Normal mitochondrial function - lecture 1

Question 1

What is the meaning of the word mitochondria?

Answer 1

2 greek words:
- MITOS= thread
- KHONDRION= granule
mitochondria have both granular and thread like structure

Question 2

What is the proposed origin of mitochondria and how did this process occur?

Answer 2

ENDOSYMBIOSIS

1) mitochondria were originally archaea or proteobacteria that were autotrophs surviving by the use of photosynthesis to generate energy
2) it was engulfed but not digested by a primitive eukaryotic cell
3) eukaryote then began a symbiotic relationship, where the eukaryote provided nutrients and protection while the prokaryote provided additional energy to the eurkaroyte via its respiratory cellular machinery

Question 3

What is the “PROOF” behind endosymbiosis?

Answer 3

No actual proof, and its believed to have occurred 2.5 thousand years ago

1) amoeba are eukaryotes that lack mitochondria so require a symbiotic relationship with an aerobic bacteria
2) mitochondria have their own DNA, RNA and ribosomes so they are pretty self-sufficient although they are not
3) mitochondria and purple aerobic bacteria both consume oxygen and produce ATP via krebs cycle and oxidative phosphorylation
4) mitochondria and bacteria are the same size- poor evidence
5) mitochondria have circular DNA like bacteria
6) mitochondria divide independently of the cell they are in- constantly dividing to make new mitochondria
7) mitochondria have a double phospholipid membrane and although purple aerobic bacteria have a single membrane they acquire a second one when they are endocytosed

Question 4

What are cristae?

Answer 4

folds and invaginations in mitochondria which increase its surface area

Question 5

What act as power sources for mitochondria ?

Answer 5

glucose or fatty acids as they produce pyruvate which can enter the mitochondria

Question 6

What happens in oxidative phosphorylation?

Answer 6

acetyl-coA is a common substrate for the krebs cycle

• it drives the production of reducing equivalents NADH and FADH2
• electrons pass through the ECT causing redox reactions that cause proton translocation across the inner mitochondrial membrane to create an electrical potential and pH gradient to drive ATP synthesis by mitochondrial ATP synthase

selective and non-elective ion channels dissipate energy and alter ionic balance and volume of matrix, partly compensated by antiporters coupled to proton movement

Question 7

What is present in the outer mitochondrial membrane?

Answer 7

• inwardly rectifying potassium channels
• voltage dependent anion channels
• BCl and Bax for apoptosis
• TOMS= transporters of the outer membrane
• SAMS= sorting and assembling machinery
• maybe AChRs

Question 8

What is present in the inner mitochondrial membrane?

Answer 8

• Ca2+ activate potassium channels
• intermediate conductance channels
• small conductance potassium mediated calcium channels
• K+ATP channel- though some people dont believe this is present
• acid sensing channels = important due to the way mitochondria function and generate energy
• Kb1.3- could be involved in survival as its thought targeting this channel leads to mitochondrial death
• adenine nucleotide transporter
• anion channels
• PTP= permeability transition pore - allows calcium to flood in when opened
• TIMS= transporters of the inner membrane - important for protein import
• magnesium sensnitive channels
• calcium uniporter- important for modifying calcium levels within the cell
• ryanodine receptor
• CLC4
• maybe NMDA receptors

Question 9

Why is calcium uniporter important ?

Answer 9

important for buffering calcium in the cytosol when large amounts of calcium enter the cytosol- almost communicates directly with the ER
- can actually move calcium from the mitochondria to the ER without it contacting the cytosol so you dont get any large rises in calcium in the cytosol

Question 10

What do ROS do to PTP?

Answer 10

a ROS surge causes PTP opening and cell death whereas the inhibition of ROS keeps the pore locked and protects cells from noxious stimuli

Question 11

What are some key facts about the genome of human mitochondria ?

Answer 11

it codes for 37 genes
first significant part of the human genome that was sequenced
inherited exclusively from the mother- unsure exactly why but the sperm mitochondria are thought to be targeted for ubiquitination
2 strands of circular DNA with a heavy guanine rich strand and a light cytosine rich strand

Question 12

What genes do the mitochondria DNA encode?

Answer 12

mainly encodes genes for tRNA but also for polypeptides and rRNA

Question 13

Why does mitochondrial DNA have a greater rate of mutations compared to nuclear DNA?

Answer 13

because of its close proximity to ROS
- thought to be involved in cause of aging as experiments in c.elegans showed that by preventing this mutations their life span was increased by 3-4 times

Question 14

What does glycolysis produce?

Answer 14

produces 2 ATP molecules which enter the citric acid cycle and another 2 ATP molecules are produced

Question 15

If cells can evoke glycolysis why do we need mitochondria?

Answer 15

because oxidative phosphorylation enables NADH to be transported into the electron transport system and this generates 32 ATP molecules
“NADH” is the currency for oxidative phosphorylation

Question 16

What does the citric acid cycle do ?

Answer 16

it is carried out all the time and it produces 4 molecules of NADH

• only complex 2 is part of the citric acid cycle because it produces fumerate from succinate
• fatty acids can also enter this cycle

Question 17

What is ubiquinone good at?

Answer 17

good at accepting electrons and being reduced to ubiquinol

Question 18

What are the 4 complexes involved in oxidative phosphorylation?

Answer 18

COMPLEX 1: NADH enters the system (it is a massive structure)
COMPLEX 2: succinate dehydrogenase- this doesn’t actually provide movement across the membrane
COMPLEX 3: carried out “Q cycle”
COMPLEX 4: oxygen is consumed
ATP synthase: energy is passed onto this complex

Question 19

What is oxidative phosphorylation vital for ?

Answer 19

vital for pumping protons into inter membranous space- this is how energy is stored = chemihypotic hypothesis

process can actually run backwards

Question 20

How does complex 1 work?

Answer 20

1) it is a large redox enzyme that oxidises NADh, reducing ubiquinone and transfers protons across the mitochondrial membrane
2) NADH oxidation occurs by a flavin mononucleotide in the hydrophilic domain and this causes intramolecular electrons to transfer towards the membrane by a chain of iron sulphur clusters
3) reduced flavin reacts with oxygen producing superoxide but it also reacts with many other molecules - of which some increase oxidative stress

it is thought that generally 4 protons are transferred for each NADH molecule oxidised but some people have suggested 3

Question 21

What does complex 1 look like?

Answer 21

it has 2 clear domains- a hydrophobic domain within the membrane and a hydrophilic domain which sticks into the matrix

Question 22

What is meant by complex 1 modulation?

Answer 22

it can exist in 2 forms - A and D
ex vivo 5-15% of it exists in D conformation at normal oxygen levels - this indicates that some of the energy released during steady state NADH oxidation in situ is for maintaining catalytcially competent A form
- maintaining some of complex 1 in D form ensures fast changes in conditions like stress, increased ATP demand and changes in oxygen availability

Question 23

What may A/D transitions be important for?

Answer 23

fine tuning enzyme activity
- under conditions such as ischemia, fine tuning would protect not only complex 1 but the entire respiratory system from oxidative damage

Question 24

What are the series of events between complex 1 and complex 2?

Answer 24

1) NADH binds complex 1 and donates 2 electrons to flavin monomucletide, FMNH2 then transfers electrons through a series of iron sulphur clusters to ubiquinone reducing it to ubiquinol
2) succinate is oxidised to fumerate reducing a bound flavin adenine dinucleotide to FADH2. the electrons are transferred though iron sulphur clusters to reduce ubiquinone

Question 25

What is complex 2 part of ?

Answer 25

part of both citric acid cycle and electron transport system

Question 26

What are the different conformations of ubiquinone?

Answer 26

ubiquinone= fully oxidised 
semiquinone= ubstable/reactive state 
ubiquinol= fully reduced

Question 27

What happens in complex 3?

Answer 27

uses 2 cycle process to transfer 4 protons to the inter membranous space

1) ubiquinol binds to Qo of complex 3 giving up 1 electron to each rieske iron sulphur protein and the BL haem producing transient free radical semiquinone
2) semiquinone can be dangerous if it is transferred to oxygen as it can produce superoxide anion but usually oxidation continues and it produces ubiquinone which is transferred to Qi site
3) electron donated to iron sulphur protein is transferred to cytochrome c1 subunit causing it to donate it to an externally bound molecule of oxidised cytochrome c that dissociates from the complex
3) electron donates to BL reduced the Bh which then reduces ubiquinone at Qi to semiquinone radical
4) as semiqunione is dangerous another q cycle occurs to so another electron transfer can occur from cytochrome Bh reducing semiqunone to ubiquinol

total of 4 protons move into intermembraneous space and a further 2 protons from the matrix

Question 28

What happens at complex 4?

Answer 28

4 electrons are removed from 4 molecules of cytochrome c and transferred to molecular oxygen producing 2 molecules of water
at the same time 4 protons are removed from the matrix but only 2 are transported across the membrane contributing to proton gradient
oxygen is consumed here

Question 29

What is ATP synthase ?

Answer 29

it is a molecular motor driven by the proton gradient

• primary function is ATP synthesis
• very large structure with a mushroom shaped protein complex
• has 2 portions F0= proton translocating region and F1= catalytic region

works by converting the energy present in the proton gradient to chemical energy stored in ATP

Question 30

What happens at the F0 portion ?

Answer 30

1) protons flow down their electrochemical gradient and enter a pore in the F0 portion which doesn’t penetrate the entire membrane
2) instead the proton binds to rotor subunits, protonating them and this causes them to rotate into the membrane away from the channel
3) once it has completed nearly a full turn and has returned to stationary channel the proton exits through another pore to the other side of the membrane

this process converts the stored energy in the proton gradient to rotational mechanical energy which is transferred to the F1 portion

Question 31

What happens in the F1 portion of ATP synthase?

Answer 31

this portion catalyses the formation of ATP
it has a 3 site alternating binding site mechanism
the central shaft of this region drives changes in the surrounding subunits so ATP can be synthesised
1) the loose subunit binds ADP and Pi
2) the energy input alters its conformation
3) New ATP forms by condensaion and the original ATP is release
4) then the cycle begins again with the knob rotated by one subunit

Question 32

Why are mitchondrial poisons important?

Answer 32

because they have aided our knowledge about the electron transport system and its functions

Question 33

What are some poisons of the different complexes?

Answer 33

comples 1: rotenone - competitive and it was used as an insecticide
complex 2: malonate
complex 3: antimycin A, myxothiazol, stigmatelin
complex 4: azide and cyanide reversible
ATP synthase: oligomycin - blocks proton pore