Lecture objectives – Electron Transport Chain

1. Describe the structure of mammalian mitochondria.

Outer membrane:
- 50 % protein, 50% lipid
- Relatively permeable due to presence of porin proteins (form channels)
- Ions and molecules up to 5000Da can readily pass
Inner membrane:
- 76% protein, 24% lipid
- Very impermeable to polar/charged molecules
o Requires transport proteins
- Highly invaginated (cristae)
o Cristae contain the enzymes of ETC and ATP-synthase
Matrix
- Contains soluble enzyme system
o TCA cycle
o Fatty acid oxidation
o Urea cycle enzymes
o Pyruvate dehydrogenase complex

4. Outline the salient features of the mitochondrial genome. How many proteins are encoded by the
mitochondrial genome?

- Mitochondria contain their own genome consisting of

o DNA and RNA polymerases
o tRNA
o ribosomes
- Transcription and translation can occur in mitochondrial matrix
- Genomes are small and circular
o Each mitochondrion has several complete copies
- Genome encodes only 13 proteins
o All are involved in oxidative phosphorylation
o All other proteins are encoded by nuclear genes and are transported into the
mitochondria

5. What is unusual about the inheritance of mitochondrial mutations?

- Mitochondrial DNA is inherited exclusively from mother

o Mothers pass mitochondrial mutations to all their children
o Fathers do not transmit mitochondrial mutations

6. Describe in general terms the structure of cytochromes and iron-sulfur centers.

- Cytochromes
o Heme prosthetic group with specific associated iron ions
 b, c1, c, a, a3 found in ETC
 Associated iron ion undergoes reversible oxidation/reduction
 Flips between Fe2+ and Fe3+
- Iron-Sulfur proteins
 o Iron ions are complexed to sulfur atoms and to cysteine sulfhydryl groups, forming
iron-sulfur centers
7. Starting with NADH, list the elements of the pathway along which electrons travel on their way to
the terminal electron acceptor, O2
- NADH
- Complex I
- Coenzyme Q
(ubiquinone)
- Complex III
- Cytochrome c
- Complex IV
- Oxygen

8. List the sites in the electron transport chain where proton pumping occurs

- Complex I (4 H+ pumped)
- Complex III (4 H+ pumped)
- Complex IV (2 H+ pumped)

9. Where does the energy to pump protons come from?

- Reduction potential is higher than the previous complex so that electrons can be passed down
- There is a release of free energy as electrons pass down to next complex
o Used to pump protons across inner mitochondrial membrane into intermembrane space

10. Outline how pumping protons across the inner mitochondrial membrane is linked to ATP synthesis

- The inner membrane is highly permeable and

allows protons to pass through, creating a proton
electrochemical gradient
o Concentration of protons is higher in
intermembrane space than in matrix
o Cytosolic side of membrane has a more
positive charge than matric
- This creates proton motive force
o Potential energy that drives return of
protons to the matrix (higher pH, more
negative)
- Protons return to matrix through ATP synthase
o F1 portion catalyzes phosphorylation of
ADP to ATP

11. Write out the net reactions catalyzed by

- Complex I
o Has several FMN-containing proteins and iron-sulfur proteins
o NADH passes electrons to FMN, which passes them on to the iron-sulfur centers, then
to coenzyme Q
 - Complex III
o Contains cytochromes b and c1 and an iron-sulfur protein
o Electrons pass from coenzyme Q to cytochrome b to iron-sulfur proteins to cytochrome
c1 to cytochrome c

- Complex IV
o Aka cytochrome c oxidase
o Contains copper ions and cytochromes a and a3
o Electrons pass from cytochrome c through copper ions and cytochromes to oxygen
o Has a high affinity for oxygen (higher than hemoglobin and myoglobin)
 Ready flow of oxygen from Hb to complex IV in blood

12. Define the roles of

- Succinate dehydrogenase (complex II)
o FAD-containing enzyme for TCA cycle
o Electrons passed from FADH2, through iron-sulfur centers, to coenzyme Q

- Electron-transferring flavoprotein/electron-transferring flavoprotein oxidoreductase

o Soluble protein of mitochondrial matrix
o Contains FAD
o Can accept electrons from several different FAD-containing dehydrogenases
 Fatty acid oxidation, amino acid metabolism
o ETF oxidoreductase reoxidizes FADH2 of ETF
 Contains FAD and iron-sulfur center
 Passes electrons on to coenzyme Q

- Glycerol 3-phosphate dehydrogenase in oxidative metabolism

o FAD-containing enzyme
o Electrons flow from FADH2 and iron to coenzyme Q
o Converts glycerol 3-phosphate to dihydroxyacetone phosphate

13. Explain why oxidation of 1 mol of NADH is estimated to yield 2.5 mol of ATP but oxidation of 1
mol of FADH2 is thought to yield only 1.5 mol of ATP

- 4 protons translocated = 1 molecule of ATP

- There is no proton translocation associated with transfer of electrons from FADH 2 to
coenzyme Q (lose the 4 translocated from complex I)
o Only 6 protons translocated per mol of FADH 2
o 1.5 mol of ATP for every 1 mol of FADH2

14. Describe how the following compounds function to inhibit oxidative phosphorylation

a. rotenone
- Inhibits complex I
- Blocks electron transfer from NADH to coenzyme Q
b. antimycin A
- Inhibits complex III
- Blocks electron transfer from coenzyme Q to cytochrome c
c. cyanide
 - Inhibits complex IV
- Binds heme iron in complex IV
- Blocks electron transfer from cytochrome c to oxygen
d. oligomycin

15. Describe, with examples, how uncoupling agents can collapse the proton electrochemical gradient.
What happens to the energy stored in the gradient when uncouplers are present?

- Proton reentry is coupled to the synthesis of ATP (passes through ATP synthase)
- If an alternative pathway is used for protons to enter matrix without passing through ATP
synthase, no ATP is made
- Energy of electrochemical gradient is dissipated as heat
- Examples
o 2,4-dinitrophenol (DNP)
 Dietary supplement used for weight loss
o UCP1 and brown adipose tissue

16. Describe the location of the mutation (mitochondrial or nuclear DNA) and the most common
mutation in each of the following oxidative phosphorylation defects. List the key pathological features
of each of these conditions.

- MERRF
o Due to point mutations in mitochondrial tRNA for lysine
 Impacts mitochondrial translation
 Multiple deficiencies in respiratory chain components
o Myoclonic epilepsy associated with ragged red muscle fibers
o Myoclonus, ataxia, generalized seizures
- MELAS
o Mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes
o Mutation in mitochondrial gene encoding tRNA for leucine
 Deficiencies in respiratory chain components
 Symptoms can overlap with those of MERRF
- Leber optic atrophy
o Sudden onset blindness in young adults
 Optic nerve dies
o Mutations impact subunits of complex 1 of respiratory chain
 Mitochondrial DNA effected
o Can result in chronic energy insufficiency or increased production of reactive oxygen
species
- Combined oxidative phosphorylation deficiency
o Autosomal recessive inheritance (nuclear DNA)
o Onset occurs soon after birth and the condition results in a fatal progressive hepato-
encephalopathy
o Severe metabolic acidosis, microcephaly, liver dysfunction, encephalopathy, growth
retardation
o Mutation in mitochondrial elongation factor 1 gene