Electron Transport Chain/Respiratory Chain Proton gradient formed Four large protein complexes...

Electron Transport Chain/Respiratory Chain

Proton gradient formed

Four large protein complexes

Mitochondria localized Energetically favorable electron flow

Mitochondrion Inner Membrane

Respiration site

Surface area for humans ca. 3 football fields

Highly impermeable (no mitochondrial porins)

Matrix and cytoplasmic sides

Standard Reduction Potentials

ΔG˚΄ = -nF Δ E˚΄ F = 96,480 J mol-1 V-1

Favorable Electron Flow: NADH to O2

Net electron flow through electron transport chain:

½O2 + 2H+ + 2e- H2O ΔE˚΄ = + 0.82V

NAD+ + H+ + 2e- NADH ΔE˚΄ = - 0.32V

Subtracting reaction B from A:

½O2 + NADH + H+ H2O + NAD+ ΔE˚΄ = + 1.14V

ΔG˚΄ = -220 kJ mol-1

Electron Transport Energetic’s

Electron Transport Chain Components

Protein complexes:

I.NADH-Q reductase

II.Succinate dehydrogenase

III.Cytochrome C reductase

IV.Cytochrome C oxidase

Bridging components:

Coenzyme Q and Cytochrome C What is the driving force for this electron flow?

Coupled Electron-Proton Transfer Through NADH-Q Oxidoreductase

FMN bridges: NADH 2 e- donor with FeS 1 e- acceptor

L-shaped Complex I

Overall reaction:

NADH + Q + 5H+ NAD+ + QH2 + 4H+

Coupled Electron-Proton Transfer Through NADH-Q Oxidoreductase

H+ movement with 1 NADHIron-sulfur clusters (a.k.a.

nonheme-iron proteins)

2Fe – 2S or 4Fe – 4S complexes

NADH-Q Oxidoreductase (Complex I) Structure

Largest of respiratory complexes

Mammalian system contains 45 polypeptide subunits; 8 Fe-S complexes; 60transmembranehelices

Different Quinone (Q) Oxidation States

QH2 generated by complex I & II

Membrane-bound bridging molecule

Overall reaction:

QH2 + 2Cyt Cox + 2H+ Q + 2Cyt Cred + 4H+

X

Oxaloacetate Enzyme Regeneration from Succinate

• Succinate Dehydrogenase

• Fumerase

• Malate Dehydrogenase

Pathways that Contribute to the Ubiquinol Pool Without Utilizing Complex I

Alternative Q-Cycle Entry PointsComplex I

Complex II (citric acid cycle)

Glycerol 3-phosphate shuttle

Fatty acid oxidation (electron-transferring-flavoprotein dehydrogenase)

Electron-Transport Chain Reactions in the Mitochondria

The Q CycleElectron transfer to Cytochrome c Reductase via 3 hemes and a Rieske iron-sulfur center Overall reaction:

QH2 + 2Cyt Cox + 2H+

Q + 2Cyt Cred + 4H+

ISP – iron sulfur protein

The Q Cycle

Cytochrome c Oxidoreductase StructureIntermembrane sideHeme-containing homodimer

with 11 subunit monomers

Functions to:

• Transfer e- to Cyt c

• Pump protons into the intermembrane space

Matrix side

Cytochrome c Oxidase: Proton Pumping and O2 Reduction

Cytochrome c Oxidase: O2 Reduction to H2OReaction shown:2Cyt Cred + 2H+ + ½ O2

2Cyt Cox + H2O

Overall reaction:

2Cyt Cred + 4H+ + ½ O2

2Cyt Cox + H2O + 2H+

Cytochrome c Oxidase

O2 to H2O reduction site

Intermembrane space

Matrix

Oxygen requiring step

13 subunits; 10 encoded by nuclear DNA

CuA/CuA prosthetic group positioned near intermembrane space

Cytochrome c Oxidase

Electron-Transport Chain Reactions in the Mitochondria

Electron-Transport Chain Reactions in the Mitochondria

Mitochondrial Electron-Transport Chain Components

ATP Synthesis via a Proton Gradient

The two major 20th century biological discoveries:

DNA structure andATP synthesis

ATP-Driven Rotation in ATP-Synthase: Direct Observation

γ rotation with ATP present

With low ATP 120-degreeIncremental rotation

Glass microscope slide

ATP Synthase with a Proton-Conducting (F0) and Catalytic (F1) Unit

Matrix side

Intermembrane side

F1 matrix unit contains 5 polypeptide chain types (α3, β3, γ, δ, ε)

Proton flow from intermembrane space to matrix

Matrix side

ATP-Synthase with Non-Equivalent Nucleotide Binding Sites

Side view

F1 contains:

α3, β3 heximeric ring and γ, ε central stalk

Central stalk andC-ring form therotor andremainingmolecule is the stator

Top view

γ-Rotation Induces a Conformational Shift in the β Subunits

Each β subunit interacts differently with the γ subunit

ATP hydrolysis can rotate the γ subunit

Proton Flow Around C-Ring Powers ATP SynthesisSubunit C Asp protonation favors movement out of hydrophylic Subunit a to membrane region

Deprotonation favors Subunit a movement back in contact with Subunit a

Proton Motion Across the Membrane Drives C-Ring Rotation

C-Ring Tightly Linked to γ and ε Subunits

C-ring rotation causes the γ and ε subunits to turn inside the α3β3 hexamer unit of F1

Columnar subunits (2 b) with δ prevent rotation of the α3β3 hexamer unit

What is the proton to ATP generation ratio?

Mitochondrial ATP-ADP Translocase

Net movement down the concentration gradient for ATP (out of matrix) and ADP (into matrix)

No energy cost

Mitochondrial Transporters for ATP Synthesis

Net movement against the concentration gradient for Pi (into matrix) and charge balance -OH (out of matrix)

Proton gradient energy cost

ATP Yield With Complete Glucose Oxidation

Heat Generation by an Uncoupling Protein UCP-1Brown adipose tissue rich in mitochondria for heat generation

Pigs nest, shiver, and have large litters to compensate for lack of brown fat

ATP Synthesis Chemical Uncoupling

What physiological effect might DNP have in humans?

Electron Transport Chain Inhibitors

Toxins (e.g. fish and rodent poison rotenone)

Site specific inhibition for biochemical studies

What impact will rotenone have on respiration (O2 consumption)?

Proton Gradient Central to Biological Power Transmission

Problems: 13, 21, 23, 31, 33