Lecture 9 - respiration/oxidative phosphorylation

18.1-18.3

Reduced fuels

carbohydrates, lipids, amino acids

• reduce NAD⁺ to NADH or FAD to FADH₂

Oxidative phosphorylation

• energy from NADH and FADH₂ used to make ATP

  • electrons are passed to proteins in the respiratory chain

  • energy of oxidation phosphorylates ADP

• inner mitochondrial membrane

  • cristae in mitochondria increase surface area

outer mitochondrial membrane

freely permeable to small molecules and ions

inner mitochondrial membrane

• impermeable to most small molecules and ions, including H⁺

• contains:

  • respiratory electron carriers (Complexe I-IV)

  • ADP-ATP translocase

  • ATP synthase (F₀F₁)

  • Other membrane transporters

mitochondrial matrix

contains:

• pyruvate dehydrogenase complex

• citric acid cycle enzymes

• fatty acid β-oxidation enzymes

• amino acid oxidation enzymes

• DNA, ribosomes

• many other enzymes

• ATP, ADP, P_i, Mg²⁺, Ca²⁺, K⁺

• many soluble metabolic intermediates

mitochondria structure

proton-motive force

• protons transported uphill against electrochemical gradient during electron transport

• each NADH oxidized drives 10 H⁺ which yields ~3 ATP molecules

Redox potentials

Δ𝜺 = Δ𝜺 (acceptor ½ rxn) - Δ𝜺 (donor ½ rxn)

ΔG_redox = -nFΔ𝜺

Half reactions

Aⁿ⁺_ox + ne^- ⇆ A_red

Δ𝜺_{(A rxn)} = Δ𝜺° - RT/nF(ln [A_red/A_ox])

• positive: reduction favorable; strong oxidant

Electron transfer cofactors

• quinone ⇆ ubiquinol

  • quinone accepts 2 e^-, 2 H⁺ to give ubiquinol

  • ubiquinol: freely diffuses in membrane, carrying e^- with H⁺ across membrane

• Heme

  • can be either ferric (Fe³⁺, oxidized) or ferrous (Fe²⁺, reduced)

• Fe/S clusters

  • transfer 1 e^- at a time

Complex I: NADH: Ubiquinone Oxidoreductase

• 40 subunits

  • complete assembly seen in EM

  • soluble part of bacterial protein crystallized

• NADH binding site: matrix side

  • bound flavin mononucleotide (FMN) accepts 2 e^- from NADH

• series of Fe/S centers pass e^- from FMN to ubiquinone binding site

• per NADH, 4 H⁺ transferred from matrix (N) to inter-membrane space (P)

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

Complex II: Succinate Dehydrogenase

• FAD accepts two e^- from succinate

• FADH2 e^- passed via Fe/S centers → ubiquinone, becomes reduced QH₂

Succinate + FAD + Q → Fumarate + FAD + QH₂

Complex III: Cytochrome bc₁

• uses 2 e^- from QH₂ to reduce two molecules of cyt c via 2 step cycle

• 4 H+ transported across membrane per 2 e^- that reach cyt c

• step 1: QH₂ give 2 out of 4 H+

• step 2: regeneration of QH₂ gives 2 H⁺ from matrix

cytochrome c

• soluble heme-containing protein in intermembrane space

• carries a single e^- from the cytochrome bc₁ complex to cytochrome oxidase

• small (12 kD), binds/unbinds quickly from Complexes III and IV

• can exit the mitochondrion as part of the apoptotic cell death pathway

Complex IV: Cytochrome c Oxidase

• 4 cyt c oxidized

  • O₂= ultimate e^- sink

• 4e^- and 4 H+ from matrix used to reduce O₂ molecule into H₂O

• 4 more H+ pumped to inter-membrane space

4 cyt c + O₂ +*H+_N → 4 cyt c(ox) + 2 H₂O + 4 H⁺_P

Cytochrome c Oxidase mechanism

• 2 heme groups, 2 Cu centers

• Heme/Cu_B center reduces O₂ to H₂O

ATP synthesis energy

• energy needed to phosphorylate ADP provided by flow of protons down electrochemical gradient (PMF)

ATP synthase structure

• two separable domains

• F₁ (ATP synthase)pocket binds ADP and stabilizes ATP formation

• F₀ (transmembrane proton channel) uses H+ translocation to force ATP out of binding pocket, bind new ADP + P_i

ATP synthase conformational change

• proton binding causes structural change in c unit, causing rotation of central shaft γ

• Rotation drives α/β conformational changes, leading to ATP dissociation

Oxidative Phosphorylation with Succinate (FADH2)

Complex I: NADH + Q + 5 H+_N → NAD⁺ + QH₂ + 4 H+_P

Complex II (FADH2): Succinate + Q → Fumarate + QH2

Complex III: 4 cyt c(ox) + 2 QH₂ + 4H⁺_N → 4 cyt c(red) + 2 Q + 8H⁺_P

Complex IV: 4 cyt c(red) + O₂ + 8H⁺_N → 4 cyt c(ox) + 2H₂O + 4H⁺_P

ATP Synthase + P_i Translocase: 4 ADP + 16H⁺_P + 4P_i(p) → 4ATP + 16H⁺_N

Net: 4 ADP + 4P_i(p) + NADH + H⁺_N + Succinate + O₂ → 4 ATP + NAD⁺ + Fumarate + 2 H₂O

Oxidative Phosphorylation per Matrix NADH

Complex I: NADH + Q + 5 H+_N → NAD⁺ + QH₂ + 4 H+_P

Complex III: 2 cyt c(ox) + QH₂ + 2H⁺_N → 2 cyt c(red) + Q + 4H⁺_P

Complex IV: 2 cyt c(red) + 0.5 O₂ + 4H⁺_N → 2 cyt c(ox) + H₂O + 2H⁺_P

ATP Synthase + P_i Translocase: 2.5 ADP + 10H⁺_P + 2.5P_i(p) → 2.5ATP + 10H⁺_N

Net: 2.5 ADP + 2.5P_i(p) + NADH + H⁺_N + 0.5 O₂ → 2.5 ATP + NAD⁺ + H₂O

Oxidative Phosphorylation per Cytosolic NADH

Complex NAHDH: NADH + H⁺ + Q → NAD⁺ + QH₂

Complex III: 2 cyt c(ox) + QH₂ + 2H⁺_N → 2 cyt c(red) + Q + 4H⁺_P

Complex IV: 2 cyt c(red) + 0.5 O₂ + 4H⁺_N → 2 cyt c(ox) + H₂O + 2H⁺_P

ATP Synthase + P_i Translocase: 1.5 ADP + 6H⁺_P + 1.5P_i(p) → 1.5ATP + 6H⁺_N

Net: 1.5 ADP + 1.5P_i(p) + NADH + H⁺_N + 0.5 O₂ → 1.5 ATP + NAD⁺ + H₂O

Oxidative Phosphorylation per FADH₂

Complex II (FADH2): Succinate + Q → Fumarate + QH2

Complex III: 2 cyt c(ox) + QH₂ + 2H⁺_N → 2 cyt c(red) + Q + 4H⁺_P

Complex IV: 2 cyt c(red) + 0.5 O₂ + 4H⁺_N → 2 cyt c(ox) + H₂O + 2H⁺_P

ATP Synthase + P_i Translocase: 1.5 ADP + 6H⁺_P + 1.5P_i(p) → 1.5 ATP + 6H⁺_N

Net: 1.5 ADP + 1.5P_i(p) + 2H⁺_N + 0.5 O₂ → 1.5 ATP + H₂O

ATP yield

• 4 H+ per ATP synthesized, 3 per turn of ATP synthase, 1 per PO4 import

• 10 H+ per NADH when e- donated to complex I, so 2.5 ATP per matrix NADH.

• 6 H+ per NADH when e- donated to quinone (4 H+ pumped by complex III, 2 H+ by complex IV), 1.5 ATP per cytosolic NADH.

• 6 H+ per FADH2 when e- donated to quinone (Succinate Dehydrogenase), 1.5

  ATP per FADH2

PMF-Driven energy systems: Common origin