Term 2 Lecture 12: Proton Pumping Flashcards
How proton pumping generates energy
Energy is generated by pumping protons across membranes in mitochondria (oxidative phosphorylation) in chloroplasts (photosynthesis) and in strange bacteria (direct light conversion)
The electrochemical gradient of mitochondrial concentrations is as follows:
Ion
/ conc. (mM) : cytoplasm &extracellular
/ Permeability coefficient (cm•s-1 10⁹)
K+ / 100 & 5/ 500
Na+/ 15 & 150/ 5
Ca²+/ 0.0092 & 2/ -
Cl-/ 13 & 150/ 10
Org ions/ 138 & 34/ 0
^ permeable to K+ and nearly impermeable to all other ions
This can be calculated by the Nernst equation:
∆Psi = R•T/F•N • Ln c out/c in
R= gas constant
T= temp. (K)
n= ion charge
F= Faraday’s constant
pH gradient also exists as there are more protons on one side of the membrane than the other causing a difference of 1pH either side of the membrane - pH is lower in intermembrane space (where H+ conc is higher)
This can be summarised as proton motive force to combine pH and membrane potential:
Proton motive force:
∆p=∆Psi - 2.303RT∆pH/F
Free energy from proton motive force:
∆GHa+ <-> H1 + = -F∆p
Types of proton pump
1) light driven proton pump transfers 2 H+ out using light energy.
Present in some prokaryotes (bacteria - rhodopsia)
2) Q cycle AKA plastaquinone cycle present in plants in photosystem ll 2H+ is transferred out via coenzyme Q and b/f- complex
3) electron driven proton pump (cytochrome oxidase) present in animals known as complex 4 in respiration transfers out 8H+ only 4 reach the cycle the rest are converted to H2O
4) proton driven ATP synthesis all 3 of the previous systems feed H+ back into the matrix via this mechanism converting ADP to ATP in the process.
ATP synthase links proton transport to ATP synthesis (it’s a specific channel for H+)
ATP synthase allows the thermodynamically favourable transport of H+ ions across the mitochondrial membrane, driven by the concentration gradient and the membrane potential to be coupled to ATP synthesis and an energy requiring process
This is the essence of the “chemiosmotic hypothesis” (Peter Mitchell 1961) showing energy could be interconverted between different forms at a biochemical level.
Before this it was thought that cells made ATP by “substrate level phosphorylation” (e.g. by enzymes such as 3-phosphoglycerate kinase in the glycolytic pathway)
Cells make some ATP this way but ATP synthase is much more efficient and productive.
In the 1960’s and 70’s scientists did not know what ATP synthase was/what it was doing.
The electron transport chain
A molecular pump that uses redox potential (∆E volts) of reduced cofactors (NADH and FADH2 mostly from TCA cycle) to power a series of molecular pumps.
In the mitochondria the molecular pumps act on H+ moving them from inside the mitochondria across the mitochondrial membrane and into the intermembrane space.
H+ cannot flow back across the membrane (it is impermeable to them) so a gradient in H+ ( and therefore pH) is produced
Some “shuttles” : coenzyme Q (ubiquinone) and cytochrome c move electrons along the chain acting as cofactors
(See diagram)
Representing electron flow through redox reactions
Linked redox reactions are represented as steps on a diagram of redox potential.
Each step represents a redox reaction that results in the upper compound being oxidised and lower compound being reduced
Sequence of reactions left to right represents electron flow.
Vertical scale in redox potential is equivalent to free energy.
Refer to a standard reduction potential table
e.g. O2/H2O
Half cell reaction: O2+ 4H+ +4e-→2H2O
E⁰’/V (reduction potential in volts):
+0.816 the more pos the redox is equivalent to more negative free energy change.
In the table any reaction above another will drive the reaction below in reverse. E.g. O2/H20 causes NADH→NAD+ + H+
The 4 complexes
Each of the 4 redox driven proton pumps is a multiprotein complex and each one uses different cofactors.
The system also contains 2 electron carriers which carry electrons between pumps via redox:
1) mobile carrier embedded in the membrane (coenzyme Q)
2) mobile carrier outside the membrane (cytochrome c)
Complex 1,3 & 4 associate together as a respirosome aka a respiratory complex
Complex 1: converts NADH→NAD+ + H+
Provides 2H+ to Q to form QH2
Releases 4H+ into the intermembrane space.
Complex 3: QH2 reduces cytochrome C and 2H+ are released into the intermembrane space.
Complex 4: 4 reduced cytochrome c are oxidised to back to cytochrome c releasing 4e- that pass through Cu→Fe→Cu then are used to combine the 4H+ with an O2 molecule to form 2H2O
Complex 2: electrons from succinate are used to oxidise FADH2 →FAD releasing electrons that are passed to FES clusters and a heme group, electrons are then transported from the heme to coenzyme Q.
Energetics of reactions catalyzed by complex ll
Complex ll aka succinate Co-Q reductase
Succinate is oxidised to fumarate (as in the TCA cycle) with concomitant reduction of FAD→FADH2
This cofactor (FADH2) remains bound to the enzyme and cannot dissociate into a soluble pool of cofactor like NADH
The reduced FADH2 transfers it’s electrons via a series of redox reactions similar to complex l to coenzyme Q reducing it to CoQH2
The reduced CoQH2 enters the pool of this electron carrier in the inner mitochondrial membrane
Succinate+ Co-Q →fumarate+ Co-QH2
The overall redox reaction of complex 1,lll and lV
2H+ + O2+2NADH <-> 2H2O+2NAD+
E°’=+1.14V
∆G°’ = -220.1kJmol-¹
This process uses molecular O2 to oxidise reduced NADH cofactor (from TCA cycle) to NAD+ + H2O
The reactivity of the O2 drives this process.
The process is strongly thermodynamically favoured (high neg ∆G)
The complex part is ensuring that the free energy is used productively to drive proton transport and not wasted as heat.
To use the free energy the process is broken down into a series of steps to release energy gradually
(Complex 2 produces less energy than the other complexes)
Energy required for proton transport.
For efficient coupling the free energy required for proton transport must be a little less than the free energy produced by the individual redox reaction driving it.
Transmembrane potential across the inner mitochondrial membrane is estimated to be -160mV
(I.e. intermembrane space is pos)
Energy required for transfer of one proton:
∆G= RTln {c in/c out} + ZF+∆E
∆G= +21.8kJmol-¹ (←transmembrane potential causes 70% of this)
Available free energy change from overall reaction:
2H+ + O2+2NADH <-> 2H2O+2NAD+
∆G= -220kJmol-¹
Enough to pump 10 protons (observed stoichiometry)
- Divided into a series of individual proton pumping steps
