulates of the Mitchell Chemiosmotic Theory of ATP Synthesis

Answer 8

1. free energy is stored as both a pH gradient and an electrical potential across the biomembrane
2. this is transduced into chemical energy via ATP formation

Question 9

Details of the Mitchell Chemiosmotic Theory of ATP Synthesis
Membrane Organisation

Answer 9

-electron carriers are arranged vectorially in an intrinsically disordered biological membrane i.e. membrane proteins are oriented, cofactors diffuse between them and bind to specific regions

Question 10

Details of the Mitchell Chemiosmotic Theory of ATP Synthesis
pH

Answer 10

• series of electron transfers between carriers and a series of linked reactions transport protons form one side of the membrane to the other
• the membrane is inherently impermeable to protons so any transport lead to a stable concentration diferrence, a ΔpH
  d) this can also lead to

Question 11

Details of the Mitchell Chemiosmotic Theory of ATP Synthesis
Potential Difference

Answer 11

• the transport of protons throught the membrane and the otherwise impermeability of the membrane to protons can also lead to an electrical potential difference ΔΨ
• it depends on the concentration of other charged species on each side of the membrane

Question 12

Details of the Mitchell Chemiosmotic Theory of ATP Synthesis
Proton Motive Force

Answer 12

-the total energy available for ATP synthesis (termed the ‘proton motive force’), Δp, is the sum of the proton chemical potential and the transmembrane electrical potential
Δp = ΔΨ + const.*ΔpH
-this gives Δp in mV

Question 13

Proton Motive Force

Chemical Potential Energy

Answer 13

-chemical potential energy for protons:
Uchem = RT ln[H+]
-and pH = -log_10_[H+]
=>
Uchem = -2.3 * R * T * pH

Question 14

Proton Motive Force

Electrical Potential Energy

Answer 14

-work in general:
Uelec = qV
-so for the protons:
Uelec = F * Ψ
-where Ψ is the transmembrane electric potential of the protons
-and F is the Faraday constant

Question 15

Proton Motive Force

Comparing Interior vs Exterior

Answer 15

-for the proton motive force across the membrane we need to compare interior vs exterior:
ΔU = Ui - Uo

Question 16

Proton Motive Force

Chemiosmotic Potential

Answer 16

Δp = U/F = -2.3RT/F ΔpH_(i-o) + ΔΨ_(i-o)
-subbing in numerical values for constants:
Δp = ΔΨ + 59*ΔpH

Question 17

Proton Motive Force

Estimate Energy Released by the Transfer of a Certain Amount of Protons

Answer 17

ΔG = nFΔp
-where:
Δp = ΔΨ + 59*ΔpH
n = number of moles
F = Faraday constant
-for 1 mole of protons under typical conditions:
ΔG = 0.223eV = 8.67kbT

Question 18

Proton Motive Force

Estimate the Amount of Protons Required to synthesize 1 mole of ATP

Answer 18

-if ATP synthesis is under standard conditions:
ΔG_atp = +51.6 kJ/mol
-and proton flow under typical Δp:
ΔG_h+ = -21.5 kJ/mol
-if we assume Δp remains constant
-mimum requirements are when ΔG=0:
0 = ΔG_atp + n*ΔG_h+
=>
n = - ΔG_atp/ΔG_h+ ~ 2.4

Question 19

What is the role of the ATP synthase complex in the energy capture cycle?

Answer 19

-the ATP synthase complex completes the energy capture cycle by transducing the energy stored in this proton gradient to chemical energy in ATP - a stable and transportable molecule

Question 20

ATP Synthase

Proton Flow

Answer 20

• protons spontaneously flow through ATP synthase form the periplasm to the cytoplasm
• this is because it is energetically favourable to dissipate this proton gradient
• proton flow occurs through the narrow space separating the stator from the motor (c-ring/a-subunit)

Question 21

ATP Synthase

Motor

Answer 21

• charge migration makes the rotor ring (c-ring) and axle (y-subunit) spin
• proton flow is converted to energy in this ‘electric motor’
• the ‘lollipop head’ (α3β3 sub-complex) behaves like a generator
• 3 in 6 protein subunits have binding sites for ADP and phosphate (α-subunits)
• the rubbing and reorienting of the proteins as the axle spins provides mechanical energy that can be converted into chemical bond energy binding the crucial 3rd phosphate to ADP forming ATP

Question 22

ATP Synthase

Biochemical Experiments

Answer 22

-biochemical experiments estimate that three or four protons must pass through ATP synthase to make one molecule of ATP
-subbing this n=3 into:
ΔG = ΔG_atp + n*ΔG_h+
-allows ΔG to be estimated as -12.9kJ/mol
-this makes sense, negative ΔG predicts the process will be spontaneous
-it can generate >100 ATP molecules per second

Question 23

Direct Observation of the Rotation of the ATP synthase Rotor Subunits
Extraction Experiment

Answer 23

• ‘lollipop head’ and stalk extracted from biological system
• β-sununits genetically tagged and then chemically bound to the glass to fix the whole α-β-α-β-α-β stator complex onto the surface in this specific orientation
• the γ-subunit was bound to a long actin filament which is fuorescently labelled so it can be observed indirectly
• reactants (ATP) were added to the solution so the system had ‘fuel’
• can observe the actin filament spinning
• because the actin filament is only attached to the γ-subunit this proves that it’s the γ-subunit that rotates and α3β3 is static

Question 24

Direct Observation of the Rotation of the ATP synthase Rotor Subunits
First In-Situ Demonstration

Answer 24

• first in situ demonstration of the motion of a single molecule biological motor
• here, ATP (high energy) is being broken down to drive the γ-subunit’s rotation whereas usually the proton gradient provides the energy for the rotation of γ-subunit which then drives ATP formation from ADP & P
• this is the reverse of normal and shows that the chemical reaction can run in both directions
• this correlates with biochemical experiments which have shown that ATP hydrolysis can be used to pump protons (against the concentration gradient)
• BUT the rotation is UNIdirectional

Question 25

Direct Observation of the Rotation of the ATP synthase Rotor Subunits
Rotatin Rate

Answer 25

• researchers have found that the rotation rate depended on the length of the actin filament attached
• can calculate the torques required to rotate a particular length filament at a certain rate
• and find the force the γ-subunit exerts on the αβ-subunits

Question 26

How is proton pumping across the membrane coupled to c-ring rotation?
Hypothetic Model

Answer 26

• developed after early crystal structures
• entry through 1/2 channel within subunit-a
• binding to c-subunits
• release through other 1/2 channel of the a-subunit
• H+ binding and release cycle generates c-ring rotation (‘rotor’) around the static a-subunit (‘stator’)

Question 27

How is proton pumping across the membrane coupled to c-ring rotation?
In More Detail

Answer 27

• a proton enters from the intermembrane space into the cytosolic half-channel to neutralize the charge on a negative amino acid (aspartate) within a c subunit
• with this charge neutralised, the c ring can rotate clockwise by one c-subunit
• this moves a (different) neutralized amino acid form the membrane into the matrix half-channel (i.e. these protonated amino acids are carrying 1H+)
• this proton can move out of the other side of the membrane, resetting the system to its initial state and transferring one proton during this rotary motion

Question 28

Results of the First Atomic-Level Simulation of Photosynthesis

Answer 28

• under low light conditions (~3% or less of bright sunlight) ATP turnover can still run at 50% of the maximum rate
• the whole process from photon to ATP is limited by the number of cyt bc1 complexes