2.2.2. ETC Flashcards
How many protein complexes are in the ETC?
4
Where are each of the complexes in the ETC found?
In the Inner mitochondrial membrane
Characterize Complex I
(Name, properties/what is in it, what it does)
Name: NADH dehydrogenase (aka NADH-Q reductase)
Properties: NADH binding site,
FMN (flavin mononucleotide) to bind electrons, chain of Fe-S electron binding centers
binding site for CoQ
What it does: Oxidizes one NADH to NAD+ and H+ and pumps 4 H+ into intermembrane space
Characterize Complex II
(Name, properties/what is in it, what it does)
Name: Succinate Dehydrogenase
Properties: Directly involved in the TCA cycle, contains FAD
What it does: Reduces FAD to FADH2, and those Hs are used by CoQ
Characterize Complex III
(Name, properties/what is in it, what it does)
**Name: **Cytochrome b-c1 (aka Cytochrome reductase)
**Properties: **CoQ binding site
**What it does: **Uses electrons from CoQ to pump 4 H+ into the intermembrane space
Characterize Complex IV
(Name, properties/what is in it, what it does)
**Name: **Cytochrome Oxidase
**Properties: **Able to grab electrons from cytochrome C
**What it does: **Pumps 2 H+ into the intermembrane space while making 1 H2O
Flavins are associated with which proteins in the ETC?
Complex I and II
Fe-S clusters are associated with which ETC proteins?
Complexes I, II, and III
CoQ (Coenzyme Q or Quinone) is associated with which proteins from the ETC?
NONE!
It is trapped inside the inner mitochondrial membrane
BUT, it does transfer electrons and protons from Complex I to Complex III
How many different Heme groups are associated with cytochromes and what are their names?
(Bonus: Name any special characteristics of each)
3
A, B, and C
A: Has 3 isoprene units
B: common structure found in hemoglobin
C: Has protein bound by two Cysteine residues at the S atom
Which complex is Heme a associated with?
Complex IV
Which complex is Heme B associated with?
Complex III
Which complex is Heme C associated with?
Complex III
Copper containing groups are associated with which Complex in the ETC, and what is its use?
Complex IV
Copper is a great metal to use for passing electrons through
How is FAD or FMN useful for testing whether mitochondria are present and or active?
Based on its oxidation state, these flavin groups will give off different wavelengths of light
FAD/FMN: Yellow
FADor FMN semiquinone (one electron): Blue or Red (depending on pH)
FADH2/FMNH2: Colorless
What shapes and colors can the Fe-S clusters take?
The Fe-S clusters can take a cuboidal or planar shape, but both of these are brown in appearance
What oxidation state(s) can Fe take in the Fe-S clusters in the ETC?
Fe3+
Fe2+
What does CoQ do in the ETC?
Carries electrons and protons
Why is CoQ trapped inside of the inner mitochondrial membrane? What keeps it there?
It is trapped in the membrane because it is highly hydrophobic. It is trapped there by an isoprene group repeated ten times, forming a 50 Carbon tail
Name the two major subunits that make up ATP Synthase
F0 and F1
What are the subunits of the F1 complex of ATP Synthase
3 alpha-beta (1 alpha with 1 beta) subunits
1 gamma subunit
1 delta subunit
1 epsilon subunit
What are the subunits of the F0 complex?
A
B (2 B subunits)
C (10 c subunits make up the proton motor)
What is the role of the b subunit within F0 of ATP Synthase?
It serves two roles:
An anchor in the lipid membrane
Stabilizing the alpha-beta subunits of F1 (which keeps them from rotating while the gamma portion of F1 rotates)
What is the role of the c subunit within F0 of ATP Synthase?
The c subunit facilitates proton movement cross the inner mitochondrial membrane through an internal hydrophilic channel
What two molecules are critical to the function of the c subunit of F0 of ATP Synthase?
Glutamyl carboxyl group (binds protons) (negative charge)
Arginyl group (positive charge)
How does the c subunit of the F0 portion of ATP Synthase rotate?
When the glutamyl group accepts a proton from the intermembrane space, it rotates the C subunit, exposing another proton accepting group to the intermembrane space.
The rotation is completed by the attraction between the glutamyl group (with a negative charge) and an arginyl group (with a positive charge)
What kind of forces are responsible for the desire of protons to enter the mitochondrial matrix? (Name two)
Electrical gradient: Electric potential of -.16V
Concentration gradient: pH difference of about 1
What do we call the “pull” protons feel to enter the mitochondrial matrix?
The Proton Motive Force (PMF)
Is the PMF a from of continuous energy, or is it quantitative?
It is a continuous form of energy
How do we establish this PMF?
By pumping protons into the intermembrane space using the ETC
How can we measure mitochondrial activity?
We can take “oxygraph” measurements to take experimental values of mitochondiral activity. This measures O2 levels while adding different substrates to our mitochondria (in a tube, or vial, or whatever)
Explain how the “oxygraph” measurements experiment works.
Think two big steps.
1) Add a reducing agent (in our cells, this is NADH) to our tube, which allows for generation of the PMF.
2) After some PMF has been generated, add ADP to allow for ATP synthesis to occur
After adding NADH to our “oxygraph” tube, what kind of change in O2 should we see?
A small drop occurs initially as we generate our beginning PMF.
See point A in graph below
Why do we see only a small drop in O2 levels after adding NADH?
NADH helps to generate the PMF, but the stronger the PMF gets, the harder it becomes to pump protons across the membrane, leading to a halt of the process. This halt is what prevents much O2 from being converted to water.
What kind of drop in O2 do we see after adding ADP to our “oxygraph” tube?
We see a large drop in O2 levels.
See point B in graph below
Why do we see a large drop in O2 levels after adding ADP to our “oxygraph” tube?
We see a large drop because the ADP allows for ATP to be synthesized, helping to dissipate the PMF. The excess NADH sitting in our tube from before may now restore the PMF as ATP is made, allowing for a sharp decline in O2 levels.
What does the P/O ratio tell us with regards to oxidative phosphorylation?
It is the amount of ATP that can be made with a certain amount of oxygen
Why is the P/O value of NADH different than that of FADH2?
They pump different numbers of protons into the intermembrane space
NADH pumps 10 (leads to 2.5 ATP)
FADH2 pumps 6 (leads to 1.5 ATP)
Recall: (4 H+ = 1 ATP)
What happens to mitochondria when there is no oxygen available to the cell?
The mitochondria swell, and given enough time, this will lead to cell lysis and death.
What do poisons do to the ETC? (In general, how do they interfere with its function?)
Poisons prevent certain enzymes from working, thus preventing the creation of their products and halting the whole process.
When a poison stops an enzyme from working in a cycle like the ETC or the TCA, what happens to the substrate?
Because the enzymes in this process are reductases, the substrates that cannot be enzymatically reduced simply get reduced over time.
NADH dehydrogenase is blocked by what substances?
Rotenone (broad spectrum insecticide)
Amytal (aka amobarbital, a barbiturate)
What substance can block the function of cytochrome b?
Antimycin A (a fungicide)
What substances block the function of a and a3?
Cyanides, Nitrates, and Carbon Monoxide
These do this by preferntially binding to the Fe that O2 normally binds to.
What do uncouplers do to the ETC and TCA cycle?
Uncouplers allow leakage of the PMF, leading to unrestrained O2 useage.
Why is uncoupling the ETC/TCA dangerous?
While the proton gradient gets degraded, the cell will try to restore it, and spend energy doing so. However, ATP may not be getting generated during this time, so the cell will uselessly use up energy, to the point of starvation.
What is DiNitroPhenol (DNP) and how does it function?
DNP is a chemical uncoupler, it functions by carrying protons across the inner mitochondrial membrane. DNP is lipophilic, but has space for protons, which it can simply carry across the inner mitochondrial membrane and drop off in the matrix. (It does this because of the varying pH levels in the two spaces.)
What do ATPase inhibitors do?
They stop the ATPase from using the proton gradient to create energy, so the proton gradient stops dissipating and ATP is not made.
Name an ATPase inhibitor
Oligomycin (an antibiotic)
Which process blocks the ETC/TCA and which process stimulates uncontrolled oxygen use?
Uncoupling causes uncontrolled oxygen use
Poisoning or drugs leads to blockage of the TCA/ETC
What is Leber Hereditary Optic Neuropathy?
A hereditary condition causing acuteoptic nerve atrophy, causing blindness
In what organelle does the cause of LHON originate?
LHON is caused by a defect in the mitochondria (i.e. it is a mitochondrial disease)
What kind of inheritance does LHON exhibit?
It exhibits maternal inheritance because it is a mitochondrial disease, so fathers cannot pass on this disorder.
What is the issue within mitochondria that causes LHON to arise?
The mitochondria have a mutation in the genes coding for Complex I subunits (in 90% of cases)
This prevents electrons from being transferred from Complex I to CoQ, therefore interfering with ETC function
Does LHON show heteroplasmy
Yes, depending on the mitochondria the mother had, cells will display varying numbers of “sick” and “well” mitochondria
When does LHON start to show up in patients?
It typically occurs around the age of 27, but may occur between 15-35.
This makes it a late onset disorder
What is Myoclonic Epilepsy and Ragged Red Fibers (MERRF)?
It is a disease caused by mutations affecting the rRNA or tRNA in the mitochondria.
What are the symptoms of MERRF and when does it show in patients?
Typically present in teens.
Presents with gradual muscle weaknessthat occurs in a progressively degenerating nature.
Which tRNA’s are typically affected the most by MERRF?
Leucine and Lysine
Mitochondrial Encephalomyopathy, Lactic Acidosis, and Strokelike episodes (MELAS)is caused by what?
Mutations in the genes encoding tRNAs or rRNAs of the mitochondria.
Histologically, what do we observe with MELAS?
Inappropriate cell proliferation (an increase) with mitochondrial aggregation.
How do we treat MELAS?
By using Quinone-like drups to bridge the gap created by these mutations, allowing for the shuttling of electrons.
In research: Use of viruses to infect MELAS cells and “donate” healthy genes to these cells
What are the three major stages of electron transport in the ETC?
1) NADH to CoQ
2) CoQ to ctyo C
3) Cyto C to Oxygen
