Electron Transport Chain

Mitochondria Structure

Inner Membrane:

• Impermeable (except O₂, CO₂, & H₂O)

• Transport proteins for pyruvate, ATP, ADP, & P_i

Outer Membrane:

• Porous (porins)

• Permeable to molecules < 10 KDa

Inner Membrane Space (Cytosol):

• Water-soluble

How is NADH transported into mitochondrion matrix

Malate Aspartate Shuttle:

• Extra NADH in cytosol binds oxaloacetate & is converted to malate via malate dehydrogenase & is transported into the matrix where it is converted back to NADH & oxaloacetate via malate dehydrogenase

Glycerophosphate Shuttle

• Cytosolic NADH reduces DHAP to 3-PG via 3-phosphoglycerol dehydrogenase

• 3-PG is oxidized by flavoprotein dehydrogenase into DHAP & FAD is reduced into a matrix FADH₂ (2 e- enter ETC)

ETC Overview

Overall delta G = -218 kJ/mol

Making ATP = 30.5kJ/ mol

Complex 1 & 2 are independent of each other

ETC Complexes

Complex 1: NADH-Coenzyme Q Oxidoreductase

• Oxidation of NADH by CoQ

Succinate- Coenzyme Q Oxidoreductase

• Oxidation of FADH₂ by CoQ

Coenzyme Q- Cytochrome C Oxidoreductase

• Oxidation of CoQ by Cyt C

Cytochrome C Oxidase

• Oxidation of Cyt C by O₂

Electron Carriers

Flavin Mononucleotide (FMN)

• Inside Complex 1

• Can accept 1 or 2 e^- (semiquinol form)

• Derived from Vitamin B2

Fe-S Clusters

• Inside Complex 1,2,3

• Accepts 1 e^- at a time

• Fe²⁺ to Fe³⁺

CoQ (Ubiquinone)

• Inside the inner membrane

• Accepts 1 or 2 e^- at a time (semiquinol form)

• Mobile Electron acceptor (complexes 1,2,3)

• Fat-soluble

Hemes

• Inside intermembrane space

• Accepts 1 e^-

• Mobile electron acceptor (Cytochrome C)

• Water Soluble

Cu²⁺

• Inside Complex 4

• 1 e^- at a time

Complex 1 (NADH-CoQ Oxidoreductase)

NADH + UQ + H⁺ → NAD⁺ + UQH₂

Large membrane protein (980 KDa) w/ 44 subunits

Electron wire:

• NADH → FMN → 9 Fe-S clusters → UQ

Proton wire:

• Matrix → Intermembrane Space

• Protons bind to His/Lys & jump from residue to residue until they enter the intermembrane space

4 H⁺ are pumped for every 2 e^-

Complex 2 (Succinate Dehydrogenase/ Succinate-Coenzyme Q Oxidoreductase)

Succinate + UQ → Fumarate + UQH₂

Soluble protein in matrix w/ a transmembrane portion

Flavin protein:

• Binds [FAD}

3 Fe-S Clusters

• 2Fe-2S

• 4Fe-4S

• 3Fe-4S

Heme-B

• Stabilizes CoQ

Electron Flow:

• Succinate → FAD → Fe-S Clusters → CoQ

No Proton Flow

Complex 3 (Coenzyme Q- Cytochrome C Oxidoreductase)

UQH₂ + Cyt C (oxidized) → UQ + Cyt C (reduced)

419 kDa, homodimer

Each Monomer:

• 9 subunits

• 12 transmembrane helices

Electron Wire:

• Q Cycle

• Heme bH (high

• Heme bL (low)

• Heme c1

• 2Fe-2S

Proton Wire:

• 4 H⁺ are pumped from matrix to inter membrane space for every 2 e^-

Q Cycle

Occurs in the inner membrane

Q_o: Site for reduced CoQ

Q_i: Site for oxidized CoQ

First Half:

1. Reduced CoQ (from complex 1 or 2) binds at Q_o site

   1. Pumps 2 H⁺ into the intermembrane space

2. a & b

   1. CoQ converts to semiquinone form

      1. 1 e^- goes to Iron Sulfur Protein (ISP) then C₁ then Cytochrome C

   2. Semiquinone CoQ converts to oxidized form

      1. 1 e^- goes to bL then bH then oxidized CoQ in the Q_i site converting it to semiquinol form

3. Reaction:

   1. UQ + UQH₂ → UQ + UQH*

Second Half:

1. Reduced CoQ (from complex 1 or 2) binds at Q_o site

   1. Pumps 2 H⁺ into intermembrane space

2. a & b

   1. CoQ converts to seminquinol form

      1. 1 e^- is transported to Iron Sulfur Protein (ISP) then Heme c1 then Cytochrome C

   2. Semiquinol CoQ is converted to oxidized form

      1. 1 e- is transported to bL then bH then to the semiquinone CoQ in Q_i site (along with 2 H⁺ from the Matrix) converting it to reduced form

3. Total Reaction:

   1. 2H⁺ + 2 UQH₂ + UQ → 4H⁺ + UQH₂ + 2 UQ

   2. Simplified:

      1. 2 H⁺ + UQH₂ → 4H⁺ + UQ

Complex 4 (Cytochrome C Oxidase)

e^- reaches the terminal acceptor O₂

Cyt C is a soluble protein in the intermembrane space that binds Complex 4

410 kDa homodimer membrane protein

• 13 subunits, 10 transmembrane subunits, 28 membrane-spanning helices

4 e^- binding sites:

• Cu_A, Heme A, Heme A₃, Cu_B

2 H⁺ are pumped to the intermembrane space per 2e^-

Electron Wire:

• Cytochrome C → Cu_A → Heme A → Heme A₃ → Cu_B

  • Reduces Heme A3 & Cu_B

• Reduced Fe in Heme A3 & Cu_B bind O₂ forming a peroxide bridge

• Cytochrome C transfers 2 more e^- which pumps 2 more H⁺ from the Matrix which cleaves peroxide bridge

  • Fe in Heme A3 & Cu_B each gain a hydroxyl group

• Addition of 2 more H⁺ causes the release of 2 H₂O

• Reaction:

  • O₂ + 4H⁺ + 4e^- → 2H₂O

  • ½ O₂ + 2H⁺ + 2e^- → 1 H₂O

Proton Wire:

• D pathway: Aspartate

• K Pathway: Lysine

• Protons hop from 1 H₂O molecule to another

ETC Creates a Proton Gradient

Protons are pulled from the matrix to the intermembrane space

• Creates a concentration & electrical potential gradient which stores potential energy for use in oxidative phosphorylation

• Matrix → intermembrane space (endergonic)

• Intermembrane Space → Matrix (exergonic)

Inhibitors of ETC

Complex 1:

• Rotenone, Amytal, Demerol

Complex 2:

• Atpenins

ATP Synthase:

• Oligomycin

Complex 3:

• Stigmatellin, Antimycin A

Complex 4:

• Cyanide Azide, Carbon Monoxide

Uncoupler:

• 2,4 dinitrophenol (DNP)

  • Leaks protons from intermembrane space back to matrix (reduces proton gradient & uncouples ETC w/ oxidative phosphorylation)

ATP Synthase

F₁ (a3b3)

• Synthesizes ATP

• in matrix

• a & b subunits

  • a subunits (inactive)

  • b subunits (active)

    • 3 conformations

      • Open (releases ATP)

      • Loose (binds ADP & P_i)

      • Tight (Clamps down on ADP & P_i & catalyzes formation of ATP)

F₀

• Proton flow

• Embedded in innermembrane

• transfers H⁺

• a, b₂, c_8-15 subunits

  • b₂ links F₀ to F₁

• C Subunit

  • 2 alpha helices

    • 1 has Asp61

  • Can become protonated via binding H⁺

    • Protonation of D61 causes conformation change that pushes against the a subunit causing F₀ to rotate

Proton Movement & F₀ Rotation

• F₀ Rotation causes conformational changes in F₁

  • AB subunits alter 3 conformations

• For every full turn of the transmembrane ring (C ring) complex, 3 ATP are released

  • Humans have 8 C subunits

    • 8H⁺ → 3 ATP

• H⁺ enters the F₀ a subunit then binds to Asp 61 in C ring

  • Protonated Asp 51 pushes against a subunit rotating C ring

• H⁺ exit a subunit & enters the matrix

ATP Translocase

ATP is produced in matrix and must be transported to cytosol

ADP in cytosol must be transported to matrix to be converted to ATP

Matrix orientation: Binds 1 ATP & releases 1 ADP -1 H⁺

Cytosol orientation: Binds 1 ADP & releases 1 ATP -1 H⁺

ATP Yield

1 NADH (Complex 1): 10 H⁺

1 FADH₂ (Complex 2): 6 H⁺

ATP Synthase + ATP Translocase:

• Cost:

  • 8 H⁺ per turn

  • 3 H⁺ per translocaton

• Yield:

  • 3 ATP

Therefore:

1 FADH₂ = 1.5 ATP

1 NADH = 2.5 ATP

Regulation of Oxidative Phosphorylation

• Most steps in ETC & oxidative phosphorylation are reversible

  • Complex 4 is the only irreversible step (rate-limiting)

• Main regulators: ATP/ADP & NADH/NAD⁺ ratios

Supply:

• Rate of Complex 4 is dependent on level of reduced cytochrome C (depends on ratio of NADH/NAD⁺)

  • More NADH = faster rate

Demand:

• During rest, ATP/ADP ratio is high (low demand)

  • Catabolic processes slow, reduced e^- supply to ETC

• During exercise, ATP/ADP ratio is low (high demand)

  • Catabolism increases, translocator transports ADP to matrix to supply ATP Synthase w/ substrate

  • ATP Synthase dissipates gradient & increases flux through ETC