Biochemistry Chapter 21: Proton-Motive Force

ATP synthase

enzyme complex embedded in the inner mitochondrial membrane which carries out synthesis of ATP, powered by the protein gradient

Chemiosmotic Theory

1. reduced substrate donates electrons

2. released energy is used to transport protons against the electrochemical gradient

3. the flow of protons down the gradient powers phosphorylation of ADP

proton-motive force

the proton gradient generated by the oxidation of NADH and FADH₂

Why does the proton gradient require an impermeable membrane?

A membrane impermeable to ions is necessary for controlled movement of protons uphill (coupled with downhill flow of electrons) and downhill (coupled with phosphorylation of ADP).

What are the structural components of ATP synthase?

F₁ subunit

• hexameric ring of alternating α₃ and β₃ subunits

•  central 𝛾ε stalk extending into the middle

• connected to F₀ by b column and δ

F₀ subunit

• proton channel c ring

• a subunit bound to outside of ring

What is the significance of cristae for ATP synthesis?

Proton pumps of the ETC can localize the proton gradient near the ATP synthases located at the tips of cristae.

What is the binding-change mechanism of ATP synthase?

The β subunit active sites rotate between 3 different conformations with different functions:

• Loose- binds ADP and Pi

• Tight- synthesizes ATP

• Open- releases ATP, allows ADP and Pi to reenter

What powers the binding-change mechanism?

120° clockwise rotations of the 𝛾 subunit drives the interconversion of the 3 forms: L → T → O → L → T → O…

a subunit

borders the c ring and sits on the periphery of the membrane; contains 2 hydrophilic half-channels which allow regulation of proton entry/exit

c subunit

made of a pair of membrane-spanning α-helices, each with a glutamate/aspartate in the middle which can bind and release protons

How does glutamate/aspartate regulated proton entry/exit?

• if glutamate is charged, the c subunit will not move into the hydrophobic interior of the membrane

• proton enters to neutralize glutamate residue → c ring rotates one subunit → residue goes into matrix half-channel → proton moves into matrix

How many ATP are synthesized from one rotation of ATP synthase?

3 ATP; for 8 c subunits each molecule of ATP requires 8/3 ~ 3 protons flowing in per ATP formed

glycerol 3-phosphate shuttle

1. transfers a pair of electrons from cytoplasmic NADH to DHAP to form G3P

2. G3P transfers electrons to FAD to reform DHAP

3. FADH₂ transfers electrons to Q to form QH₂

malate-aspartate shuttle

involves two antiporters and a transamination reaction (glutamate + oxaloacetate → aspartate + α-ketoglutarate) where cytoplasmic NADH is consumed and reformed inside the matrix

ATP-ADP translocase

antiporter coupling the entry of ADP to the exit of ATP; contains a single nucleotide-binding site that alternately faces the matrix and cytoplasmic sides of the inner membrane

How many molecules of ATP are formed from 1 molecule of glucose?

~30 ATP from 1 glucose completely oxidized to CO₂

• Glycolysis: 2 ATP + 2 cytoplasmic NADH*1.5 = 5 ATP

• Pyruvate to acetyl CoA: 2 NADH*2.5 = 5 ATP

• CAC: 2 ATP + 6 NADH*2.5 + 2 FADH₂*1.5 = 20 ATP

regulator/acceptor control

regulation of the rate of oxidative phosphorylation by the ADP level; electrons do not usually flow through the ETC unless ADP is simultaneously phosphorylated to ATP

How does ADP level regulate the rate of the citric acid cycle?

Low ADP → NADH + FADH₂ not oxidized in ETC → less NAD⁺ and FAD → citric acid cycle slows