ETC, ATP synthase and Shuttle

3 key ratios to consider for ATP synthesis:

H/O ratio

• # of protons pumped to IM space per oxygen atom reduced (or NADH/FADH2 oxidized)

H/P ratio

• Number of protons required per ATP made by ATP synthase

• Determined by the enzyme's rotary mechanism

P/O ratio

• ATP produced per O atom reduced

• Yield is determined by the combination of H/O ratio and H/P ratio

 

Calculation: Theoretical ratio for NADH calculated by H+ cost for synthesis and transport

1. Protons for synthesis (H/P)

   1. 1 circle of rotation of the gamma-stalk synthesizes 3 ATP molecules

   2. Rotation is driven by the flow of 8H+ c-ring

   3. Another proton is needed for the transport of inorganic phosphate into the matrix for each ATP made, 3H+

   4. Total cost per 3 ATP is 11 phosphate

   5. H/P ratio is 11/3 = 3.6667 3.7 but round to 4

2. P/O Ratio for NADH

   1. Assuming H/O ratio is 10 for NADH oxidation

   2. P/O ratio is calculated as H/O / H/P = 10/4 = 2.5 ATP per NADH

Mitochondrial Transport Mechanisms

Import substrates and export products across the inner mitochondrial membrane and driven by the proton-motive force, utilizing difference in chemical or in electrical potential

ADP/ATP Antiporter (Adenine Nucleotide Translocase)

• ADP moves in to matrix and ATP moves out into IM space

ADP has a -3 charge and ATP has a -4 charge

• Net charge of -1 moving out

  • Exploits electric driving force because the IM space is positively charge

  • Only disrupts the electric potential

  • ATP export and ADP import is energetically favorable, determines the directionality of nucleotide exchange

Phosphate Translocase Pi/H+ symporter

• Specifically transport H2PO4- with H+ from the IM space to the matrix

• No net charge movement

• Only exploits the concentration gradient driving force (-4kJ.mol)

 

ATP synthase structure and function

ATP synthase structure and function

• Made of 2 main sectors that couple proton flow to ATP syntehsis by rotational catalysis

  • F naught is membrane embed

    • Proton flow from P to  occurs through the c-ring

  • F1 is matrix facing

    • 3 beta subunits are capable of ATP synthesis

F1 Sector

• Gamma subunit, the central stalk

  • Fixed to c-ring, it rotate relative to the fixed F1 alpha+beta subunits

    • c-ring with stalk is pushed in a circle by protons as it flow through F naught

  • Stalk pushes each sector of F1 successively to drive synthesis and eject ATP

    • 3 ATP produced per full 360 revolution

  • Associates with one alpha-beta dimer at a time

    • Gamma rotates and causes sequential conformational changes of each 3 alpha/beta dimers

• Alpha-beta subunit

  • The beta subunit has a nucleotide binding site and rotates through different conformations

F naught Sector

• c-ring

  • Made of vertical subunit (number varies with species)

  • Provides a channel for protons to flow from P-side to N-side

  • Rotates as protons move through it

  • Aspartate in each c subunit undergoes protonation/deprotonation during ATP catalysis

• Alpha subunit

  • Works with c-ring to provide a transmembrane channel for proton passage

• 2 beta2-subunits

  • Anchors F naught and F1 in the membrane

  • Hold alpha/beta dimers in place

 

Rotational catalysis

• Gamma subunit associates with the alpha/beta hexamer and rotates as protons flow through a and c subunits of F naught. Rotation induces conformational change in each alpha-beta dimer to drive ATP synthesis

• Alpha/beta subunits are stationary, anchored be b2(stator) of F naught

• Delta subunit secures F1 head to the stator

• Epsilon stabilizes the gamma shaft and has a regulatory role in controlling rotation

 Conformations of alpha-beta pair

• O, open

  • Nucleotide-binding site open

    • ATP is released and new substrates (ADP + Pi) can enter

• L, Loose

  • Substrates (ADP + Pi) are loosely bound and positioned for catalysis

• T, tight

  • Active site closes tightly, catalyzes the condensation fo ADP and Pi to form ATP

• Every 120 degree rotation the gamma subunit rotates, each alpha pair changes conformation, O>L>T>O

  • 3 ATP molecules are synthesized and release per full turn, one from each pair

  • Each 120 degree rotation requires 3 H+ inflow (P>N)

ATP stabilization on the F1 catalytic surface

ATP synthase alters the energetics of ATP formation, on the enzyme surface the equilibrium constant is

• Enzyme-ATP <>Enzyme-(ADP-Pi)

• Keq = 2.4

• Free energy = 0

Keq is much lower than ATP hydrolysis in solution

• Keq= 10^5 and free eneryg is -30.5kJ/mol

ATP synthase stabilizes ATP relative to ADP and Pi, ATP synthase has a high affinity for ATP but binds to ADP weakly

 

Thermodynamics

• Major energy barrier is forming the transition state between substrate and product

• Rate limiting step is the ATP release from the catalytic site

• Rotational catalysis of the gamma subunit drives the enzyme's conformational change and allows the alpha/beta subunits to trap substrates, synthesize ATP then release it efficiently

Proton to ATP stoichiometry in ATP synthase

• 1 full rotation of the central gamma stalk, 3 ATP molecules are synthesized while 8 protons flow through the c-ring

  • H+(P side) + 3ADP + 3Pi > 8H+(N side) + 3 ATP

• Proton  stoichiometry depends on the number of protons binding sites in the c ring  (varies between organisms)

  • More binding sites increase H+/ATP ratio and decrease P/O ratio

  • Lose 1 proton from the IM space to transport Pi (H2PO4-) per ATP synthesis, 8+3=11 11 total protons needed per full turn

• H/P ratio (H+ (P side) per ATP = 11/3 = 3.6667 round to 4

• H/O, ratio of H+ (P side) to H2O = 10 for NADH

• P/O ratio for ATP to H2Ofor NADH

  • 10/4 - 2.5 ATP per NADH

Chemical Uncoupling

• Uncouples block ATP synthesis without directly inhibiting the ETC or ATP synthase

• Dissipate proton gradient across IM, prevents PMF from driving ATP production

• Energy form electron transport is release as heat instead of being conserved in ATP

2,4-dinitrophenol (hydroxyl group)

• Pka 4.1, at intracellular pH phenolic hydroxyl is deprotonated

• Near IM it is partially protonated and increase hydrophobicity and it diffused across the IM

• Inside the matrix (high pH) it deprotonates and release the proton to complete the cycle and collapse the proton gradient

• Allows respiration (TCA and ETC) to continue but prevents ATP synthesis, etc works harder to reestablish lost gradient

Physiological Roles of mitochondrial uncoupling

• Cold adapted, hibernating and new born generates lots of heat by uncoupling electron transport from oxidative phosphorylation

  • Adipose tissue contains lots of mitochondrial that it is called brown adipose tissue

  • IM of brown adipose tissue mitochondrial as an endogenous protein thermogenin

• Thermogenin

  • Passive proton channel  so protons flow form the cytosol to the matrix

Regulations of Oxidative phosphorylation

• Mitochondrial respiration is regulated by substrate availability

  • ADP

  • Pi

  • O2

• And  oxidizable metabolites to generate NADH and FADH2

• Coupling

  • Respiration is tightly coupled to ATP synthesis

  • Electron flow to oxygen occurs when ADP is available to drive ATP production

  • ATP levels are 4-10 times higher than ADP

• Control

  • High ATP consumption increases ADP, stimulating respiration and ATP synthesis

  • When cell is resting, ADP is depleted, ATP has accumulated and electron transport chains

 

Shuttle systems for reducing equivalents

NADH/NAD+ cannot cross the mitochondrial Inner membrane directly, requires the shuttle system to transfer electron into the matrix for the ETC

 

1. Glycerol 3-Phosphate Shuttle Steps

Occurs in skeletal muscle and brain

1. Reduction of DHAP

   1. Electrons are transferred by the DHAP/G3P shuttle

   2. DHAP in the cytosol is reduced to G3P by NADH, transfers electrons from NADH to DHAP

   3. Reduces DHAP ketone to secondary alcohol in G3P

2. Transport

   1. G3P moves through the outer mitochondrial membrane to the outer surface of the inner membrane

3. Re-oxidation

   1. Mitochondrial G3P dehydrogenase deoxidizes G3P to DHAP (on inner mmebrane outer surface)

   2. Transfer electrons to FAD into FADH2 which reduces Q to QH2 and feed into complex 3

   3. DHAP diffuses back to the cytosol

Purpose and Electron Flow of Glycerol 3-Phosphate Shuttle

Key point

• Shuttle allows cytosolic NADH electron to enter ETC indirectly

• Produces less ATP then NADH entering by the malate-aspartate shuttle

Electron flow through G3P shuttle

• Uses 2 G3P dehydrogenase

  • Cytosolic one to make DHAP into G3P using NADH

  • Mitochondrial re-oxidizes G3P to DHAP which passes electrons by FAD to CoQ to QH2

• Protons used to form QH2 is from the IM space (P side) so no net proton pumping for the 2 protons attached to QH2

  • Lower P/O ratio compared to NADH entering complex 1 or succinate at complex 2

• ATP yield by G3P shuttle

  • Complex 3: Net 2H+ as QH2 delivered was protonated on the P-side

• 1 QH2 is on DHAP shuttle and another QH2 from another entry

 

Malate-aspartate shuttle, Purpose

Connects cytosolic NADH to the mitochondrial ETC

• Produces NADH that enter ETC at complex 1, better energy efficiency

• Uses malate (4C) to transport hydride (alpha hydroxy succinate)

  • Requires rebalancing cytosol vs matrix pools of 4C and 5C dicarboxylates using aspartate 4C (alpha-amino succinate) and glutamate 5C (alpha-amino glutarate)

• In aerobic conditions, NADH from glycolysis isn't removed by lactate dehydrogenase, but it can't be transported across the mitochondrial inner membrane for ETC

  • Solution is to use twin forms of malate DH one on either side of the IM

Steps of the malate-aspartate shuttle

1. Hydride loading

   1. Malate DH outise (cytosol): OAA + NADH > Malate + NAD+

2. Hydride Transport (on malate) into the matrix

   1. Use the malate/alpha ketoglutarate antiporter

3. Hydride unloading

   1. Malate DH inside (matrix): malate + NAD+ > OAA + NADH (TCA cycle)

• Achieves net transfer of NADH into matrix

• We must replace matrix alpha-KG and cytosolic OAA to keep the shuttle running

Balancing amino and keto acids by aspartate aminotransferase in the malate-aspartate shuttle

• Alpha amino acids and alpha keto acis are rebalance

  • Alpha-KG (5C keto) + aspartate (4C amino) > glutamate  (5C amino) + OAA (4C keto)

  • Send alpha-KG back to matrix as Glu and OAA back to cytosol as ASP using the Glu/Asp antiporter in the inner membrane

1. Rebalancing

   1. Aspartate amion transferase in the matrix transfers amino group from glutamate to oxalacetate to form aspartate and alpha-ketoglutarate

2. Glu-Asp Antiporter

   1. Exchange across the inner membrane: glutamate (protonated) enters the matrix to replenish the 5C alpha-KG

   2. Aspartate exits to the cytosol to replenish the OAA that was lost (4C)

3. In cytosol

   1. Aspartae aminotransferase reverse the reaction: aspartate + alpha-KG > glutamate + OAA

   2. Completes the cycle and reloads both sides for another round of reducing equivalent shuttling

ATP yield of glycolysis depends on NADH shuttle used

Malate-aspartate shuttle

• Electrons enter at complex 1

• Full proton pumping at Complex 1,3 and 4

• 2.5 Atp per NADH or 5 ATP for 2 NADH

G3P shuttle

• Transfer electrons to CoQ by mitochondrial G3P DH, and reduce to QH2 go to the IM space

• Fewer protons contribute to ATP synthesis

• Only 4 protons, so 1 ATP per NADH or 2 ATP for 2 cytosol NADH

Key point

• Tissue-specific shuttle usage determines the effective ATP yield from glycolysis

  • Malate-aspartate is maximal energy

  • G3P is lower energy

Glycolysis Final ATP Yield

• 2 or 5 final ATP

  • 2 NADH (cytosolic) and 2 ATP as direct product

Pyruvate final ATP Yield

• 2 NADH (mitochondrial matrix)

• 5 final ATP

Acetyl-CoA, 2 per glucose ATP yield

• 6 NADH (mito. Matrix)

  • 15 ATP

• 2 FADH2

  • 3 ATP

• 2 ATP or 2 GTP

Total yield per glucose

29 or 32 ATP