ETC & MITOCHONDRIAL OXIDATIVE PHOSPHORYLATION

Energy Metabolism

Acetyl CoA

Common product of macromolecules

Recycle NADH/H+ & FADH2

Mitochondria

Redox reaction = transfer of electron to O2

Cytosol to mitochondria

Respiratory Chain / Electron Transport Chain

Produces ATP

Oxidative Phosphorylation

Free energy trapped as high energy phosphate

Proton Gradient

Stored potential energy; basis of coupling in oxidative phosphorylation / chemiosmotic coupling

Reducing equivalents of ETC

NADH: cytosol to mitochondrial matrix

FADH2: mitochondrial matrix to inside mitochondria

Heat Production / Proton leak / Mitochondrial uncoupling

Protons enter mitochondrial matrix w/o contributing to ATP synthesis

Unharnessed potential energy of proton electrochemical gradient = heat

Components and Enzymes in Mitochondrial Oxidative Phosphorylation

Complexes

II- Succinate Dehydrogenase

III- Q- Cyt C Oxidoreductase

I - NADH-Q Oxidoreductase

IV- Cyt C Oxidase

Enzyme

Fe-S Centers, FMN

Prosthetic Groups

Coenzyme Q

Acceptor

No. of H+ transported

4

FAD, Fe-S Protein

Coenzyme Q

0

Fe-S Protein, Cytochrome I

Cytochrome C

4

Copper, Heme

Oxygen

2

1. Flavoproteins

2. Fe-S

3. Mobile Proteins

4. Enzymes

Accepts 1 electron = forms semiquinone intermediate

Oxidized FMN/FAD can be reduced

Complex 1

Complex 2

FMN --> FMNH2

FAD --> FADH2

Single electron transfer

Redox between Fe2+ & Fe3+

Coenzyme C

Coenzyme Q

Transfer 1 e- from Complex 3 to 4

Heme undergoes redox

Transfer 2 e- from 1 to 2 or 2 to 3

3 Forms

Quinone / Ubiquinone

Hydroquinone / Ubiquinol

Semiquinone

Oxidized Q

Reduced Q

Free radical; intermediate between oxidized & reduced Q

Hydroxyperoxidases

Oxygenases

Oxidases

Dehydrogenases

Removes hydrogen using oxygen

Forms H2O or H2O2

Cytochrome Oxidase Complex 4, Flavoproteins

Transfer 1 hydrogen from 1 substrate

Cannot use O2

NAD & FAD

Hydrogen peroxide / Organic peroxide as substrate

Direct transfer of O2 to substrate

Electron Transfer

Shuttle Systems

Inhibitory Toxins

Electron Transport Chain

NADH ➡️ FMN ➡️ Fe-S Centers ➡️ Q

Respiratory Chain Complexes

COMPLEX 2️⃣

COMPLEX 4️⃣

COMPLEX 1️⃣

COMPLEX 3️⃣

NADH binds

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

NADH Dehydrogenase / NADH-Ubiquinone Oxidoreductase

Transports 4H in intermembrane space

Major source of ROS

Produces 2.5 ATP per 1 mol NADH

Steps

  1. 4 protons translocated
  1. e- transferred via Fe-S centers
  1. e- transferred from NADH to FMN = FMNH2

FADH2 produced during conversion of succinate to fumarate in TCA

Succinate Q reductase

e- passed via Fe-S centers to Q

G3P & Acyl CoA pass e- to Q

G3P: generated in TAG breakdown / glycolysis

Succinate Dehydrogease / Succinate Ubiquinone Oxidoreductase

Succinate + Q --> Fumarate + QH2

Does not transport H+ in intermembrane

Produces 1.5 ATP per 1 mol FADH2

Bypass complex 1

Steps

  1. 2 e- & 2 p+ transferred to complex 2 FAD = FADH2
  1. FADH2 transfers e- to ubiquinone via Fe-S centers
  1. Fe-S centers tranfer e- to CoQ = CoQH2

Q cycle: e- passed from QH2 to cytochrome

Oxidation of 2 QH2 to Q: releases 4H

Reduction of Q to QH2: takes 2H

Cytochrome bc1 complex / Ubiquinol Cytochrome C Oxidoreductase

QH2 + 2 Cyt C + 2 H --> Q + 2 Cyt C + 4H

Transports 4H in intermembrane

3 Forms

Q: Quinone / Ubiquinone

QH2: Hydroquinone / Ubiquinol

QH: Semiquinone

Steps

  1. CoQH2 donates e- to Fe-S centers
  1. Fe-S centers transfer e- to cytochrome C
  1. 4 protons transferred

Oxidizes reduced cytochrome C

Cytochrome C Oxidase

2 e- used ; 1 H2O produced per cycle

4 Cyt C + O2 + 8 H --> 4 Cyt C + 2 H2O + 4H

Transports 2H in intermembrane

Cycle occurs 2x bec. of 2 Cyt C

Steps

  1. Complex IV transfer e- from Cyt C to copper
  1. e- passed to Cyt a, a3, CuB
  1. CuB transfer e- to O2 = H2O
  1. 2 protons transferred

4 electrons needed to reduce 1 mole O2 to 2 mol H2O

Oxidative Phosphorylation at Respiratory Chain

Clinical Correlation

Source of NADH: fat oxidation, glycolysis, TCA

Any movement in F0 will lead to F1 movement

ATP Synthase: generates ATP

Y stalk: attached F0 to F1

Complex V Domains

F0 subunit

F1 subunit

Hydrophobic; inner mitochondrial membrane

Movable subunit = main rotatory movement

Alpha subunit

Beta subunit

Ball-shaped around an axis

Binding site of substrates for ATP synthesis

For catalytic activity of enzyme

ATP synthesis

each has opening

3 B-subunits in between a-subunits

Malate Aspartate Shuttle

Glycerol 3 Phosphate Shuttle

Phosphocreatine Shuttle

Mechanism

Liver, Heart, Kidney

Enters through complex 1

Transfers reducing equivalent of NADH

Yields 2.5 ATP

  1. NADH enters via porins

Malate dehydrogenase: transfer reducing equivalent of NADH to oxaloacetate = yields malate

  1. Malate enters mitochondria via malate-asparate shuttle

Malate a-KG antiporter

  1. Malate dehydrogenases forms NADH & Oxaloacetate
  1. Transaminase & Glutamate: transaminates oxaloacetate to aspartate

NADH pass directly to respiratory chain

  1. Aspartate cross inner mitochondria via glutamate-aspartate transporter

Glutamate becomes a-KG

Aspartate aminotransferase: Aspartate converted to oxaloacetate

  1. Oxaloacetate regenerated in cytosol
  1. Aspartate = converted to oxaloacetate a-KG = glutamate
  1. Malate dehydrogenase responsible for entrance of NADH proton

Muscles, Brain

Forms FADH2

Yields 1.5 ATP bec. complex 1 bypassed

Mechanism

  1. G3P dehydrogenase with NADH: Reduce DHAP to G3P
  1. G3P donates to FAD via mitochondrial G3P dehydrogenase
  1. FADH2 reduces Q --> binds to Complex 2
  1. DHAP crosses outer mitochondrial membrane

Creatine

Creatine smaller than ADP = faster transport

Fast transfer system for new ATP

Transfers energy to active muscles (hearts/skeletal)

Carrier of high energy phosphate group

Replenishes ATP

ETC Inhibitors

Uncouplers of Oxidative Phosphorylation

Oxidative Phosphorylation Inhibitors

Complex II

Complex III

Complex I

Complex IV

Rotenone

Piericidin, Amytal, Barbiturates

Binds to ubiquinone binding site

blocks e- transfer from Fe-S to ubiquinone

Carboxin, Thenoyltrifluoroacetone

Malonate

Succinate analog / Competitive inhibitor

Binds to ubiquinone binding site

Antimycin

Myxothiaxol, Stigmatellin

Binds to Qo site

Binds to Qi site

Cyanide, Axide, H2S

CO

Binds to oxidized heme = Fe3+

Binds to reduced heme = Fe2+

Inhibits phosphorylation of ADP w/o affecting ETC

O2 reduced to H2O, no ATP produced

Ionophores

Thermogenin / Uncoupling protein

Toxic = RR not controlled by ADP & Pi

Generates heat

2,4 dinitrophenol

Valinomycin

Generates body heat

For newborns during hibernation

Atractyloside

Carbonyl cyanide m-chlorophenyl hydrazone (CCPP) & 2,4 dinitrophenol

Oligomycin

Prevents proton movement via ATP synthase

Inhibits exchange of ATP/ADP

Used as poison / weightloss

Disrupts proton gradient, causes uncoupling of proton pump

Brown Adipose Tissue / Cold-induced thermogenesis

Leber Hereditary Optic Neuropathy

MELAS: Mitochondrial Encephalopathy, Lactic Acidosis, Stroke

Reactive Oxygen Species (ROS)

Fatal Infantile Mitochondrial Myopathy and Renal Dysfunction

Absence/ deficiency of oxidoreductase enzyme

NADH-Q oxidoreductase (Complex 1) or Cytochrome oxidase (Complex 4) deficiency

Mutation in mitochondrial DNA

Diabetes mellitus & Alzheimers

Uncoupling protein (UCP-1) carries protons and uncouples ATP synthesis

Blindness due to optic nerve death

Due to single base mutation in Complex 1

Oxygen accepts 1 or 2 e- = forms superoxide anions/peroxide

Antioxidants protect against radical damage

Glutathione reductase: maintains reduced form of glutathione