2- Mitochondria ETC/Respiratory Chain/Oxidative Phosphorylation

Question 1

what happens to most of the energy released from glycolysis and TCA?

Answer 1

its captured by NADH and FADH2

Question 2

what happens (big picture) to the electrons released from NADH and FADH2?

Answer 2

they flow through the mitochondrial electron transport chain to eventually reduce O2 to H20 as the final electron acceptor (this is why we breathe in O2… >95% of the O2 we use is used by the electron transport chain)

the energy of those two are stored as a proton gradient established across the mitochondrial inner membrane

Question 3

final equation

Answer 3

NADH + 1/2 O2 + H+ —> NAD+ + H2O

Question 4

how many atp does glycolysis/oxidative phosphorylation produce?

Answer 4

glycolysis - 2

oxidative phosphorylation ~36

Question 5

what will stop the ATPase from pumping?

Answer 5

1. dont have ATP
2. if there is no longer a concentration gradient you are working against
3. build up of ADP
4. build up of protons inside the vesicle that use the ATPase

ATPase will keep working as long as you have a source of protons

Question 6

given an impermeable membrane and an ATPase pump that can translocate H+, what happens if you add H+ inside the vesicle?

Answer 6

ATP will be made from ADP

Question 7

if the proton circuit is uncoupled and there is a leak then what happens?

Answer 7

the gradient will bleed out and it will destroy the energy used for the reaction

Question 8

what do you need to build a proton circuit to make ATP?

Answer 8

1. impermeable membrane
2. e- carriers (to hand off e-)
3. proton pumps (to make graident)
4. ATPase

Question 9

Explain the impermeable membrane that the mitochondrial inner membrane has

Answer 9

low sterol, cardiolipin, TONS of proteins (60-70% of weight)

has a lipid bilayer

Question 10

explain the electron carriers in the e- transport chain

Answer 10

more than 20 redox carriers exist

• ubiquinone (Coenzyme Q)
• flavoproteins w/ tightly bound FAD/FMN
• cytochromes (a, b, and c)
• Fe/S proteins (Fe3+ +e- —> Fe2+)
• protein bound Cu (Cu(II) + e- —> Cu(I))

Only mobile carriers are CoQ and cyt c which shuttle around and are not bound by proteins

Question 11

is e- flow down a thermodynamic gradient favorable?

Answer 11

YES! it is favorable and youre going from one e- carrier to another just handing it off

Question 12

O2 oxidant or reductant?

Answer 12

O2 is a STRONG oxidant which means it has a high affinity for electrons (0.82)

Question 13

which complexes do not pump H+ from the matrix to the IMS

Answer 13

Complex II is the only one that does not

I, III, and IV pump H+ to the inner mitochondrial space

all of these are thermodynamically favorable and youre using this to move protons across an unfavorable gradient

Question 14

proton pumps

Answer 14

complexes I, II, III, IV, and V

V is ATP synthase

Question 15

ATPase vs. ATP synthase

Answer 15

ATPase- hydrolyses ATP

ATP synthase- makes ATP

Question 16

Proton pump: Complex I

Answer 16

NADH-CoQ reductase. Transfers two e- to ubiquinone which is a mobile carrier that floats b/w complex 1 and 3 and transfers those e- to complex 3.

in the process of doing that it pumps 4 protons into the inner-membrane space

Question 17

Proton pump: Complex III

Answer 17

complicated cycle referred to as the q cycle

takes 2 protons from ubiquinole (reduced version of ubiquinone) and takes two protons from the matrix and pumps all 4 of these into the inner-membrane space

Question 18

cytochrome c

Answer 18

skates along surface of outer membrane and dumps electrons from complex 3 onto complex 4, which is where O2 is consumed and H2O is made

Question 19

Proton pump: Complex IV

Answer 19

gets e- from cytochrome c and O2 is the final e- acceptor here. it also takes 4H+ from the matrix and pumps 2H+ into the inner membrane space from the matrix and uses the other two to reduce O2 to H2O

Question 20

where does the TCA cycle occur?

Answer 20

mitochondrial matrix

Question 21

where does oxidative phosphorylation occur?

Answer 21

in the inner membrane space

Question 22

Complex II

Answer 22

Succinate dehydrogenase

succinate-CoQ reductase

forms fumarate from succinate and also generates ubiquinole (reduced form of ubiqunone) and transfers those e-

this complex is especially important if you have a complex I deficiency cause you might be able to bypass complex 1 in order still make ATP (youd just wanna stay away from a ton of carbs but youd want protein/fatty acids so you can form succinate straight away)

Question 23

megacomplexes

Answer 23

mitochondrial respiratory complexes can form megacomplexes that have I,III,IV all attached together and another megacomplex that has I,II,III, and IV attached.

this supercomplex allows for efficient transfer of e- and pumping of protons
-important for quick energy demand

Question 24

Ubiquinone/ubiquinol pool

Answer 24

can be fed into from Complex I or II which then goes to complex III which goes to cytochrome c which goes to complex IV which gives the e- to O2

Question 25

what are the other ways to get e- into the electron transport chain without using the proton pump complexes?

Answer 25

1. B-oxidation
2. pyrimidine biosynthesis
3. glycolysis

All of these dump e- into the ubiquinone/ubiquinol pool

Question 26

Complex I diseases

Answer 26

LHON
MELAS
Leigh’s syndrome

Question 27

Complex II diseases

Answer 27

leigh’s syndrome

Question 28

Complex III diseases

Answer 28

Cardiomyopathy

leigh’s syndrome

Question 29

Complex IV diseases

Answer 29

ALS-like syndrome

leigh’s syndrome

Question 30

Complex IV inhibitors

Answer 30

These block heme groups in complex IV

Cyanide (CN-)
Azide (N3-)
Carbon Monoxide (CO)
Nitric Oxide (NO)

Question 31

Treatment of cyanide poisoning

Answer 31

Essentially giving them a treatment that will shift the equilibrium from Hb binding to cyanide to Hb binding to O2 again.

-administering nitrite, which converts Hb to metHb to bind cyanide and then giving thiosulfate to allow metabolism of cyanide to less toxic thiocyanate by rhodanese

Question 32

inner mitochondrial membrane is impermable, so how do we get substrates/products across?

Answer 32

1. ATP-ADP translocase (takes ATP out)

and a ton of other carriers so this problem has been solved and these carriers are linked to metabolism based on what the body needs.

Question 33

How do you get NADH from cytoplasm into inner mitochondrial matrix?

Answer 33

1. Malate- Aspartate Shuttle

2. Glycerol Phosphate Shuttle

Question 34

Malate- Aspartate Shuttle

Answer 34

NADH gives up e- to oxaloacetate which forms malate

malate then gets transported into the matrix (a-ketoglutarate goes in the opposite direction)

then malate becomes oxaloacetate again turning NAD+ into NADH

Irreversible but forms NADH in the matrix again

There is also a aspartate/glutamate side where glutamate goes into the matrix and aspartate goes out.
-glutamate transfers its amino group to oxaloacetate, making OAA into aspartate and converting glutamate to a-ketoglutarate

Question 35

outer mitochondrial matrix

Answer 35

very permeable in comparison to the inner mitochondrial membrane.

Question 36

Glycerol Phosphate Shuttle

Answer 36

irreversible and yields FADH2 and then reduced Coenzyme Q (rather than NADH)

Shuttle makes FADH2 and it then gives 2e- to CoQ to form CoQH2 which continues electron transport by giving e- to Complex III

Question 37

Electron Transport Chain vs. Oxidative Phosphorylation

Answer 37

ETC- complex I-IV
OP- Coupling e- transport to ATP Synthase, the process as a whole

ETC is part of OP

Question 38

big picture of oxidative phosphorylation (using chemiosmotic hypothesis)

Answer 38

protons are pumped then flow back into the matrix to drive the unfavorable reaction, which happens in the cristae (invaginations of the inner mitochondrial membrane)

electron transport generates a proton gradient across an impermeable membrane. that gradient supplies the energy for ATP synthesis

about 35% efficient from P/O ratio

Question 39

proton-motive force (delta.p)

Answer 39

2 parts

1. membrane potential (electrical)
2. chemical gradient (concentration)

Note that with 10 protons pumped/NADH oxidized, virtually all the energy of NADH (52.6 kcal/mol) is captured in the proton gradient

“making of the battery” is incredibly efficient

Question 40

ATP synthase and its subunits

Answer 40

Key regulatory factor: levels of ADP (low ADP = free L site, rotation of gamma subunit stops and no ATP synthesis occurs)

one full rotation makes 3ATP and uses 9H+ ions

Fo: in the membrane and pumps H+ into the matrix

F1: On the inner matrix side, ATP synthesizing unit, has L.O.T subunits which spin and create ATP from ADP and Pi

Question 41

Uncouplers

Answer 41

ETC and ATP synthesis are very tightly coupled!

dinitrophenol is an uncoupler along with valinocycin and gramicidin (the last two are antibiotics)- these uncouple mitochondria by rapidly replacing ejected H+ by K+ or Na+, thus dissipating the electrical component of teh proton-motive force.

Question 42

Respiratory Control Index/Ratio (RCI or RCR)

Answer 42

RCI is a measure of the degree to which ETS and OxPhos are coupled. it reports on health of the mitochondria 
state 3(ADP)/state 4 WHICH IS active/resting

Question 43

Phosphorylation efficiency

Answer 43

P/O ratio. refers to number of high energy phosphate bonds formed per O reduced to water. NADH = 2.5, FADH2 = 1.5

P/O ratio = molecules of ATP made / atoms of oxygen reduced

• each ATP made requires 3H+ translocated
• 1 H+ required for transport of Pi, ADP, and ATP
• Net: 1 ATP requires total of 4 H+

Question 44

P/O for NADH

Answer 44

10/4 = 2.5 ATP/O

Question 45

P/O for succinate/FADH2

Answer 45

6/4 = 1.5 ATP/O

Question 46

Physiological Uncoupling

Answer 46

• Brown adipose tissue (brown fat/BAT) in newborns or hibernating mammals is important in thermogenesis
• rich in mitochondria that contain Uncoupling Protein-1 (UCP1) or thermogenin
• this protein allows protons to reenter the matrix without making ATP; the energy of the proton gradient is instead released as heat
• such mitochondria have low RCI
• Lipid catabolism is accelerated with a greater heat/ATP ratio than is observed in other tissues.
• results in NON-SHIVERING THERMOGENESIS

Question 47

why is uncoupling important?

Answer 47

1. metabolic regulation
2. limit oxidative damage in mitochondria thought to be main generators of ROS
3. Confounds certain clinical tests (eg. PET)
4. Relationship to obesity (drugs stimulating UCP would cause weight loss/ possible connection b/w low UCP activity and obesity)