Lecture 28: Oxidative Phosphorylation and ATP Synthase

True or False: Biological oxidations are mostly dehydrogenation.

true

How does catabolism prepare organic compounds for energy usage?

by stripping off energy-rich electrons—releasing CO₂

What is the overall combustion/dehydrogenation reaction?

C₆H₁₂O₆ + 6O₂ → 6CO₂ + 6H₂O

In metabolism, where do the O atoms of CO₂ come from?

water or the substrate (none of the atoms come from oxygen)

What is the chemical reaction for biological oxidations?

C₆ H₁₂O₆ + 6H₂O → 6CO₂ + 24[H]

In respiration, reducing equivalents are transferred to oxidants and the free energy is used to what kind of work?

• ATP synthesis

• oxidative phosphorylation

• photophosphorylation, or ET phosphorylation

What are the major sources of electrons for oxidative phosphorylation?

NADH and FADH₂

What are the substrates for NADH?

1. a-ketoglutarate

2. succinyl-CoA

3. isocitrate

4. a-ketoglutarate

5. pyruvate

6. acetyl-CoA

7. B-OH-acyl-CoA

8. B-ketoacyl-Coa

What is the E’⁰ for NADH?

-0.32 V

What are the substrates for FADH₂?

1. succinate

2. fumarate

3. glycerol

4. DHAP

5. acyl-CoA

6. trans-enoyl-CoA

What is the E’⁰ for FADH₂?

0.22 V

What is the E’⁰ for O₂/H₂O?

0.82 V

True or False: In mitochondria, reduced pyridine nucleotides from glycolysis and the TCA cycle generate free energy through electron transfer-coupled proton translocation, ultimately leading to the reduction of atmospheric O₂ to water.

true

What are the common prosthetic groups involved electron transfer processes?

• flavoproteins: FAD or FMN

• cytochromes: iron porphyrins/heme

• copper irons

• iron-sulfur proteins

What are the central electron carriers in the membrane?

quinones

Describe flavoproteins.

• FAD and FMN

• can act as 1 e^- or 2 e^- carriers

• FMN has a 1 e^-/semiquinone intermediate state

• reduction steps involve protonation:

  • oxidized FMN is aromatic and reduced FMN is non-aromatic (higher energy)

Describe cytochromes.

• prosthetic group: heme (iron hepatocyte)

• pyro structure 

• normally are 1 e^- carriers

• redox potentials range from -400 mV to +450 mV depending on the heme type and microenvironment

Describe iron-sulfur centers in proteins.

• act as 1 e^- carriers no matter how many Fe atoms

• type of prosthetic group

• redox potentials range from -650 mV to +450 mV, which depends on oxidation state of Fe and microenvironment

Describe quinones.

• ubiquinone:

  • carries electrons in the ETC in the mitochondria

  • also called coenzyme Q, Q10, or simply Q

• benzoquinone:

  • lipid soluble

  • contains a decisoprenoid chain (very hydrophobic)

  • free diffuses within the IMM

• general info:

  • acts in 1 e^- or 2 e^- transfers

    • 2 e^- transfers are involved with the semiquinone intermediate state

  • both reduction steps also involve protonation

Are you familiar with the quinone reaction from oxidized to reduced?

yes!

What is the natural downhill electron flow?

1. NADH

2. Q

3. b

4. c₁

5. c

6. a

7. a₃

8. O₂

How can the order of e^- transfer be determined experimentally?

• kinetically using spectroscopy:

  • chromophores absorb UV and visible light differently depending on their redox states

• using site-specific inhibitors:

  • rotenone

    • inhibits Q and down

  • antimycin A

    • inhibits Cyt c₁ and down

  • CN^- or CO

    • inhibits O₂/complex IV

True or False: Each step in electron transfer involves large, multi-enzyme membrane protein complexes.

true

What is the net transport/reaction equation for complex I?

NADH + Q_m + 4H⁺_in →/← NAD⁺ + Q_mH₂ + 4H⁺_out

State the enzyme for complex I.

NADH-ubiquitnone

What is the net transport/reaction equation for complex II?

succinate + Q_m →/← fumarate + Q_mH₂

State the enzyme for complex II.

succinate-ubiquinone oxidoreductase

What is the net transport/reaction rate for complex III?

Q_mH₂ + 2c⁺_out + 2H⁺_in → / ← Q_m + 2c_out + 4H⁺_out

State the enzyme for complex III.

ubiquinol-cytochrome c oxidoreductase

What is the net transport/reaction equation of complex IV?

½ (4c_out + O₂ + 8H⁺_in → / ← 4c⁺_out + 2H₂O + 4H⁺_out)

State the enzyme for complex IV.

cytochrome c-dioxygen oxidoreductase 

How many protons are pumped by each complex?

• complex I: 4

• complex II: none

• complex III: 4

• complex IV: 2

Describe the mitochondrial complex I.

• extremely large transmembrane protein complex

• 42 polypeptide chains

• an FMN-containing flavoprotein

• at least 6 iron-sulfur centers

True or False: There is an electrostatically positive charge on the intermembrane space, and there is an electrostatically negative charge on the matrix.

true

What is the entry point for e^- from NADH?

complex I

Describe the process for complex I.

• step I:

  • exergonic transfer of hydride from NADH to ubiquinone (Q), together with H⁺ from the matrix (N-side)

  • reaction:

    • NADH + H⁺ + Q → NAD⁺ + QH₂

• step 2:

  • endergonic transfer/”pumping” of 4 H⁺ from the matrix to the intermembrane space (P-side)

  • overall reaction:

    • NADH + 5H⁺_N + Q → NAD⁺ + QH₂ + 4 H⁺_P

Describe the process for complex II.

• the complex couples succinate oxidation via FAD and Fe-S centers, directly to the ETC at the point of the membrane pool of ubiquinone (Q)

• no additional protons are being pumped by this complex

True or False: There are many homologs of SDH in bacteria; different substrate specificities and opposite directionality (such as fumarate reductase).

true

What are other examples of direct input of e^- to Q/bypassing complex I?

• FAD-containing enzymes that connect substrates to the Q pool in the IMM

  • succinate

  • fatty acyl-CoA

    • none of these other entry points for e^- into the ETC result in proton translocation

True or False: The ubiquinone (Q) pool is the common junction for all entry points of e^- into the ETC. Complex I and II is important for Q reduction, and complex III and complex IV is important for QH₂ oxidation.

true

Describe mitochondrial complex III.

• exists as a homodimer

• each monomer has 8-12 subunits

• only 3 of the subunits actually catalyze e^- transport reactions (and many bacteria only have 3 subunits)

  • cyt b

  • cyt c₁

  • Rieske-type iron-sulfur protein

What are the 2 quinone (Q) binding sites on complex III?

• Q_PQ₀: near heme b_L

• Q_NQ_i: near heme b_H

True or False: The two Q binding sites function in a “Q cycle” that encompasses two stages.

true

What is the goal of the Q cycle?

• to transfer e^- from QH₂ to Cytochrome c

• pump protons out of the mitochondrial matrix

How many electrons does Cytohrome c carry at a time?

1 e^-

Describe the first stage of the Q cycle.

• QH₂ binds to the Q_P site and transfers:

  • 1 e^- → Rieske 2Fe-2S center → c₁ → Cyt c

  • 1 e^- → b_L → b_H → Q (in Q_N site), creating -Q^- radical

• protons from QH₂ are released to the intermembrane space (P-side)

Describe the second stage of the Q cycle.

• a second QH₂ exchanges with Q at the Q_P site and transfers:

  • 1 e^- → Rieske 2Fe-2S center→ c₁ → Cyt c

  • 1 e^- → b_L → b_H → -Q^- (in Q_N site), creating QH₂ after picking up two protons from the matrix

• again, protons from QH₂ are released into the intermembrane space (P-side)

What is the net equation of complex III.

QH₂ + 2 cyt c₁ (oxidized) + 2H⁺_N → Q + 2 cyt c₁ (reduced) + 4H⁺_P

Describe the process for complex IV.

• e^- are transferred from cytochrome c oxidase to O₂, with substrate proton pumping

• O₂ gets reduced to H₂O using e^- that were carried from complex III by reduced Cyt c

• O₂ + 4 e^- + 4H⁺(in) → H₂O is coupled to 4H⁺(in) → 4H⁺(out)

• protons are consumed in forming water (substrate or chemical protons) and protons are pumped across the membrane

Describe complex IV.

• 12-16 subunits, but only subunits I and II are directly involved in catalytic activity

• bacteria usually have 2-4 subunits

• cofactors:

  • Cu_A (2Cu that share electrons equally, cycling from (Cu-Cu)²⁺ to (Cu-Cu)³⁺

  • cytochrome/heme a

  • cytochrome/heme a₃

  • Cu_B (a single Cu ion near Heme a₃)

True or False: It takes 4e^- and 4H⁺ to reduce one O₂ to two H₂O.

true

delta p (proton-motive force) has which 2 components?

electrical and H⁺ concentration

What is the net result for the proton-motive force?

NADH + 11H⁺_N + ½ O₂ → NAD⁺ + 10 H⁺_P + H₂O

Why is ATP synthesis driven b proton-motive force?

• the proton pumping sets up a difference in [H⁺] between matrix and intermembrane space

• these 2 compartments differ in pH and charge distribution (yielding an electrochemical H⁺ gradient)

• the energy in this electrochemical proton gradient across the IMM constitutes the proton-motive force

• this drives ATP synthesis as protons flow back into the matrix through the ATP synthase

• FAD creates a smaller proton gradient, which is why less ATP is produced

What is the net equation for NAD-linked proton-motive force?

NADH + 11H⁺_N + ½ O₂ → NAD⁺ + 10H⁺_P + H₂O

What is the net equation for FADH₂-linked proton-motive force?

FADH₂ + 6H⁺_N + ½ O₂ → FAD + 6H⁺_P + H₂O

What is the chemiosmotic hypothesis?

• the free energy of e^- transport is conversed by pumping H⁺ from the mitochondrial matrix to the intermembrane space

• creates electrochemical H⁺ gradient across the IMM

• electrochemical potential of this gradient is harnessed for work and used to synthesize ATP

What is important to know about the chemiosmotic mechanisms?

• uncouplers allow H⁺ to leak back down the gradient

• the proton pump driven by the electron transport (respiration or photosynthesis)

• exchange transporters allow metabolites in and out

• proton gradient drives a proton-pumping ATPase in reverse, to make ATP

Describe complex V (ATP synthase).

Components:

• F₁

  • aces the matrix

  • contains the alpha-beta subunits

  • peripheral membrane protein complex

  • isolated F₁ portion will catalyze ATP hydrolysis

• F₀

  • integrated in the internal membrane

  • “stick”

  • integral membrane protein complex

  • this portion rotates

• b₂

  • peripheral stalk

  • connects the F₁ to the F₀

The synthesis of ATP by ATP Synthase is fully reversible. What is the experimental evidence for it?

• incubating the F₁ portion of ATP Synthase with ATP and H₂¹⁸O yields P_i with 3 or 4 ¹⁸O atoms

• this is because the gamma-phosphate of ATP is joined and cleaved repeatedly, leaving the P_i free to tumble in the active site

• this inserts ¹⁸O randomly into the four O atoms of P_i

Why is ATP synthesis by ATP synthase reversible?

• the enzyme greatly stabilizes the binding of the product (ATP) to the enzyme’s active site

• there’s multiple conformations available to the Beta subunit

What does ATP Synthase use to eject the newly synthesized ATP from the enzyme?

the proton-motive force

How does ATP synthase use the proton-motive force?

• ATP synthase functions as a rotary motor, driven by the protons moving from the intermembrane space to the matrix

• H⁺ pass through F₀ via hemi-channels at interface of subunit a and c-ring

• once an H⁺ is bound, electrostatics allow c-ring to rotate freely only in one direction

• each revolution of F₀ rotor drives ATP synthesis in 3 catalytic sites in F₁

Discuss ATP synthesis in relation to ATP Synthase’s rotating movement.

• synthesis takes place in the three Beta subunits, which have different conformations at any given moment

• rotation of the gamma subunits (attached to the rotating c-ring) sequentially alters the conformations of the three Beta subunits

• for each 360 degree turn of the ATP synthase rotor, 3 ATP molecules are synthesized and released

The electrochemical gradient favors transport of what out of the matrix and what into the matrix?

favors transport of ATP out of the matrix and ADP into the matrix

True or False: ATP^4- moves out of the matrix, and ADP^3- moves into the matrix, which is favored by the proton-motive force.

true

True or False: Phosphate translocation is also favored by the H⁺ gradient.

true

What does the mitochondria need for ATP synthesis?

an energy source (such as succinate) and ADP + P_i

What happens when CN^- is applied to the ETC?

• O₂ binding is inhibited

• the proton pumping stop

• no electrochemical gradient

• ATP synthesis and ET both stop

• cytochrome c will be reduced and block of the rest of the cycle

What do uncouplers do? What does this do to the proton gradient?

• short-circuit the ETC by allowing H⁺ to re-enter the mitochondrial matrix and bypass ATP synthase

• this results in the collapse in the H⁺ gradient

Name the two mentioned uncouplers and what their functions are.

• DNP

  • allows H⁺ a direct path back into the matrix

• UCP

  • thermogenic (generates heat)

  • allows H⁺ back into the matrix in a controlled way in brown fat to generate heat

What does oligomycin inhibit?

ATP synthase

What happens when oligomycin inhibits ATP synthase? What are the downstream effects?

• O₂ consumption is slowed because H⁺ pumping is coupled to ATP synthesis

• DNP carries H⁺ across the IMM, which collapses the H⁺ gradient

True or False: O₂ consumption will resume after adding DNP to oligomycin-poisoned mitochondria.

true

What do the mitochondrial shuttle systems do?

convey reducing equivalent from cytosolic NADH into the mitochondrial matrix

What are the 2 mitochondrial shuttles?

• malate-aspartate shuttle

• glycerol 3-phosphate

True or False: In the glycerol 3-phospahte shuttle, the electrons enter the ETC at Coenzyme Q and bypass complex I, so fewer protons are pumped per electron pair.

true

What does the overall ATP yields depend on?

depends on which mitochondrial shuttle is used

What type of shuttle does the liver, kidney, and heart use?

the malate-aspartate shuttle

What type of shuttle does skeletal muscle and the brain use?

the glycerol 3-phosphate shuttle