ATP Synthase and Completing Photosynthesis Flashcards
Phosphoanhydride Bond
- bonds between phostphate groups
- formed during ATP synthesis and broken in hydrolysis
- sometimes called ‘high energy’ phosphate bond
ATP Hydrolysis Reaction
ATP (-4) + H20
->
ADP (-3) + HPO4(-2) + H+
-where numbers in bracket indicate charge
What is the standard free energy change for ATP hydrolysis?
-the STANDARD free energy change (at 1M concentrations, pH7, 298K, 1atm) is:
ΔGo’ = -30.5 kJ / mol
-this means the standard reaction is spontaneous
What is ATP hydrolysis used for?
- 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
Actual Free Energy Change of ATP Hydrolysis
ΔG = ΔGo’ + RT ln( [ADP][Pi]/[ATP] )
-where [X] indicates concetration of X in M
How do you find the free energy change of the backwards reaction from the forward reaction?
-the free energy of the reverse reaction is just the negative of the free energy of the forward reaction
Chemiosmosis
Definition
-the movement of ions across a semipermeable membrane, down their electrochemical gradient (i.e. from high to low concentration)
Postulates of the Mitchell Chemiosmotic Theory of ATP Synthesis
- free energy is stored as both a pH gradient and an electrical potential across the biomembrane
- this is transduced into chemical energy via ATP formation
Details of the Mitchell Chemiosmotic Theory of ATP Synthesis
Membrane Organisation
-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
Details of the Mitchell Chemiosmotic Theory of ATP Synthesis
pH
- 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
Details of the Mitchell Chemiosmotic Theory of ATP Synthesis
Potential Difference
- 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
Details of the Mitchell Chemiosmotic Theory of ATP Synthesis
Proton Motive Force
-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
Proton Motive Force
Chemical Potential Energy
-chemical potential energy for protons: Uchem = RT ln[H+] -and pH = -log_10_[H+] => Uchem = -2.3 * R * T * pH
Proton Motive Force
Electrical Potential Energy
-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
Proton Motive Force
Comparing Interior vs Exterior
-for the proton motive force across the membrane we need to compare interior vs exterior:
ΔU = Ui - Uo
Proton Motive Force
Chemiosmotic Potential
Δp = U/F = -2.3RT/F ΔpH_(i-o) + ΔΨ_(i-o)
-subbing in numerical values for constants:
Δp = ΔΨ + 59*ΔpH
Proton Motive Force
Estimate Energy Released by the Transfer of a Certain Amount of Protons
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
Proton Motive Force
Estimate the Amount of Protons Required to synthesize 1 mole of ATP
-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
What is the role of the ATP synthase complex in the energy capture cycle?
-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
ATP Synthase
Proton Flow
- 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)
ATP Synthase
Motor
- 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
ATP Synthase
Biochemical Experiments
-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
Direct Observation of the Rotation of the ATP synthase Rotor Subunits
Extraction Experiment
- ‘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
Direct Observation of the Rotation of the ATP synthase Rotor Subunits
First In-Situ Demonstration
- 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
Direct Observation of the Rotation of the ATP synthase Rotor Subunits
Rotatin Rate
- 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
How is proton pumping across the membrane coupled to c-ring rotation?
Hypothetic Model
- 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’)
How is proton pumping across the membrane coupled to c-ring rotation?
In More Detail
- 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
Results of the First Atomic-Level Simulation of Photosynthesis
- 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
