Lecture 9 - ATP synthesis

ATP synthesis: ADP vs ATP

ADP - very low affinity

ATP - very high affinity

ATP synthase

Enzyme that makes ATP

• 2 complexes: F1 (in the mitochondrial matrix) and F0 (at the membrane)

F0

Part of ATP synthase

• 3 subunits

  • A (1)

  • B (2)

  • C (10-15)

B subunit

Part of F0 complex

• keeps F1 complex in place

A subunit

Part of F0 complex

• proton channel (in from IMM, out into matrix)

• Mechanism:

  • 1) proton binds to Asp (bc it is acidic)

  • 2) Arg repels proton → pushes c disk

c disk

Part of F0 complex

• proton binding site (proton binds to Asp or Glu)

• only turns one way → bc the pKa for proton on the outside of the membrane is higher than the inside

• counterclockwise

F1

Part of ATP synthase

• polypeptide chains combine to form a ring structure that catalyzes the synthesis of ATP

• Polypeptide chains:

  • α (3)

  • β (3)

  • 𝜸 (1)

  • 𝜹 (1) scaffolding

  • 𝛆 (1)

𝜸 subunit

Part of F1 complex

• shape: alpha helical coil coil

• attached to c-ring → as it rotates, the ratcheting causes confirmational changes that influence binding for ATP/ADP

• 10 protons = 1 full rotation

• 1 full rotation = 3 ATP

𝛆 subunit

Part of F1 complex

• Connects catalytic domain to axel (does this by interacting with 𝜸 and α3β3)

• compact state: ATP synthesis proceeds

• extended state: rotation of 𝜸 subunit blocked → ATP synthesis slowed

α3β3

Part of F1 complex

• 3 confirmations:

• Open (O): releases newly synthesized ATP

• Tight (T): catalysis (ADP and Pi brought close together)

• Loose (L): Binds ADP and Pi

ATP synthase mechanism

1. 𝜸 rotates → β subunit converts from tense state to open state

2. ATP released from β subunit

3. ADP and Pi enter the open β subunit → 𝜸 rotates

4. 𝜸 rotation causes the conversion from open state to loose state

Adenine nucleotide translocase

Protein that pushes finished ATP out of the matrix, pulls ADP in (ATP^4- → ADP³-)

• powered by proton-motive force

Phosphate translocase

Protein that brings phosphate in, along with H+

• powered by proton-motive force (symporter)

Acceptor control ratio

Example of oxidative phosphorylation regulation.

Tells you how much the rate of oxygen consumption depends on the substrate

• high ratio: Respiration depends on ADP availabiltiy

• Ratio close to 1: something is wrong, respiration does not depend on ADP

Mass-action ratio

Example of oxidative phosphorylation regulation.

• ATP/(ADP)(Pi) → (energy it has vs. the energy it has potential to make)

Hypoxia

When there is not enough oxygen for cells

• enzymes with prolines sense hypoxia → HIF activated and gets expressed → activates genes that respond to low oxygen (ex. complex 4-2)

Complex 4-2

Enzyme that gets activated by HIF when there is low oxygen

• efficient during low oxygen

• not as efficient as normoxia (4-1 dominates instead)

UCP-1

Allow pumping of protons to happen without ATP synthase

• it does this by uncoupling oxidative phosphorylation from ATP synthesis → this generates heat (heat is released)

What kind of energy is the proton-motive force?

Electrochemical

Electrochemical energy formula

ΔG=nfΔψₘ

• n=number of electrons per mole of product

• f = faraday constant (96.48 kJ/mol)

• Δψₘ = change in volts across membrane

*this equation considers flow from outside to inside the membrane

How to interpret ΔG=nfΔψₘ answer?

Positive answer - favorable (to flow from inside to outside of the membrane)

Negative answer - favorable (to flow from outside of the membrane to inside)

Apoptosis

Cell death - happens when cytochrome c is in the cytosol

• passive - mitochondrial damage causes cytochrome c to go to the cytosol

• active → bax channels form → cytochrome c goes to cytosol

MitoTracker

Fluorescent dye that goes into mitochondria → changes color based on H+ concentration

Apoptosomes and caspases

Apoptosome turns on caspases → there is a cascade where caspases are turned on → degrade/chops proteins