2- Mitochondria ETC/Respiratory Chain/Oxidative Phosphorylation Flashcards
what happens to most of the energy released from glycolysis and TCA?
its captured by NADH and FADH2
what happens (big picture) to the electrons released from NADH and FADH2?
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
final equation
NADH + 1/2 O2 + H+ —> NAD+ + H2O
how many atp does glycolysis/oxidative phosphorylation produce?
glycolysis - 2
oxidative phosphorylation ~36
what will stop the ATPase from pumping?
- dont have ATP
- if there is no longer a concentration gradient you are working against
- build up of ADP
- build up of protons inside the vesicle that use the ATPase
ATPase will keep working as long as you have a source of protons
given an impermeable membrane and an ATPase pump that can translocate H+, what happens if you add H+ inside the vesicle?
ATP will be made from ADP
if the proton circuit is uncoupled and there is a leak then what happens?
the gradient will bleed out and it will destroy the energy used for the reaction
what do you need to build a proton circuit to make ATP?
- impermeable membrane
- e- carriers (to hand off e-)
- proton pumps (to make graident)
- ATPase
Explain the impermeable membrane that the mitochondrial inner membrane has
low sterol, cardiolipin, TONS of proteins (60-70% of weight)
has a lipid bilayer
explain the electron carriers in the e- transport chain
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
is e- flow down a thermodynamic gradient favorable?
YES! it is favorable and youre going from one e- carrier to another just handing it off
O2 oxidant or reductant?
O2 is a STRONG oxidant which means it has a high affinity for electrons (0.82)
which complexes do not pump H+ from the matrix to the IMS
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
proton pumps
complexes I, II, III, IV, and V
V is ATP synthase
ATPase vs. ATP synthase
ATPase- hydrolyses ATP
ATP synthase- makes ATP
Proton pump: Complex I
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
Proton pump: Complex III
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
cytochrome c
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
Proton pump: Complex IV
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
where does the TCA cycle occur?
mitochondrial matrix
where does oxidative phosphorylation occur?
in the inner membrane space
Complex II
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)
megacomplexes
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
Ubiquinone/ubiquinol pool
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
what are the other ways to get e- into the electron transport chain without using the proton pump complexes?
- B-oxidation
- pyrimidine biosynthesis
- glycolysis
All of these dump e- into the ubiquinone/ubiquinol pool
Complex I diseases
LHON
MELAS
Leigh’s syndrome
Complex II diseases
leigh’s syndrome
Complex III diseases
Cardiomyopathy
leigh’s syndrome
Complex IV diseases
ALS-like syndrome
leigh’s syndrome
Complex IV inhibitors
These block heme groups in complex IV
Cyanide (CN-)
Azide (N3-)
Carbon Monoxide (CO)
Nitric Oxide (NO)
Treatment of cyanide poisoning
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
inner mitochondrial membrane is impermable, so how do we get substrates/products across?
- 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.
How do you get NADH from cytoplasm into inner mitochondrial matrix?
- Malate- Aspartate Shuttle
2. Glycerol Phosphate Shuttle
Malate- Aspartate Shuttle
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
outer mitochondrial matrix
very permeable in comparison to the inner mitochondrial membrane.
Glycerol Phosphate Shuttle
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
Electron Transport Chain vs. Oxidative Phosphorylation
ETC- complex I-IV
OP- Coupling e- transport to ATP Synthase, the process as a whole
ETC is part of OP
big picture of oxidative phosphorylation (using chemiosmotic hypothesis)
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
proton-motive force (delta.p)
2 parts
- membrane potential (electrical)
- 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
ATP synthase and its subunits
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
Uncouplers
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.
Respiratory Control Index/Ratio (RCI or RCR)
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
Phosphorylation efficiency
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+
P/O for NADH
10/4 = 2.5 ATP/O
P/O for succinate/FADH2
6/4 = 1.5 ATP/O
Physiological Uncoupling
- 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
why is uncoupling important?
- metabolic regulation
- limit oxidative damage in mitochondria thought to be main generators of ROS
- Confounds certain clinical tests (eg. PET)
- Relationship to obesity (drugs stimulating UCP would cause weight loss/ possible connection b/w low UCP activity and obesity)
