3. ATP Synthesis

Proton Gradient

The net equation of a transport of a pair of electron from NADH to O2:

NADH +  H+  +  1/2 O2 —> NAD+  + H2O

NAD+ + H+ + 2e —> NADH, E´ = -0.32V (donor)
1/2 O2 + 2H+ + 2e —>H2O, E´ = +0.82V (acceptor)

Δ E’0 = Eacceptor – Edonor = 1.14V, n=2, F=96.5 kJ/Vmol

ΔG’0 = -220.1 < 0  Super exergonic!

Energetics of a Proton Gradient

Free Energy = Chemical potential + Electrical potential

ΔG = RT ln(CI/CM) + ZFΔΨ

CI = [H+]IMS
CM = [H+]MX

Z = absolute value of charge === 1 = charge of a proton

ΔΨ = transmembrane difference in the electrical potential. Negative charge in the matrix. Experimentally determined to be ~0.15-0.20V

Since ln x= 2.3 log(x) => ln(CI/CM) = 2.3*(pHM – pHI) = 2.3ΔpH

ΔG = 2.3 RT ΔpH + FΔΨ (when Z=1)

matrix pH > IMS pH, ΔpH ~0.75

Energy cost to pump one proton across the membrane: ΔG = 20 kJ/mol

To move 10 protons would require 200 kJ/mol; however, that 220kJ is released, and so we have enough! This, you’ll note, is a very efficient process then–only a 10% energy loss.

Role of Proton Gradient in ATP Synthesis

There have been studies on isolated mitochondria from human cells (grown in cell culture) to see how various disruptions in both the gradient and the transport chain impact oxidation and the creation of ATP.

Not sure I’m explaining this correctly but…

In one such study an isolated mitochondria was placed in a pH 9 solution with a small amount of KCl while keeping [K+] and [Cl-] levels fixed inside the matrix. This created a “slow leakage” of ions, which erased the pH gradient between the matrix and the inner membrane space.

Next, the solution was changed to a pH of 7 (sans KCl) returning the pH gradient. The buffer also included valinomycin. Valinomycin has an affinity for K+and it can move freely across the membrane. Thus valinomycin transports K+ across the membrane to create an electrical gradient. This creates an proton motive force. From this ATP synthesis can resume.

In detail here.

Experiment #1 using purified mitochondria

  1. Add the substrate of ATP production (ADP + Pi) but no source of electrons. Result: no O2 is consumed, no ATP produced
  2. Add Succinate, an electron source, and O2 consumptions starts, as does ATP synthesis
  3. Add Cyanide, which blocks ETC, O2 consumption halts, as does ATP synthesis => ATP synthesis requires an active electron transport

Experiment #2 also using purified mitochondria

  1. Start with Succinate, no ADP or Pi. No ATP produced, negligible O2 consumption
  2. Add ADP, Pi and now ATP production increases, as does O2 consumption
  3. Now block ATP synthesis via Oligomycin => the production will stop as will O2 consumption

An obligatory coupling exists between electron transport and the synthesis of ATP. This two can be uncoupled via DNP and FCCP

DNP, FCCP: uncouple ATP synthesis from ETC. O2 consumption can continue.

Chemiosmotic Hypothesis

  • If ΔpH =0, no protein gradient will be formed
  • if [H+]IMS = [H+]MX, no proton motif force will exist

So, if ATP synthesis stops, gradient gets out of whack, jams the ETC.

Note: low pH => high [H+] and vice versa. Molecule will be a cation if pH < pKa and vice versa

FCCP uncouples proton gradient but ETC continues, O2 still reduced

Cyanide inhibits ETC, O2 not reduced, no gradient formed, no ATP produced

Oligomycin inhibits ATP Synthase, proton gradient builds up until ETC overwhelmed (free energy of transport becomes too great to sustain), no O2 reduced


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