Respiratory Chain and ATP synthase I

Question 1

What are the four steps during pyruvate oxidation where NADH is produced?

Answer 1

1. pyruvate dehydrogenase (NADH)
2. isocitrate dehydrogenase (NADH)
3. alpha-ketoglutarate dehydrogenase (NADH)
4. malate dehydrogenase (NADH)

Question 2

What step during pyruvate oxidation is FADH2 produced?

Answer 2

1. succinate dehydrogenase (FADH2)

Question 3

What must happen to NADH and FADH2 for the TCA cycle to continue?

Answer 3

They are reduced so they must be re-oxidized

There are limited amounts of these coenzymes in the mitochondria and they must be continuously recycled between the oxidized and reduced states

Question 4

T or F. NADH and FADH2 are energy rich compounds

Answer 4

T. ATP is an energy rich chemical compound and we have made it from NADH and FADH2, so these must also be energy rich compounds.

Question 5

Why are NADH and FADH2 considered high-energy?

Answer 5

The electrons in NADH and FADH2 are at a very high negative reduction potential. Their tendency will be to move to more positive reduction potentials and release free energy as they move.

Question 6

Do we get more energy out of NADH or FADH2?

Answer 6

NADH (~2.5ATP) vs FAD2H (~1.5ATP).

Question 7

What is Complex 1 of the ETC?

Answer 7

NADH Dehydrogenase complex. (contains 45 subunits in humans)

Question 8

How many of the subunits of complex 1 are encoded by mitochondrial genome?

Answer 8

Seven of its subunits are coded in the mitochondrial genome. This is highly significant, since only 13 protein coding genes are found in the mitochondrial genome and more than half of them are for complex I

Question 9

What are the two domains of complex I?

Answer 9

two domains- the membrane arm and the peripheral arm

These seem to be assembled separately.

Question 10

What does the peripheral arm contain?

Answer 10

most of the redox active centers. contains FMN (flavomononucleotide) site

Question 11

What does the membrane arm contain?

Answer 11

all 7 of the mitochondrially encoded subunits.

Question 12

What is rotenone?

Answer 12

(a fish poison) an inhibitor that binds to complex I and competes at one of the ubiquinone binding sites (there are two). When rotenone binds, electron transfer from complex I is blocked.

Question 13

Describe the ubiquinone binding sites of complex I.

Answer 13

There are two.

One is tightly bound and does not seem
to come off in purified preparations. The other is loose and presumably is the one that can transfer electrons from complex I to complex III

Question 14

How many electrons and protons are pumped for each NADH oxidized?

Answer 14

2e- and 4 protons (H+)

Question 15

What are the subunits of complex I’s membrane sector?

Answer 15

NuoM, NuoN, and NuoL (similar in structure) and a long helix HL (that runs perpendicular to the other three subunits)

The movement might be passed on to the long helix HL causing it to shift the angle of the red helices. These helices have a conserved glutamate in the middle where the helix is disrupted. Movement of this key glutamate may alter its exposure to the two membrane surfaces and shift its pK to allow protons to be pumped.

Question 16

Why is flavin needed between NADH and iron sulfur centers?

Answer 16

Flavins are needed as a “middle man” between the transfer of electrons from NADH to iron sulfur centers. Flavins (like FMN on complex I and another on complex II) accept the electrons first as a pair. NADH is an obligatory 2 electron acceptor and cannot directly donate its electrons to iron because iron is an obligatory 1 electron acceptor. Therefore, having flavin as an intermediate electron acceptor allows for the iron sulfur centers to accept the electrons one at a time.

Question 17

What is the role of Ubiquinone? Where does it exist?

Answer 17

it is a lipid soluble electron carrier. It exists in the mitochondrial inner membrane and it carries electrons between complexes (from complex I and complex II to complex III) in the electron transport pathway

Question 18

How many electrons can ubiquinone accept at one time?

Answer 18

one electron at a time (like iron clusters)

Question 19

What is ubiquinone called when it accepts and is then bound to a single electron? How is this different from fully reduced (bound to two e-) or fully oxidized (bound no 0 e-) ubiquinone?

Answer 19

a semiquinone. Semiquinone is unlike fully oxidized or reduced ubiquinone in that it is stabilized by binding to protein sites and does not float around freely in the mitochondrial membrane

Question 20

How many protons are pumped at complex I?

Answer 20

4 protons per NADH

Question 21

How many protons are pumped at complex II?

Answer 21

No protons.

Question 22

How many protons are pumped at complex III?

Answer 22

4 protons per NADH/ FADH2

Question 23

How many protons are pumped at complex IV?

Answer 23

2 protons per NADH/ FADH2

Question 24

What are Iron-sulfur clusters?

Answer 24

redox active centers that can accept and then donate electrons in the electron transfer pathway

These act as a conduit for the electrons to travel from the FMN to ubiquinone

Question 25

What is Complex II composed of? How many proteins does it contribute to the pump system?

Answer 25

(aka Succinate dehydrogenase) a tetramer of non-identical subunits. It contains FAD and three iron sulfur clusters

None of its subunits are coded in the mitochondrial genome, and it cannot pump protons across the inner membrane, so it does not contribute to the proton gradient like complexes I, III and IV

Question 26

disease in humans that is caused by a defect in iron sulfur cluster formation in proteins would cause deficit in which complexes?

Answer 26

complex I, aconitase in the TCA cycle, complex II and complex III

Question 27

Where is FMN found on complex I?

Answer 27

on the peripheral arm (The fact that FMN is bound to Complex I makes this a flavoprotein)

Question 28

What are the 4 Flavoproteins that reduce ubiquinone in the mammalian electron transport pathway?

Answer 28

1. Complex I
2. Complex II
3. Electron transfer flavoprotein dehydrogenase (ETF-QO)
4. sn-glycerophosphate dehydrogenase (an NADH shuttle)

Question 29

What do all flavoproteins have in common? Why?

Answer 29

the flavin gets the electrons first and then passes them on to the iron-sulfur centers

This is absolutely required since NADH is an obligatory 2 electron donor and Fe3+ is an obligatory one electron acceptor. So, NADH cannot donate its electrons directly to iron.*

There has to be an intermediate electron acceptor that can accept two electrons and give them to iron one electron at a time. That is why flavins get the electrons first.

Question 30

What is the only TCA cycle enzyme to be an integral membrane protein?

Answer 30

succinate dehydrogenase (aka complex II of protein pump complex)

Question 31

Describe the subunits of Complex II and the flow of electrons

Answer 31

Complex II has two hydrophobic membrane
subunits that are fairly small (may have a heme in the center between the two subunits).

The electrons flow from succinate to FAD then to the iron sulfur clusters and on to the ubiquinone. Notice the cardiolipin associated with the membrane sector.

Question 32

The mitochondrial sn-glycerophosphate dehydrogenase shuttle contributions electrons to which complex?

Answer 32

complex II. Electrons from cytosolic NADH are transferred to DHAP to form glycerol 3-phosphate and then back to the membrane bound glycerol 3-phosphate dehydrogenase FAD coenzyme and then on to ubiquinone in the membrane

Question 33

Complex III is aka?

Answer 33

the bc1 complex

Question 34

How many subunits does complex III have?

Answer 34

11 in humans but only three are redox subunits.

Question 35

What are the subunits in complex III that are not redox centers called?

Answer 35

supernumerary subunits

Question 36

What are the important components of complex III?

Answer 36

a) two b type hemes that are contained in the same subunit and are directly above one another in the membrane. They are perpendicular to the membrane.
b) At the cytosolic side of the membrane there is the Rieske iron-sulfur protein.
c) Also on the cytosolic side of the membrane is cytochrome c1

Question 37

How does complex III work?

Answer 37

the protonmotive Q cycle, or just the Q cycle

Question 38

What is the initial step of the Q cycle involving ubiquinone?

Answer 38

At the start, fully reduced ubiquinone diffuses to the cytosolic side of the membrane where it binds to a site known as center P (P for positive side of the membrane) or the oxidation center, because the ubiquinone will become oxidized here

Question 39

What happens to the released electrons from oxidized ubiquinone in complex III?

Answer 39

The two electrons will be split into two different paths, one going to the Rieske iron sulfur center (reduction potential +290 mV) and the other going to the first b heme called BL (for its low potential -20 mV).

As we discussed earlier, electrons will prefer to choose the pathway toward the most positive reduction potential so they would prefer to both go to the iron-sulfur center, one at a time rather than split up and go both ways.

Question 40

If the electrons from oxidized ubiquinone in complex III would both prefer to go to the Rieske iron sulfur center, why do this split?

Answer 40

The complex has a structure that forces this splitting to take place. Iron can only accept one electron at a time, so both the Rieske protein and the BL cytochrome can only take on one electron. The protein forces them to go different ways by requiring that both electrons are released from ubiquinone simultaneously. This way they have to go to two different acceptors because each acceptor can take only one electron

This bifurcation of electron flow is unique to the bc1 complex.

Question 41

How many ubiquinone molecules can bind at the P center at complex III? What role(s) do each play?

Answer 41

two. One is ten times more tightly bound than the other and acts as a prosthetic group, never leaving the enzyme. This tightly bound ubiquinone is oxidized at the start of the cycle. In this paired arrangement, the major energy barrier to reaction is deprotonation of the loose ubiquinone. This occurs first before the electrons move.

Then in a more speculative part of this model, one electron moves from the deprotonated ubiquinone to the tightly bound form and they both become semiquinones. It is these two semiquinones that simultaneously give up their electrons to the two different pathways. Only the loosely bound form is released into the membrane as a fully oxidized ubiquinone

Question 42

What happens to the electron that goes to the Rieske protein from ubiquinone?

Answer 42

It goes from the Rieske iron-sulfur protein to
cytochrome c1 and is finally passed on to cytochrome c. This electron eventually is used to reduce oxygen in complex IV, cytochrome c oxidase

Question 43

What happens to the other electron from oxidized ubiquinine (the one that goes to the first b heme)?

Answer 43

This non-productive electron passes on to the second b heme BH (for high reduction potential +50 mV). Finally this electron is given to a fully oxidized ubiquinone bound near the matrix side of the membrane at a site called the N center (N for negative) or the reduction center, because ubiquinone becomes reduced here. This is a different site than the P center and the ubiquinones are different. This electron transfer forms a semiquinone that stays tightly bound to this site. In a second reaction, another ubiquinone binds at center P and undergoes the same reactions as before. The low potential electron moves down through the two b hemes to the semiquinone and fully reduces it. This ubiquinone must pick up two protons from the matrix side of the membrane then it is released.

Question 44

The net movement of protons out of the mitochondrion from complex III has to be paid for by free energy release of the electrons flowing down their pathways. The majority must come from transfer to the ____ since it has by far the biggest drop in reduction potential.

Answer 44

Rieske iron-sulfur protein

Question 45

What are the main inhibitors that binds to the bc1 complex and how does it work?

Answer 45

a) Antimycin binds very tightly to the N center at the matrix side of the membrane and prevents electrons from reaching ubiquinone from the b hemes.

b) Stigmatellin binds at the P center, at the interface between the iron-sulfur protein and
cytochrome b. It prevents entry of the loosely bound ubiquinone and stops electron flow through the complex.

Question 46

How many subunits does Cytochrome c oxidase have? How many are from the mitochondria genome?

Answer 46

13. Three subunits are from the mitochondrial genome and these contain all the redox centers. The 10 nuclear coded subunits seem to be embellishments

Question 47

T or F. the channel for oxygen to reach the active site appears to open in the lipid bilayer.

Answer 47

T

Question 48

How do the electrons get to cytochrome oxidase?

Answer 48

They come on the carrier cytochrome c. Cytochromes can only deliver one electron at a time

Question 49

How many electrons does it take to reduce oxygen? So how many cytochrome c carriers are needed?

Answer 49

4; 4 carriers are need since Cytochromes can only deliver one electron at a time

Question 50

What is charge compensation?

Answer 50

Adding a charge to a protein interior, such as placing an electron into cytochrome c oxidase is thermodynamically expensive. One way to compensate for the cost is to take up a proton

cytochrome c oxidase is an extreme example of charge compensation. Four electrons are taken in and four protons are taken up

Question 51

What are the redox centers on complex IV?

Answer 51

There are 2 copper centers called copper A and copper B. Copper A actually has two copper ions near one another. There are two hemes of the a type. These are called heme a and heme a3. There is also a

magnesium ion and a zinc ion, though the role these play is not understood

Question 52

What is the first place the electron goes upon leaving cytochrome c?

Answer 52

The copper A site

Question 53

Are both copper A and copper B sites vital to making complex IV work?

Answer 53

Yes. Experiments have been done that modify this site so it binds only a single copper ion instead of two. The enzyme activity is nearly completely lost, so both coppers are required for function

Question 54

Where do electrons go (one at a time) from the copper A site?

Answer 54

From the copper A site the electron migrates to heme a then to heme a3

NOTE: There is no obvious reason to have two heme “a”s in the enzyme. Both hemes are about equal distance from the copper A site and theoretically, the electron from Copper A could go directly to heme a3, but it does not. The structure suggests a network that would favor electrons moving to heme a first

Question 55

Heme a3 and copper B form what?

Answer 55

the binuclear center. The heme a3 Fe is very close to the copper B ion. This is critical in the chemistry of the enzyme.

Question 56

How is copper B involved with proton pumping?

Answer 56

One site that takes up a proton during the entry of the four electrons is the copper B. When an electron moves from heme a to the heme a3, copper B binuclear center, the proton that was associated with copper B is forced to leave. This might be part of the driving force for proton pumping.

Question 57

Since 4 electrons have to come in to reduce oxygen, what is the order of addition?

Answer 57

Experiments with various types of spectroscopy show that 2 electrons enter the heme a copper B site, one on each metal ion, then oxygen binds between them and both electrons are added to the dioxygen molecule at the same time. This forms a peroxy bridge between the two metal ions Fe-O-O-Cu. Now two protons come in and one more electron and the O-O bond is broken. Another 2 protons and another electron finish the reduction to water. The two waters leave through a water channel.

Note that while this is going on 4 more protons are being pumped across the membrane through the protein.

Question 58

Notes on the higher level structure in the electron transport pathway

Answer 58

Recent work has shown that complex I, a dimer of complex III and complex IV are associated together in a supercomplex. This allows efficient transfer of electrons down the pathway. The binding sites for ubiquinone on complex I and complex III are close so diffusion between the sites is nearly direct. The same is true for the cytochrome c binding sites on complex III and IV. They are adjacent. Cytochrome c probably does not have to leave the protein complex.