Electron Transport Chainand

Oxidative Phosporylation

CJW

Learning Objectives & Outcomes

• This lecture will emphasize on the following topics; which the students are expected to understand at the end of this lecture.

1. Know the course of electron transport chain in mitochondria

2. Know how the electrical and pH gradient lead to the production of ATP in oxidative phosphorylation

3. Know the various inhibitors and uncouplers for them

Review of glycolysis and the TCA cycle

• There are three main pathways that generates energyin the body

• Energy production pathways:-

1. Glycolysis

2. TCA cycle

3. Electron transport chain & oxidative phosphorylation

• All three of them are inter-connected

• So far, we have learn two of them

• Next is the coupled reaction of electron transport chain and oxidative phosphorylation

Recap from Glycolysis and TCA cycle

• Synthesized in Glycolysis/TCA cycle

• NADH, Nicotinamide adenine dinucleotideFADH2, Flavin adenine dinucleotide

• Energy rich reduced co-enzymes

• Utilized in ETC and OP

• H+ gradient

• ATP synthase

• NADH and FADH2 ; get oxidized

• ADP get phosphorylated

Review of glycolysis and the TCA cycle

• Electron carrying nucleotides (NADH and FADH2) generated from glycolysis and TCA cycle are funneled to the electron transport chain to generate MORE ATP

Food generates power

• Various sources of food are used to generate ATP

• The electrons are being carried as NADH and FADH2

• In the end oxygen is reduced

Tales of the Two Systems that Generate Energy

1. Electron transport chain (ETC)

• Electron pass through an electron transport chain

• Loose free energy, but generates a H+ gradient

2. Oxidative phosphorylation (OP)

• H+ pass through ATP synthase; produce ATP

• Both processes are highly integrated / coupledprocess

• All cells that have mitochondria

Mitochondrial structures

• Location, location, location!

• Outer membrane

Inter-membrane space (H+ accumulates)

Inner membrane(Electron transport assembly + ATPsynthase)

• Cristae

• Matrix

• The chain has 4multiprotein complexes (IIV)

• Note where NADH / FADH2 donates the electron

• Too complex?

1

2

34

FMN – Flavin mononucleotide

FeS – Iron-sulphur protein

Q – Coenzyme Q10

Cyt – Cytochrome (heme)

FAD – Flavin adenine dinucleotide

Oxidation and Reduction

• The key to understand this chapter

• Reduced

1. Addition of H

2. Removal of O

3. Additional of electrons

• Oxidized

1. Removal of H

2. Additional of O

3. Removal of electrons

Reduced Form(eg. NADH, FADH2)

Oxidized Form(eg. NAD+, FAD+)

Simpler ?

5

1

2

3

4

Chemi-osmotic theory

There is a need for reducing agents

• To generate the proton gradient in the intermembrane region, the mitochondria require a constant supply of reducing agents (NADH and FADH2) from Kreb Cycle

• And next door (cytosol), they have plenty of NADH (from glycolysis) transport in via:-

1. Malate-aspartate shuttle

2. Glycerophosphate shuttle

A deal is a deal: Malate-Aspartate shuttle

• Transfer of NADH from cytosol to Mitochon.

• Malate is used to shuttle H+ into Mitochon. (like TCA)

Ketoglutarate

transporter

Glutamate-

aspartate

transporter

E:

E: E:

E:

A deal gone bad: Glycerolphosphate shuttle

• Short-changed, as NADH has been replaced by FADH2

• FADH2 has less reducing power compared to NADH

• NADH = 3 ATP ; FADH2 = 2 ATP

Everyone is an accountant

18 ATP 4 ATP

6 ATP

4 ATP

2 ATP 2 ATP

6 ATP

• Famous trick question (Ans: Campbell & Reece)

NADH = 3 ATPFADH2 = 2 ATP

Keeping up with the demand

• As ATP synthase generates more ATP, it has to be exported out

And there’s a demand for ADP

E: adenine nucleotide transporter / translocase (2)

• Inorganic phosphate is required as well; phosphate transporter (1)

Uncouplers & inhibitors

• UncouplersAllow protons to flow back into the matrix of the mitochondria from the intermembrane region, without ATP synthesis, and with the consumption of oxygen

• InhibitorsInhibitors blocks the transfer of electron within the ‘electron transport chain’ (ETC), denying the pump of proton at each complexes, thus reducing the proton gradient between the intermembrane region and the matrix of the mitochondria

Uncouplers (saboteur)

• ETC and OP is a coupled reaction

• The most evil one. What do they do to the ETC?

• It allows proton to flow back into the matrix of the mitochon., without making ATPs

• Rendering the whole electron transport and reduction of oxygen, POINTLESS

• Eg:

1. Dinitrophenol

2. Long-chain fatty acids

3. Valinomycin – is a K+ ionophore disrupt the electrochemical gradient, also prevent ATP synthesis

Dinitrophenol

• Amphipathic

• Increases the permeability of H+ through the inner-membrane

• Reducing the electrochemical potential (proton gradient)

• ‘Short-circuiting’ the ATP synthase

• Leaked proton can be oxidized ( H2O) without phosphorylating ADP

Dinitrophenol uncouples oxidative phosphorylation, causes release of calcium from mitochondrial storesand prevents calcium re-uptake. This leads to free

intracellular calcium and causes muscle contraction and hyperthermia.

Are you slim enough ?

Inhibitors

• Each type of inhibitor block a specific site of the electron transfer

Complex IV

Complex IComplex III

1

2

3

RTC e- transfer inhibitors

• Complex IRotenone – pesticide/insecticideAmytal (Amobarbital) – barbiturate class, sedative, analgesic

• Complex IIIAntimycin – produced by bacteria (Streptomyces), as fish poison

• Complex IVCyanide – used primarily in industriesCO – used primarily in industriesreleased during fires

RTC non-e- transfer inhibitors

• ADP-ATP phosphorylation (E: ATP synthase)Blocks phosphorylation

Oligomycin – antibiotic; result in high levels of lactate (due to regeneration of NAD+, in glycolysis)pyruvate lactate

• ADP-ATP transport (E: translocase)Blocks ADP-ATP transportation cytosol-matrix

A-trac-ty-lo-cide - from Mediterranean thistle, a glycoside Bong-kre-kate - antibiotic produced by Pseudomonas cocovenenans

Inhibitors & uncouplers

e- carrier (reduced) before [INHIIBITOR] after e- carrier (oxidized)

1

2 3

Cyanide poisoning

Jewelery & textile industry

usually attempted suicide

Symptoms:-

1. neurological impairment

2. severe acidosis

Antidote:-

1. sodium thiosulphate

2. hydroxocobalamin

Cyanide poisoning

• Cyanide poisoning is also seen in:-

1. Survivors from fires in buildings with fittings that produces HCN gases upon combustion

2. Sewerage workers

3. Petroleum refineries

4. Gold extraction

Cyanide poisoning

• LD=1.5mg /kg

• If 70kg = 105 mg = 0.1 g

• Sad but true. Used for gas chambers – mass killing; industrial or household fires

• Best murder weapon: HCN, colorless, faint bitter almond scent (but most ppl can’t detect it by smell)

• [read more – LMS]

• Firstline: Amyl nitrite

Antidote biochemistry

• Amyl nitrate stimulates the formation of methemoglobin which has high affinity for cyanide forming cyanomethemoglobin

• Sodium thiosulphate induces the E: Sulfur transferase activity; which converts the formed cyanomethemoglobin to thiocyanate (excreted in urine)

• Hydroxocobalamin (has a cobalt ion) which has high affinity for cyanide; binding of cyanide, forms cyanocobalamin (Vit B12)

Cyanide can be extracted from the pits of apricot as amygdalin; 1 kernal = 0.5 mg cyanide

References

• Harper RK, Granner DK, Mayes, PA, & Rodwell, VW (2003). Harper’s Illustrated Biochemistry (26th Ed). McGraw-Hill. Chapter 12: The respiratory chain and electron transport chain.

• Champe, PC., Harvey, RA & Ferrier, DR. (2008). Lippincott’s Illustrated Review, 4th Ed. LWW. Chapter 30 – RNA structure, synthesis and processing.

• Lieberman & Marks (2009). Marks’ Basic Medical Biochemistry: A clinical approach, 3rd Ed. Chapter 14 –Transcription: Synthesis of RNA