BIOS 301 - ATP Synthesis

What is proton motive force?

• IMM separates two compartments of different [H+], resulting in differences in chemical concentration (delta pH) and charge distribution (delta psi) across the membrane

• Net effect is the proton motive force (delta G)

What are three means by which electrochemical proton gradient is generated?

• Actively transporting protons across the membrane (Complex I and Complex IV)

• Chemically removing protons from the matrix (reduction of CoQ and O2)

• Releasing protons into IMS (oxidation of QH2 by Complex III)

Ionophores (function)

• Ionophores freely pass through membranes and transport ions

• Can dissipate electrical gradient without altering chemical gradient significantly

  • Electric potential is extremely sensitive to charge, only a small fraction of ions must move to collapse voltage and leave bulk concentration almost unchanged

Artificial proton gradient experiments

• Scenario 1: Matrix and IMS have equimolar concentrations of KCl and H+

  • No ATP synthesis occurs due to lack of both concentration and electrical gradients

• Scenario 2: [H+] in IMS is increased and K+ is removed from IMS. Add valinomycin to start flow of K+ from matrix to IMS

  • ATP synthesis begins without electron transport and is driven by two components:

    • Chemical gradient due to difference in pH

    • Electrical gradient due to asymmetry in [K+] and [Cl-]

F1 subunit

• Soluble complex in matrix

• Individually catalyzes hydrolysis of ATP

• Natural direction of motor rotation leads to ATP hydrolysis

F0 subunit

• Integral membrane complex

• Transports protons from IMS to matrix, dissipating proton gradient

• Energy transferred to F1 to catalyze phosphorylation of ADP

• Natural direction of motor rotation (opposite of F1 leads to ATP synthesis)

What determines function of ATPase vs. ATP synthase?

• If F1 dominates rotation (due to high matrix [ATP]), then ATP is hydrolyzed

• If F0 dominates rotation (due to high electrochemical gradient), then ATP is made

F1 conformations

• Hexamer arranged in three alpha-beta dimers

• Dimers can exist in three different conformations:

  • Open: empty

  • Loose: binds ADP and Pi

  • Tight: catalyzes ATP formation and binds product

Binding change model

• Proton translocation causes rotation of the F0 subunit and the central gamma shaft

• This creates a conformational change that differentially affects each dimer

• The conformational change in one of the three pairs promotes condensation of ADP and Pi into ATP

How many H+ ions crossing the membrane does it take to synthesize 1 ATP in net?

• Each conformational change requires 3 H+ to be translocated from IMS to matrix

• So 1 complete rotation of the 3 alpha/beta dimers requires 9 H+ but 3 ATPs are made per complete cycle

  • So 3 H+ per ATP

• But translocation of a fourth H+ per ATP is required to facilitate cotransport of substrates into and products out of the mitochondria

  • So ultimately net 4 H+ are transferred from IMS to matrix

ATP transport into/out of mitochondrial matrix

• Facilitated by adenine nucleotide translocase

• Passive antiport (driven by electrical gradient)

  • Matrix is negatively charged compared to IMS, so ATP4- is transferred out and ADP3- is transferred in

Phosphate transport into/out of mitochondrial matrix

• Facilitated by phosphate translocase

• Secondary active symport (driven by concentration gradient)

  • H+ concentrated in IMS is transferred into matrix, along with H2PO4-

What causes variation in the net production of ATP via oxidation of glucose?

• In eukaryotes, organellar segregation prevents NADH produced in cytosol (e.g. by glycolysis) from directly entering ETC at Complex I

• Two methods are used to feed electrons from NADH in the cytosol into the mitochondria

  • Malate aspartate shuttle

  • Glycerol 3 phosphate shuttle

• Different method used = different amounts of ATP produced

Malate-aspartate shuttle

• Used in liver, kidneys

• Uses NADH to convert oxaloacetate into malate

• Malate crosses IMM and is converted back to oxaloacetate in matrix (NADH is regenerated and can donate to Complex I of ETC)

• Advantages: doesn’t result in loss of ATP generated

• Disadvantages: complex (lots of steps, enzymes = scope for error)

Glycerol 3 Phosphate Shuttle

• Used in skeletal muscle, brain

• Dihydroxyacetone phosphate is converted to Glycerol 3 phosphate and NADH is oxidized to NAD+

• G3P reduces FAD to FADH2 in the glycerol 3 phosphate dehydrogenase enzyme attached to IMM

• FADH2 reduces QH2 which enters Complex III

• Advantages: less complex

• Disadvantages: results in less ATP yield (bypassing Complex I results in missed opportunity for proton pumping)

ATP yield from complete oxidation of glucose

• Glycolysis produces:

  • 2 NADH → 3 or 5 ATP (depending on use of malate-aspartate vs. glycerol 3-phosphate shuttle)

  • 2 ATP

• Pyruvate (2 per 1 glucose) oxidation produces:

  • 2 NADH → 5 ATP

• Acetyl CoA (2 per 1 glucose) oxidation in TCA produces:

  • 6 NADH → 15 ATP

  • 2 FADH2 → 3 ATP

  • 2 GTP → 2 ATP

• Total yield per glucose is 30-32 ATP

Uncouplers

• Dissipate proton and charge gradients

• Are similar to ionophores but specialized for proton transport

What is one reason we observe 2.5 ATP/NADH instead of 3 ATP/NADH?

• Ubiquinone is naturally leaky and sometimes performs single electron transfers to O2 to result in free radicals

• Some of the NADH produced during TCA/glycolysis is used to reduce NADP+ to NADPH, which in turn replenishes reduced glutathione reductase, which neutralizes ROS