6. ETC Flashcards
Up until the ETC, all electron acceptors (oxidizing agents) were _________ and ___________
NAD+ and FAD
So far we have discussed the catabolism involving oxidation of 6 carbons of glucose to CO2 via glycolysis and CAC without any __________ molecule directly involved.
oxygen
Before ETC, the free energy released in these oxidation reactions were stored as reduced compounds (NADH and FADH2) and synthesis of net _____ ATP molecules.
4
When did we see substrate-level phosphorylate in the catabolism of glucose?
- 1,3-bisphosphoglycerate to 3-phosphoglycerate to make ATP after the GAPDH reaction
How many ATP by SLP and how many ATP by oxidative phosphorylastion?
SLP –> 4
Major chunk of ATP molecules are generated by a special biochemical machinery in which oxidation of NADH and FADH2 is coupled to ATP synthesis in the ________ mitochondrial membrane. This process is called Oxidative phosphorylation.
inner
How much ATP is made per glucose in anaerobic conditions and what does this support?
2 ATP - Pasteur’s effect –> anaerobic requires higher amount of sugar
What drives the production of ATP in ETC?
The formation of the electrochemical gradient, driving H+ thru the channel and electrical energy is converted to ATP by rotating the conformation.
In most aerobic organisms, mitochondria is the major site for what 3 important processes?
PDC, CAC, and ETC
Mitochondria also have their own transcription and translation machinery. The mitochondrial ribosomes similar to that of bacteria.
Okay.
Why does mt have a convoluted double mb structure?
increase surface area
ETC ultimate electron acceptor
O2
How many H+ is pumped out to intermembrane space from the matrix per NADH? per FADH2?
10 H+
Why does the reduction potential increase at every cytochrome despite same Fe 3+ ion?
Differing AA side groups have different electron densities surrounding the porphyrin ring
What does complex I do?
Catalyzes oxidation of NADH by CoQ
What does complex II do?
Catalyzes oxidation of FADH2 by CoQ (gets reduced)
What does complex III do?
Catalyzes oxidation of CoQ by Cyt C (gets reduced)
What does complex IV do?
Catalyzes oxidation of Cyt C by O2 (gets reduced)
Provide the reaction that takes place at complex IV
CytC + 1/2O2 –> CytC + H2O
Electrons are transferred through different electron-carriers sequentially in mitochondrial membrane. Most of electron-carriers (except ____________ ) are proteins with ____________ ____________ capable of accepting or donating electrons.
CoQ
3 ways electrons are transferred:
1.Electrons can be transferred directly: e.g. reduction of Fe3+ to Fe2+ or by
What are the 3 forms of CoQ?
ubiquinone (fully oxidized) takes a pair to become semiquinone radical, then another pair to fully reduce to ubiquinol
True or false?
TRUE
What are flavoproteins?
Proteins tightly (or covalently) bound to Flavin nucleotides FMN or FAD.
What complexes have the most flavoproteins?
I and II
What does reduction potential of the flavoproteins depend on?
The reduction potential of flavin nucleotides depends on the protein it is bound to. Local interaction of functional groups of amino acids distorts the electron orbitals of flavin ring, thus changing the stability of oxidized or reduced forms.
Flavoproteins are capable of accepting or donating one or two electrons. True or false?
TRUE
What are iron sulfur proteins?
These are proteins containing Fe atom co-ordidated to sulfur atoms of either Cys residues of the proteins or also with inorganic sulfur atoms. The Fe atom is oxidized or reduced
There are ___ different Fe-S proteins that function in mitochondrial electron transfer.
8
What affects the reduction potential of the proteins?
The electron density of the AAs around dictate reduction potential
What are cytochromes?
Cytochromes are proteins with a Fe-containing heme prosthetic group.
There are ________ classes of cytochromes depending of the type of heme group they have.
three
Cyt-a and b are ____________ proteins whereas cyt-c in mitochondria is a ____________ protein associated with the outer surface of membrane by electroststic interaction.
a
What does cyt-c do in the ETC?
moves from complex III to IV depending on oxidation state
What is an another function of cyt-c?
If something bad happens in cell and membrane of mitochondria leaks, CytC comes out and serves as an apoptotic factor that results in formation of apoptosome to cause cell death
Describe the absorption spectra differences seen between oxidized and reduced cytochrome c:
Reduced form has higher absorbance of gamma peak (at 400nm), and also includes beta and alpha peaks at higher wavelengths (between 500-600nm)
What wavelength peaks only exist in reduced form of cytc?
beta and alpha
get beginning of slide 15 info
okay
So oxygen consumption can be used as what?
rate of reactions
Indicate the process in which oxygen consumption was messed with using different compounds (6):
1.Added beta-hydroxybutarate to produce NADH (which gets oxidized on complex I and electrons go to II then III), so as time goes on, oxygen concentration goes down as it is converted to water
What is the effect of using antimycin A
Adding antimycin A, oxygen consumption will be blocked forever bc doesn’t matter if from I or II, CoQ cannot be reduced
TMPD/Ascorbate together do what?
TMPD/Ascorbate together transfer electrons to complex IV for electron transfer to oxygen.
Name complex I
NADH-Coenzyme Q oxidoreductase
Just read
okay
So CoQ10 is oxidizing agent at complex I, but they don’t jump directly bc we don’t want all the energy to be released all at once and then we lose it. true or false?
TRUE
Describe what happens at complex I
2e- from NADH to FMN to 2 Fe-S centres (Fe3+ to Fe2+) to N-2 (iron sulfur) centre, then 2e- alone to Q and then 2h+ comes in and makes QH2
What blocks the complex I pathway? (2)
rotenone and paraquat
What are the 3 enzymes that can produce FADH2 and transfer electrons to CoQ10?
Succinate dehydrogenase
Describe the pathway of electron transfer for succinate dehydrogenase:
FAD to FADH2, then transferred to multiple Fe-S centres then ubiquinone
Describe the pathway of electron transfer for Glycerol 3-phosphate dehydrogenase:
fadh2 direct transfer to CoQ10
Describe the pathway of electron transfer for Fatty acyl-CoA dehydrogenase:
Fatty acyl has electrons being transferred to FAD then to electron transferring flavoprotein (ETF) then to ETF:ubiquinone oxidoreductase which passes electrons to ubiquinone.
Name and describe structure of complex III
`Complex III: Cytochrome bc1 complex or Ubiquinone:cytochrome c oxidoreductase complex.
What is unique about the electron transfer of complex III?
CoQ10 gets oxidized and reduced
Describe the Q cycle
- QH2 gives up one electron to Fe-S centre, which gives to CytC1, giving to CytC, which when reduced, it leaves to complex IV
Briefly name and describe complex IV:
Cytochrome c oxidase: It catalyses the transfer of electron from cyt. C to oxygen leading to the reduction of oxygen to H2O.
Describe each of the 3 subunits found on complex IV
Subunit I contains two heme groups and a Cu ion (CuB).
What is the importance of subunit III on complex IV?
subunit III doesn’t have a copper centre, but it is believed that it is important for complex IV by binding to oxygen and only allowing for its release if it gets reduced to water first
Describe electron transfer at complex IV
Not all 4e- comes at the same time. One O2 is trapped and CytC brings 1e- to CuA at subunit II, then Fe-S a and a3, then CuB all on subunit I. Once all 4 electrons have done this, O2 gets reduced to 2H2O
What is complex IV affected by?
CO and CN-
__________ __________ occurs as energy released from oxidation at complexes leads to formation of ATP
energy coupling
Indicate how Slater believed oxidation of NADH and FADH2 is coupled to phosphorylation of ADP and the problems with his theory:
Slater thought it was like gapdh coupling of oxidation to phosphorylation.
Indicate how Boyer believed oxidation of NADH and FADH2 is coupled to phosphorylation of ADP and the problems with his theory:
Conformational-coupling hypothesis by Boyer
Indicate how Mitchell believed oxidation of NADH and FADH2 is coupled to phosphorylation of ADP:
Chemiosmotic hypothesis by Mitchell
Provide the 4 components of Mitchell’s chemiosmotic hypothesis:
- Intact inner mitochondrial membrane is required for oxidative phosphorylation
How did they find proof of proton gradient generation?
They noticed that the pH is lower in the intermembrane space compared to the matrix
Why is ATP from NADH and FADH2 2.5 and 1.5 respectively instead of 3 and 2 as the 3H+ per ATP would indicate?
Some of the gradient is lost by leakage through the membrane
What indicators are present for electron transport/oxidation and phosphorylation?
Oxygen consumption is used to measure the amount of oxidation aka electron transport
Describe the experiments that proved that oxidation and phosphorylation are coupled
Oxidation and phosphorylation were found to be interdependent
Describe experiments with isolated mitochondria
They isolated mitochondria first
Explain the proton pump mechanism
reduction causes conformational change, reoxidation restores original conformation
Explain the proton pump mechanism in complex I
Reduction of iron sulfur centre of complex I to change conformation, then CoQ10 is oxidizing agent as it is reduced to oxidize the complex so it goes back to original conformation.
Name and describe the support for the proton pump mechanism:
Bacteriorhodopsin proton pump that uses light energy to drive conformational change to release H+ outside
Inside it is more ____________ and ____________, outside it is more ____________and ____________.
basic, negative
How did they do atp synthase tests?
purified F1 bc it hangs out and salt could break protein interactions with innermb bound f0
Which complex of ATP synthase is water soluble and which is not?
F0 is water insoluble and F1 is water soluble
What does F1 do when isolated?
F1 is able to HYDROLYZE ATP in isolation (cannot synthesize ATP)
What is used to block ATP synthesis and how?
DCCD blocks F0 - so blockage of H+ transport.
Describe ATP synthase movement to produce ATP
Protons rotate the barrel (F0), which moves “eta?”, moving gamma, which changes conformation of alpha-beta that makes ATP
Describe structure of F1
Three alpha beta dimers
What are the 3 conformations of ATP synthase and in what order does it move?
T –> tight - ATP is made and holds to it tightly
get info for bottom half of slide 40
okay
How much atp is made as the cycle turns?
When 3 protons come through the barrel, 120 degrees of this cycle on the right occurs.
What drives the ATP synthase activity forward specifically?
Adding energy opens conformation to let ATP go, but once atp leaves and adp binds, the atp synthesis occurs right away.
Describe the experiment that showed that ATP synthesis and hydrolysis occur at surface of F1 and what this means in terms of energy of synthesis:
When they studied deltaG of atp hydrolysis (bc f1 by itself)
How did they prove that this synthase movement is how ATP is made?
Did the reverse experiment.
Describe what the electrochemical gradient is used for: (3)
- Rotate ATP synthase for ATP synthesis
Why is the antiport of ATP outside and ADP inside favourable?
Transports net negative charge out which is a favourable reaction.
Pyruvate also gets transported in from cytosol. True or false?
TRUE
Transport of various metabolites in and out of mitochondria uses electrochemical gradient energy. true or false?
TRUE
get info from slide 46
okay
add nadh hydride ion info on slide 46 too
okay
Describe the glycerol 3 phosphate shuttle:
In skeletal muscle and brain
How much ATP does skeletal muscle and brain get from glucose
So we said to write 32 ATP from glucose
How is oxidative phosphorylation decoupled by chemicals?
gradient can be uncoupled by DNP and FCCP
Just read
okay
Describe the physiological dissipation of H+ gradient:
thermogenin - in brown adipose tissue of newborn - produces heat
Why do babies need to produce heat by BAT?
Our body has our own uncoupler
Describe how thermogenin is an uncoupling protein:
When proton passes through thermogenin channel, it converts gradient energy into heat.
Describe heat production in Skunk Cabbage (incomplete)
Uses short circuit of electron transport
Under emergency situation, is ETC up regulated?
Not really, glycolysis is
Talk to me about meat.
White meat –> very few mitochondria
Describe regulation of F1 of ATP synthase
If gradient across mb is low then F1 unit is crazy guy. Cannot synthesize ATP. Start hydrolyzing ATP. IF1 protein binds to neighbouring F1 units and inhibits hydrolysis when we need to save the ATP.
IF1: Inhibitor of F1, a peptide of 84 amino acid residues, binds to two F1 subunits and blocks ATPase activity during Ischemia or hypoxia. true or false?
TRUE
I have given up at slide 54. 55-61 just read slides and dont be dumb.
Okay
