Electron Transport Chain

the final stage of aerobic respiration

converting reducing power to ATP

substrate level phosphorylation

• direct transfer of phosphate from a high-energy substrate to ADP

• does not require oxygen

oxidative phosphorylation

ATP synthesis driven by electron transport and the proton gradient

location of oxidative phosphorylation

inner mitochondrial membrane

oxidative phosphorylation process

1. electrons from NADH/FADH2 flow through ETC complexes

2. energy released pumps H+ across membrane

3. H+ gradient (proton-motive force) drives ATP synthase

4. ATP synthesized as H+ flows back into matrix

oxidative phosphorylation yield per NADH

2.5 ATP

oxidative phosphorylation yield per FADH2

1.5 ATP

mitochondrion structure

inner and outer membranes, cristae (folds), matrix (low [H+]), and intermembrane space (high [H+])

inner mitochondrial membrane and cristae

location of ETC complexes and ATP synthase

mitochondrial structure: outer membrane

• permeable to small molecules (<5 kDa)

• contains porins

mitochondrial structure: intermembrane space (IMS)

• high [H+] - acidic

• similar composition to cytosol

• contains cytochrome c

mitochondrial structure: matrix

• low [H+] - alkaline

• contains TCA cycle enzymes

• contains PDH complex

• contains mtDNA and ribosomes

Why is the inner membrane’s impermeability in mitochondria essential;?

it maintains the proton gradient

mitochondrial structure: Inner membrane

• impermeable to most molecules

• contains ETC complexes I-IV

• contains ATP synthase

• highly folded into cristae (increase surface area)

Complex I

NADH-ubiquinone oxidoreductase (NADH dehydrogenase)

Complex II

succinate dehydrogenase

Complex III

ubiquinone-cytochrome c oxidoreductase

Complex IV

cytochrome c oxidase

mobile carries

ubiquinone (CoQ)

cytochrome c

ubiquinone (CoQ, coenzyme Q)

lipid-soluble, (moves within membrane)

accepts e- from complex I and II

delivers e- to complex III

cytochrome c

water-soluble, in IMS

shuttles e- from complex III to complex IV

NADH-Ubiquinone Oxidoreductase function

transfer e- from NADH → CoQ

pumps 4 H+

Succinate Dehydrogenase

transfer e- from FADH2 → CoQ

no H+ pumping

Ubiquinone-Cytochrome c function

transfer e- from CoQ → Cytochrome c

pumps 4 H+

Cytochrome c Oxidase function

transfer e- from cytochrome c → O2 (reduc O2 to H2O)

pumps 2 H+

Electron flow through ETC

NADH → complex I → CoQ → complex III → complex IV → O2

FADH2 → complex II^(CoQ)

overall reaction for ETC

NADH + H+ + ½ O2 → NAD+ + H2O

-220 kJ/mol (highly exergonic)

What is the key to ATP synthesis

chemiosmosis

chemiosmosis

the movement of ions across a selectively permeable membrane, down their electrochemical gradient, coupled to ATP synthesis

The Chemiosmotic Hypothesis

1. electron transport pumps H+ from matrix to IMS

2. this creates a proton-motive force (Δp):

   • chemical gradient (ΔpH)

   • electron gradient (Δψ)

3. H+ flows back through ATP synthase

4. This flow drives ATP synthesis

chemical gradient (ΔpH)

more H+ outside

electron gradient (Δψ)

positive outside, negative inside

proton-motive force

Δp = Δψ - (2.3RT/F) ΔpH ≈ 200 mV

uncouplers

• molecules that dissipate the H+ gradient

• electron transport continues, but no ATP is made

• energy is released as heat

characteristics of uncouplers

• weakly acidic (can accept/donate H+)

• hydrophobic (can cross membrane)

• Ex: 2,4-dinitrophenol, dicumarol, and FCCP

What results from the ETC complexes pumping H+ out of the IMM

a H+ gradient that drives ATP synthesis

chemical uncoupler (U^-)

• catalyzes a protonation/deprotonation cycle that facilitates a proton influx, which does not generate ATP

• act as proton shuttles, bypassing ATP synthase and releasing energy as heat

ETC complex I

NADH-Ubiquinone Oxidoreductase (NADH Dehydrogenase, NADH-CoQ Reductase)

What is the function of NADH-CoQ reductase

transfers electrons from NADH to CoQ

NADH-CoQ Reductase structure

largest ETC complex subunits: 44 subunits, 980 kDa

L-shaped: membrane arm + matrix arm

NADH-CoQ Reductase prosthetic groups

• 1 FMN (Flavin Mononucleotide)

• 6-8 Fe-S clusters

NADh-CoQ Reductase electron path

NADH → FMN → Fe-S clusters → CoQ (ubiquinone)

NADH-CoQ Reductase Mechanism

1. NADH binds and transfers 2 e- to FMN

   • FMN → FMNH2

2. e- pass through Fe-S clusters one at a time

   • Fe³⁺ <=> Fe²⁺ (one e- transfers)

3. e- reduce ubiquinone (Q) to ubiquinol (QH2)

   • Q → QH (semiquinone) → QH2

NADH-CoQ Reductase coupling to proton pumping

• conformational changes drive H+ translocation

• 4 H+ pumped per NADH oxidized

Whats the point of the semiquinone intermediate? (QH•)

allows CoQ to accept e- one at a time from Fe-S clusters

What are the most common forms of Fe-S clusters found in biological systems

• 2Fe-2S

• 3Fe-4S

• 4Fe-4S

Fe-S clusters

one electron carriers

transfers electrons one at a time

Fe³⁺ + e^- <=> Fe²⁺

What is the key point of Fe-S clusters

bridges the gap between 2-electron carriers (NADH, FADH2) and 1-electron carriers (cytochromes)

Rotenone

• natural isoflavonoid from plant roots

• potent complex I inhibitor

• blocks e- transfer from Fe-S to CoQ

ETC Complex II

Succinate-CoQ Reductase (Succinate Dehydrogenase)

What is the only enzyme in both TCA cycle and ETC

Succinate Dehydrogenase

Succinate Dehydrogenase reaction

Succinate + Q → Fumarate + QH2

Succinate Dehydrogenase structure

4 subunits (smallest ETC complex)

succinate dehydrogenase prosthetic groups

• FAD

• Fe-S clusters

Succinate dehydrogenase electron path

Succinate → FAD → Fe-S → CoQ (QH2)

What is the critical difference between Succinate Dehydrogenase and NADH-CoQ Reductase

succinate dehydrogenase has no proton pumping

Why does succinate dehydrogenase not have any H+ pumping

• less energy released (ΔG less negative)

• FADH2 has higher reduction potential than NADH

• this is why FADH2 yields less ATP than NADH

ETC Complex III

Q-Cytochrome C Oxidoreductase (Cytochrome bc1 Complex)

Q-Cytochrome C Oxidoreductase function

transfer e- from CoQ to cytochrome c

Q-Cytochrome C Oxidoreductase Structure

• dimeric complex (functions as a dimer)

• contains: 3 heme groups + 1 Fe-S cluster (Rieske)

Q-Cytochrome C Oxidoreductase structure

• Heme bL and bH (cytochrome b)

• Heme c1 (cytochrome c1)

• [2Fe-2S] Rieske iron-sulfur protein

Q-Cytochrome c Oxidoreductase overall reaction

QH2 + 2 Cyt c (ox) + 2 H+ (matrix) → Q + 2 Cyt c (red) + 4 H+ (IMS)

Q-Cytochrome c Oxidoreductase proton pumping

4 H+ per 2 e-

Q-Cytochrome c Oxidoreductase electron path

QH2 → Q → Fe-S → Cyt c

complex IV

cytochrome c Oxidase

cytochrome c oxidase function

transer e- from Cyt c to O2 (final acceptor)

Cytochrome c oxidase proton movement

• 4 H+ used to reduce O2 to H2O (“chemical” protons)

• 4 H+ pumped to IMS (“pumped” protons)

• total: 8 H+ consumed from matrix per O2

cytochrome c oxidase structure

13 subunits in mammals

cytochrome c oxidase prosthetic groups

• 2 Heme groups (a and a3)

• 2 Copper centers (CuA and CuB)

cytochrome c oxidase electron path

Cyt c → CuA → Heme a → Heme a3-CuB → O2

complex IV inhibitors

• Cyanide (CN-)

• Carbon Monoxide (CO)

• Sodium Azide (N3-)

Why are lethal poisons inhibitors to complex IV

• they block the final step of e- transport

• all upstream complexes back up

• no ATP production

• cells die within minutes (especially brain, heart)

Treatments for cyanide poisoning

• nitrates (form methemoglobin to bind CN-)

• thiosulfate (converts CN- to thiocyanate)

• hydroxocobalamin (binds CN-)

The proton Motive force

drives ATP synthesis

Two components of proton-motive force

1. chemical gradient (ΔpH): 0.75 pH units

2. electrical gradient (Δψ): 150-200 mV

Two main subcomplexes F0 and F1. Which are the components?

1. Rotor

2. Headpiece

   1. Stator

Rotor

made up of lambda, gamma and epsilon subunits, and the c-subunit ring, which function as a single unit, rotating as H+ enter and exit the ring

Headpiece

containing the hexameric alpha 3 beta 3, site of ATP synthesis in the intact complex

Stator

the alpha subunit embedded in the membrane, has 2 half-channels for H+ to enter and exit the F0 component, and a stabilizing arm made up the b, d, h, and OSCP subunits

Complex V

ATP Synthase (the molecular motor)

OSCP (Oligomycin Sensitivity-Conferring Protein)

a single subunit of mitochondrial F0F1-ATP synthase

Rotational Catalysis

H+ flow drives ATP synthesis via rotational catalysis

Rotational Catalysis: Three beta subunit conformations

Open (O): empty - releases ATP

Loose (L): ADP + Pi bind - binds substrates loosely

Tight (T): ATP is bound - catalyzes ATP formation

Rotational catalysis stoichiometry

3-4 H+ per ATP

How does Rotational Catalysis work

1. H+ flows through Fo, rotating the c-ring

2. c-ring rotation turns the gamma subunit

3. gamma rotation causes conformational changes in beta subunits

4. each beta cycles: O → L → T → O

5. one full rotation (360) = 3 ATP synthesized

Complex I inhibitors

• rotenone

• piercidin A

• amytal

complex III inhibitors

antimycin A

Complex IV inhibitors

cyanide

CO

azide

ATP synthase inhibitors

oligomycin

Uncouplers inhibitors

DNP

FCCP

(dissipate gradient)

what is the point of ATP-ADP translocase

getting ATP out of mitochondria

ATP-ADP translocase mechanism

• antiporter: exchanged ATP^4- (out) for ADP^3- (in)

• electrogenic: net export of 1 negative charge

• driven by membrane potential (Δψ)

Why is ATP-ADP translocase favorable

• matrix is negative relative to IMS

• exporting ATP^4- (more negative) is favored

• importing ADP^3- (less negative) is favored

ATP-ADP translocase energy cost

• 1 H+ equivalent per ATP exported

• this is factored into ATP yield calculations

The NADH problem (How do cytosolic e- enter mitochondria?)

• glycolysis produces 2 NADH in the cytosol

• complex I is in the inner mitochondrial membrane

• the inner membrane is impermeable to NADH

What is the solution to the NADH problem (How do cytosolic e- enter mitochondria?)

Shuttle systems

• transfer e- (not NADH itself) into mitochondria

• regenerate NAD+ in cytosol to keep glycolysis running

What are the two main shuttles to fix the NADH problem

1. glycerophosphate shuttle

2. malate-aspartate shuttle

glycerophosphate shuttle

• faster but less efficient

• yields 1.5 ATP per cytosolic NADH

• location: muscle, brain (where speed matters)

malate-asoartate shuttle

• slower but more efficient

• yields 2.5 ATP per cytosolic NADH

• location: primarily in liver, kidney, heart

Key Features of glycerophosphate shuttle

• irreversible

• fast and efficient at low NADH

• yields only 1.5 ATP per cytosolic NADH (e- enter at CoQ level, bypassing complex I)