10 Oxidative Phosphorylation 2014-2015

Oxidative Phosphorylation

- Glycolysis + TCA cycle produces very little ATP directly (substrate level): Glycolysis – 2 ATPs and TCA cycle – 2 ATPs per glucose

- The rest of the energy is stored in the reduced cofactors: 10 NADH + 2 FADH2 per glucose

- Oxidation of the reduced co-factors releases energy:

(G' = -220 kcal/mol)

(G' = -182 kcal/mol)

- Consider that: C6H12O6 + 6 O2 6 CO2 + 6 H2O (G' = -2870 kcal/mol)

FADH2 + 1/2 O2 FAD + H2O

NADH + H+ + 1/2 O2 NAD+ + H2O

- Thus, the reduced co-factors from oxidation of 1 mole of glucose store 10 X 220 + 2 X 182 = 2564 kJ of energy, accounting for about 90% of the total realizable energy (under standard conditions)

NAD+/NADH and NADP+/NADPH

- Diffusible 2 electron carriers - Accept or donate 1 hydride (H-) ion (1 proton and 2 electrons)

FAD/FADH2 and FMN/FMNH2 - Bound in enzymes as prosthetic groups - 2 electron-carriers - May transfer 1H+ + 1e- at a time

The mitochondrion - Aerobic oxidation of biomolecules

Alligator jaw muscle - white muscle Flight muscle - red muscle

Electron transport chain (ETC) in mitochondria

Four integral membrane protein complexes:

Electron transfer reactions

- The driving force is expressed as a difference in the standard redox potentials of the components, and this is equivalent to the change in free energy:

G’ = -nF E' n = no. of e- transferred F = 96.4 kJ mol-1 V-1

E‘ = EA' - ED'

- Electron flow is favorable from donors of low potential (i.e. more negative E values) to acceptors of high potential.

Complex I

Complex II Complex III Complex IV

NADH dehydrogenase (Complex I)

• Cofactors (electron carriers): riboflavin coenzyme FMN, iron-sulfur clusters, • transfers electrons from NADH to unbound ubiquinone (Q) • the unbound ubiquinol (QH2) carries electrons diffuses through inner membrane to cytochrome bc1 complex (Complex III)

Overall: NADH + H+ + Q NAD+ + QH2

Proton pumping

(matrix)

(intermembrane space)

FMN

- Protein-bound co-factor - Accepts/donates 1 hydride ion

(1 electron + 1 proton) at a time - 2 electron-carrier

Single electron carriers: Fe2+ Fe3+ + e-

4Fe-4S: 2Fe-2S:

- only the inorganic S is counted in the designation - each Fe is always coordinated by 4 S - 4 cysteine residues from the protein also contribute 4S to the cluster - it is also posible to have a single Fe coordinated by 4 cysteine residues

Iron-sulfur (Fe-S) clusters

Protein

Protein

Q (or CoQ)/QH2

- Diffusible through inner membrane

- Accepts/donates 1 electron + 1 proton at a time

- A carrier of both electrons (2) and protons (2)

• The only membrane-bound enzyme in TCA cycle • Contains an internal chain of electron transfer cofactors • No proton pumping in Complex II

Succinate dehydrogenase (Complex II)

(matrix)

(intermembrane space)

Cytochrome bc1 complex (Complex III) • Electron carriers: Fe-S clusters, cytochrome b and cytochrome c1

• Transfers electrons from ubiquinone to cytochrome c

• QH2 + 2 cyt c1 (oxidized) + 2 H+ Q + 2 cyt c1 (reduced) + 4H+

• Cytochrome c is a water-soluble protein coenzyme in the intermembrane space.

• Complex III pumps 4 protons per 2 electrons transferred

(Iron-sulfur protein)

(matrix)

(intermembrane space)

Cytochromes - Protein coenzymes - Heme as a tightly bound co-factor - Heme is a tetrapyrrole coordinating a single

atom of Fe(II/III) - Single electron-carrier (Fe2+ Fe3+ + e-)

Cytochrome a - Contains heme A - Membrane-bound Cytochrome b

- Contains heme B - Membrane-bound

Cytochrome c - Contains heme C - Membrane-bound or

diffusible

The Q cycle in Complex III

• Electron carriers: 2 Cu ions and 2 heme A groups (cytochrome a proteins)

• Transfers electrons from cytochrome c to oxygen

• 4 cytochrome c (reduced) + 8H+ in + O2

4 cytochrome c (oxidized) + 4H+ out + 2 H2O

• The redox centers only transfer electron one at a time

• Incompletely reduced intermediates (e.g. hydrogen peroxide and hydroxyl free radicals) remain tightly bound until complete reduction to water.

Cytochrome oxidase (Complex IV)

(x2)

(matrix)

(intermembrane space)

Electron transport and proton pumps

inner membrane

• A protein gradient is then established across the inner membrane

• The energy stored in the gradient, proton-motive force, has two components: chemical potential energy and electrical potential energy

• The electrochemical energy released when protons flow spontaneously down the gradient can be used to drive the synthesis of ATP from ADP and Pi

Proton gradient

(matrix) (intermembrane space)

ATP synthesis by ATP synthase • ATP synthase: F0F1 complex in the inner membrane

• Protons flow through the F0 unit down the gradient

• ATP is synthesized by the F1 unit (ATPase) from ADP and Pi

• For every 2 electrons donated by NADH, 2.5 ATPs are synthesized.

• For every 2 electrons donated by FADH2, 1.5 ATPs are synthesized.

NADH + H+ NAD+

(1) Glycerol 3-P shuttle - Skeletal muscle and brain

(DHAP)

Shuttle systems for NADH generated in cytoplasm

• Inner mitochondrial membrane is impermeable to NADH

• Cytosolic NADH is shuttled indirectly into the mitochondria as reducing equivalents

• Reducing equivalents are molecules that can be transported into the mitochondria

Complex III

Complex IV

(2) Malate-aspartate shuttle - Liver, kidney, and heart

(Complexes I, III, IV)

Net profit of aerobic metabolism

If we start from glucose:

- Glycolysis to 2 pyruvate yields: 2 ATP 2 NADH

- Conversion of 2 pyruvate to 2 acetyl-CoA: 2NADH

- Oxidation of 2 acetyl-CoA in the TCA cycle: 20 ATP

Net yield = __________ ATP for complete oxidation of glucose to CO2

Cytosol

2 X 2.5 (malate-aspartate shuttle) = 5 ATP or 2 X 1.5 (glycerol 3-P shuttle) = 3 ATP

Mitochondria = 2 X 2.5 = 5 ATP

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