Biochemistry - ATP Synthesis (Ch.21)

List the process that generates NADH

• Glycolysis

• Pyruvate dehydrogenase complex

• TCA cycle

• Fatty acid degradation

• Amino acid degradation (some)

List the processes that generate FADH2

• TCA cycle

• Fatty acid degradation

• Amino acid degradation (some)

Define the proton-motive force. Define it’s two components

• The unequal distribution of protons is called the proton motive force and it;s composed of two components

  • A chemical (pH) gradient for the protons

  • A charge gradient due to the unequally distributed, positively charged protons

Define the chemiosmotic hypothesis. Who what it proposed by?

• Chemiosmotic hypothesis: Electron transport and ATP synthesis are coupled by a proton gradient across the inner mitochondrial membrane

  • Proposed by Peter Mitchell

Describe the chemiosmotic hypothesis in the mitochondria

• Protons are pumped across this membrane as electrons flow through the respiratory chain

• pH outside the matrix is 1.4 pH units lower

  • Corresponds to a 0.14V membrane potential

  • Corresponding free energy is 21.8 kJ/mol of protons

Describe the structure and function of the F₀ subunit of ATP Synthase

• F₀ is composed of 3 different polypeptides: a,b, and c

• F₀ is the proton channel of the complex

  • in the membrane

Describe the function and structure of the F₁ subunit of ATP synthase

• F₁ is composed of 5 different polypeptides: alpha, beta, epsilon, sigma, and y

• F₁ subunit contains catalytic activity

  • The a and B subunits (three of each) are homologous, but the symmetry of the hexamer is broken in the way each B subunit interacts with a different face of the y chain

  • Y,E, and c rotate (rotor), other portions of the molecule are stationary (stator)

Does ATP formation occur in the absence of the proton-motive force?

• Yes - ATP formation occurs readily in the absence of proton-motive force

• The role of the proton gradient is actually to release ATP from the synthase

Describe the loose state of ATP synthase

• Loose: binds ADP and Pi

Describe the tight state of ATP synthase

• high affinity for ligands - binds ATP with very high avidity, such that ADP and Pi are converted to ATP

Describe the Open state of ATP synthase

• almost no affinity for ligands - releases bound nucleotide

What are the roles of the a (alpha) subunits?

• The a subunits bind ATP, but appear to be non-catalytic and are generally involved in supporting the structure and ATP binding of the B subunits

What is the first step of ATP synthase moving from one configuration to another?

1. ADP and Pi are transiently combining at the T-form site to form ATP, but the ATP can’t be released

Describe the second step of the ATP synthase

• A 120 degree rotation of subunit y converts the T-form site into an O-form site

Describe step 3 of the ATP synthase mechanism

• ATP at the O-form site can now depart

Describe the fourth step of the ATP synthase mechanism

• The ATP at the O-form site is replaced by ADP and Pi

How does the proton flow drive the y subunit?

• The subunit abuts the membrane-spanning c units, and apears to contain two hydrophilic half channels where protons can enter, but cannot pass through the entire membrane

• A proton eneters the a subunit from the intermembrane space (high H+) of the membrane and binds a specific ASP (or GLU) residue on the associated c subunit

• The c subunit rotates to a more hydrophobic environment with its now uncharged Asp (of Glu), rotating all the other c subunits with it

• The c subunit that has rotated all the way around to exposure with the matrix half channel, can release its proton to the proton deficient matrix - deprotonated Asp is regenerated

Understand the coordination between the ETC and ATP Synthase

What is required for the transport of electrons through the electron-transport chain

• Electrons do not flow through the electron-transport chain unless ADP is available to be converted into ATP (shuttle: ATP-ADP translocase)

Define the process of Oxidative phosphorylation (OXPHOS). Can it be regulated?

• Overall, the process of Oxidative phosphorylation is the process of oxidizing nutrients to release energy, which is used generate ATP from ADP and P_i

  • The regulation of oxidative phosphorylation by ADP is called acceptor or respiratory control

What does the drug retenone do?

• Blocks the transfer of electrons from Complex I to CoQ

  • ETC is partially blocked - No ATP synthesis from NADH

What does the drug Antimycin A do?

• Blocks the transfer of electrons from cytochrome b to cytochrome c1

  • Block the ETC - No ATP synthesis

What does the drug oligomycin do?

• Block the proton channel (F₀) subunit

  • No proton translocation back to the matrix - No ATP synthesis - slow down the ETC