EFB325 Cell Physiology

Aerobic respiration (oxidative phosphorylation)

Through glycolysis and the citric acid cycle, the current tally of energy yield is:

Glucose (6 C) has been oxidized to 6 CO₂, to yield 4 ATP + 10 NADH + 2 FADH₂

If a fatty acid were being oxidized, for every 2 carbons in the fatty acid (which are cleaved off as acetyl CoA), the yield would be 2 CO₂ + 1 ATP + 4 NADH + 2 FADH₂

This does not represent much energy in the form of ATP (what we call our energy currency), plus we need to oxidize NADH to regenerate NAD⁺ to keep glycolysis and the citric acid cycle running. However, through the oxidation of glucose, a great deal of reduced carrier molecules (NADH) were generated.

NADH and FADH₂ carry a great deal of energy

• to "burn" (oxidize) NADH directly (NADH + 1/2O₂ + H⁺ -> NAD⁺ + H₂O) yields 52.4 kcal/mol
• to oxidize FADH₂ = 45.9 kcal/mol
• to hydrolyze ATP (ATP + H₂O -> ADP + Pi) = 7.3 kcal/mol

-biological oxidation of NADH releases that energy in stepwise reactions, capturing it efficiently

In overview:

• oxidation of NADH and FADH₂ leads to ATP synthesis by a process of electron transport (a series of oxidation-reduction reactions) that is coupled to production of a H⁺ electrochemical gradient across the inner mitochondrial membrane; flow of H⁺ down the electrochemical gradient and through a protein drives the enzymatic synthesis of ATP

The system of mitochondrial electron transport and ATP synthesis can be thought of as three linked processes

1) A redox reaction involving the oxidation of NADH and FADH₂ and the reduction of oxygen to form water

2) The use of the energy released by that oxidation to drive active transport of H⁺ producing a H⁺ electrochemical gradient

3) The use of the H⁺ electrochemical gradient by the ATP synthase enzyme to synthesize ATP from ADP + P_i

-all of this is accomplished by membrane-bound, multi-subunit protein complexes in the inner mitochondrial membrane

Oxidation of NADH (or FADH₂) releases high-energy electrons passed through a series of redox carriers, finally reducing oxygen to water

• a redox carrier is a molecule that can act as an electron acceptor, then donor
• the acceptor & donor=a redox pair, such as NAD⁺ (acceptor) and NADH (donor)
• acceptors of different redox pairs can act better or worse as electron acceptors=reduction potential (expressed in volts)
• each carrier can only donate electrons to a "weaker" electron donor (with a higher reduction potential), which means going from higher energy to lower energy levels
• in mitochondrial electron transport, NADH is the best electron donor (lowest reduction potential), while O₂ is the weakest electron donor (it is the best electron acceptor, with the highest reduction potential)
• Therefore, the series of oxidation/reduction reactions between electron carriers in electron transport are always cascading down from higher to lower energy levels (and from lower to higher reduction potentials)

The redox carriers in electron transport are prosthetic groups (mostly metals) bound to 3 multi-subunit, membrane-bound protein complexes and two mobile electron "shuttles"

• the prosthetic groups, which may be oxidized and reduced, include: Fe-S centers, heme groups (heme has an Fe atom), and copper atoms

The path of electrons from NADH:

• NADH dehydrogenase (also called Complex I): this protein complex oxidizes NADH to NAD⁺, then passes those electrons through Fe/S and other carriers to then reduce ubiquinone
• Ubiquinone: this is a quinone (Q), not a protein, which can accept and donate 2 electrons. Ubiquinone is hydrophobic, so it moves in the lipids of the membrane, "shuttling" electrons between proteins
• Cytochrome b-c₁ complex (also called cytochrome c reductase or Complex III): this protein complex oxidizes ubiquinone, then passes those electrons through heme and Fe/S carriers to reduce cytochrome c
• Cytochrome c: this is a small, single subunit protein that is peripheral to the membrane. It shuttles electrons from complex III to complex IV using a heme group
• Cytochrome c oxidase (also called Complex IV): this protein complex oxidizes cytochrome c, passing those electrons through heme groups, then through an Fe/Cu center that reduces oxygen to water
• cytochrome c oxidase can hold O₂ and 4 electrons in those Fe, Cu atoms, so that O₂ is reduced to 2 H₂O all at once - this way no O₂^- superoxide radical is produced - this would be very reactive and destructive

The path of electrons from FADH₂:

• Succinate dehydrogenase (also called Complex II) in the citric acid cycle: this protein complex oxidizes succinate to form fumarate and to reduce FAD to FADH₂. FADH₂ is oxidized by this protein and the electrons are passed through Fe/S centers to reduce ubiquinone.
• Reduced ubiquinone passes its electrons to the cytochrome b-c₁ complex and the path continues as above

Oxidation of NADH/FADH₂ and electron transport drives the unidirectional transport of H⁺ from the matrix to the intermembrane space

• as two electrons are passed through the three complexes-each protein complex transports a H⁺ from the matrix (inside of mito) to the intermembrane space=they act as pumps to generate an electrochemical gradient for H⁺
• the electrons from the oxidation of NADH drive the transport of H⁺ at 3 sites during electron transport (finally being used to reduce O₂ to water)
• the electrons from the oxidation of FADH₂ drive the transport of H⁺ at only 2 sites since NADH dehydrogenase is bypassed (electrons are used to reduce ubiquinone directly)

H⁺ flow down the electrochemical gradient through another membrane-bound protein complex=F₀F₁ ATP synthase and drives the synthesis of ATP

• the ATP synthase protein has two parts=the F₀ part forms a pore in the membrane, allowing H⁺ to pass through, the F₁ part sits on top of the pore and synthesizes ATP from ADP + Pi as H⁺ passes through
• the use of the H⁺ electrochemical gradient to drive ATP synthesis by the ATP synthase was proposed by Peter Mitchell (in 1961) as the chemiosmotic model
• the pumping of H⁺ during electron transport is said to be coupled to ATP synthesis
• the ATP synthase can run in reverse and act as an ATPase and pump H⁺ across the membrane under certain circumstances
• compounds that disrupt the inner mito membrane to allow free flow of H⁺ (not through the ATP synthase, so no ATP synthesis), thus diffusing the electrochemical gradient are called uncouplers

The net maximum theoretical yield of ATP from glucose

• 2 ATP from glycolysis, 2 ATP from the citric acid cycle, 4 ATP from 2 FADH₂, and 30 ATP from 10 NADH= 38 ATP
• compare this to the 2 ATP from glycolysis & fermentation
• this represents an efficiency of close to 50% in the capture of energy (stored as ATP) from glucose (by comparing the free energy of glucose to the free energy of 38 ATP)
• this maximum yield is probably never attained, because not all of the energy is used for ATP synthesis-actually only yield about 30 ATP per glucose, because some energy is used to transport NADH, pyruvate, Pi, and ATP in and out of the mitochondria

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