Oxidative Phosphorylation Flashcards
What enzymes are present in the mitochondrial matrix?
pyruvate dehydrogenase,
TCA cycle enzymes (except succinate dehydrogenase),
Enzymes for beta-oxidation of FAs,
parts of the urea cycle
What is the permeability of the outer mito membrane?
-permeable to small molecules (contains porins)
What is the permeability of the INNER mito membrane?
TIGHTLY REGULATED!
-impermeable to small molecules EXCEPT O2, CO2, H2O, and NH3 (transporters)
-site of the respiratory chain
Oxidation
substance loses electrons (LEO)
Reduction
substance gains electrons (GER)
What is the point of redox rxns in the mito?
The transfer of e- during chemical rxns releases energy stored in organic molecules, which is ultimately used to synthesize ATP
Complete oxidation of glucose is coupled with the reduction of oxygen to water.
Explain.
Glucose (C6 H12 O6) loses electrons and hydrogen atoms (oxidation), forming CO2 (and donating H+ to form water)
Oxygen (6 O2) gains electrons and hydrogen atoms (from glucose) to form water (6 H2O)
The reaction is EXERGONIC (ATP is formed)
Glucose + 6 O2 –> 6 CO2 + 6 H2O + ATP
What is the point of a stepwise ETC?
NADH passes HIGH ENERGY e- to the electron transport chain —> as e- move down, they lose energy until low energy (at end of chain) –> water
Stepwise creates a controlled reaction (instead of one explosion).
O2 pulls e- down the chain in an “energy-yielding tumble” –> energy yielded is used to make ATP (instead of losing so much energy as heat)
What does E’o measure?
Standard ox-red potential E’o (V) = measure of the affinity for a compound to accept or donate electrons.
Negative E’o: take away electrons (more negative = more oxidized)
Positive E’o is to gain electrons (more positive = more reduced)
What happens to ETC compounds’ affinity for electrons as we move down the chain?
NAD+/NADH has the lowest deltaE’ (most negative) so it has a lower affinity for e-), thus it gives them up easily to a redox pair with a higher deltaE’o (because higher deltaE’o means they have a higher affinity for e-.
ie: 1/O2/H2O (completely reduced) has E’o (V) = +0.82
In ATP production by cellular respiration, how much of the energy is captured and how much is released as heat?
40% of the energy is captured to produce ATP from ADP
60% is released as heat
What are the components of the ETC?
- 4 large protein complexes in INNER mito membrane: I, II, III, IV
- 2 mobile carriers: lipid-soluble CoQ, water soluble protein Cyt-c
What is the course of electron transfer?
NADH –> complex I –> CoQ –> Complex III –> cyt c –> complex IV –> O2
What happens in Complex I?
hint: 3 parts
Key enzyme: NADH dehydrogenase
1) NADH (carrying electrons from food) donates electrons and protons (entering from mito matrix)
2) 4 protons get pumped through into inter membrane space, increasing the acidity of the intermembrane space
3) 2 electrons get passed through the lipid bilayer of the inner mito membrane to CoQ (aka:Ubiquinone or Q10)
(Note: complex 1 has FMN and Fe-S core)
What happens in Complex II?
1) Complex II = Succinate dehydrogenase (from TCA cycle)
2) Pumps 2 e- to CoQ (in lipid bilayer of inner membrane) using FADH2 generated during
a) fatty acid oxidation via fatty acyl-CoA dehydrogenase
b) mitochondrial/cytosolic (?) glycerol-3-phosphate dehydrogenase of glycerol phosphate shuttle
Bypass reaction: uses FADH2 instead of NADH (bypass complex I)
*NO PROTONS PUMPED BY COMPLEX II
What happens in Complex III?
Complex III: cyt-bc1
1) electrons move one at a time from Complex III to Cyt-c
2) Proton is pumped into intermembrane space FROM ___ in the mitochondrial matrix
What is cyt-c?
ONLY water-soluble protein of ETC; contains single heme group
cytochrome c gets reduced by complex III and reduces complex IV.
What happens in Complex IV?
Complex IV = cytochrome oxidase
Accepts electrons from cyt c, and then coordinates the transfer of 4 electrons to oxygen, reducing it to water.
Steps:
cyt c –> Cu (2 copper ions) –> Cyt a –> Cyt a3 (heme group of cyt a3 in reduced state binds O2 and transfers 4 electrons to O2) –> reducing O2 to 2 molecules of H2O (H2O ends up in mitochondrial matrix)
How much ATP is generated from NADH in ETC?
3 ATP
How much ATP is generated from succinate in ETC?
2 ATP
What drives ATPsynthase of complex 5?
Proton gradient:
ADP + Pi –> ATP
How was the proton gradient formed?
large amts of energy are given off at 3 sties of ETC: complex I, II, and IV
This energy is used to pump H+ from matrix to outer side of inner mito membrane, establishing the proton gradient
What are the 5 prerequisites for oxphos?
1) availablility of the reducing agents NADH or FADH2 (glycolysis, FA oxidation, TCA cycle
2) pH (low pH in intermembrane space)
3) Presence of a terminal oxidizing agent (in form of Oxygen)
4) ADP/ATP ratio (the ratio needs to be high bc you run oxphos when you need energy. If you have low ration then why run oxphos)
5) sufficient qty of intact mito (with enzymes and machinery needed)
Where does the ADP come from? How do you get it into mito matrix?
ADP/ATP antiport pumps ADP into the mito matrix from the intermembrane space while pumping the formed ATP out of matrix into intermembrane space
*driven by pH gradient and membrane potential
How does hypoxia affect ATP production?
Complex IV would not work properly, so ATP production would decrease.
Thus the Na+/K+ ATPase would not work properly, so Na+ would be retained int he cell —> CELLULAR SWELLING –>
increase of Calcium –> increase mito permeability –> increase PM permeabability –> cell death
What is the effect of atractyloside on ATP synthesis?
blocks ADP/ATP antiport:
Atractyloside prevents transport and availability of ADP in matrix for ATP synthesis
(stops ATP synthesis; stops ETC - since H+ gradient builds up and cannot be used to make ATP)
How does amytal (amobarbital) affect the ETC?
reverse inhibitor of NADH DH (complex I)
- inhibits Complex I in high doses, so can only produce ATP from FADH2 (via complex II)
- also, allows buildup of reactive oxygen species (bc not generating as many e- for complete O2 reduction)
(therapeutic for cardiac ischemia in low doses)
How does rotenone affect the ETC?
potent inhibitor of NADH DH (complex I)
- no NADH can be oxidized but succinate can still enter the chain and lead to some electron transport
- thus REDUCED AMTS OF ATP
How does ANTIMYCIN affect the ETC?
- inhibitor of complex III: tightly binds to cyt b in reduced state
- STOPS ETC
- NO ATP is produced (cannot use bypass)
- Complex I, CoQ and Complex II are fully reduced
- cyt C and complex IV are fully oxidized
How does cyanide affect the ETC?
CN prevents O2 reduction
(bc binds to oxidized (ferric: Fe3+) form of iron in heme of cytochrome-a3 component of Complex IV)
*mito respiration and ATP production cease –> rapid cell death
How is cyanide treated?
Nitrate followed by thiosulfate
1) Hb + Nitrate –> MetHb –> add CN —> CN Met Hb
2) S2O2 –> SCN- + SO3(2-) –> CN soluble to urinate out
How does oligomycin affect the ETC?
blocks the Fo portion of ATP synthase, preventing re-entry of protons into matrix of mito
*NO ATP produced;
H+ gradient builds up;
Everything remains oxidized once ETC stops
(ETC will soon stop bc of difficulty pumping H+ against an increasingly steep proton gradient. Thus, inhibition of ATP formation by oligomycin also prevents oxidation-reduction rxns)
What are uncouplers in ATP synthesis?
GENERATE HEAT AND INHIBIT ATP SYNTHESIS:
molecules that allow protons to re-enter the mito matrix from the intermembrane space, independent of the proton-channeling fxn of ATP synthase
What happens with reduction of the proton gradient (ie due to uncouplers)>
resp rate:
- very fast always (bc no longer controlled by ADP/ATP concentration levels
- limited only by availability of substrates NADH, succinate, and O2
Also:
- acceleration of both TCA cycle and ETC to O2
- inhibition of ATP synthase
- generation of heat due to flow of H+ into the matrix
What are 3 sources/reasons for uncoupling?
A) Membrane damage (ex: AraC, AZT)
B) Proton carriers (grab H+ that is waiting in inner membrane space and bring into matrix; ex: DNP; Aspirin)
C) Protein Channels (thermogenin = UCP-1: channels in certain tissues that allow dissipation of H+ gradient to keep our body warmer as needed)
What is the different between brown adipose tissue and white adipose tissue
BAT: thermogenic, highly vascularized, relatively many mito, UCP-1
WAT: non-thermogenic, relatively few mito and capillaries; leptin (appetite suppressing hormone)
What signaling mechanism is used for UCP-1 expression?
Activation of SNS: norepinephrine signaling
(NE released when cold –> activates cAMP –> TAG degradation –> FFA within BAT activates UCP-1 –> release H+ gradient –> dissipate heat and not make ATP
What is the fxn of malate-aspartate and glycerol phosphate shuttles?
transport cytoplasmic NADH into mito matrix in order to participate in oxphos
Complete this sentence for Malate-Aspartate Shuttle:
It transfers the reducing equivalents of ___________ in the cytoplasm to _______ in the mitochondrial matrix
It transfers the reducing equivalents of __NADH__ in the cytoplamsm to ___NADH____ in the mitochondrial matrix
Complete this sentence for Glycerol Phosphate Shuttle:
It transfers the reducing equivalents of ___________ in the cytoplasm to _______ in the mitochondrial matrix
It transfers the reducing equivalents of __NADH__ in the cytoplamsm to ___FADH2____ in the mitochondrial matrix
What are the conditions for using the Malate-Aspartate Shuttle?
Only works if [NADH]/[NAD+] is higher in cytoplasm than in matrix
What are the conditions for using the Glycerol Phosphate Shuttle?
can be used under essentially all conditions
bc irreversible operates in skeletal muscle and brain
Which shuttle is reversible: malate-aspartate OR glycerol phosphate shuttle?
malate-aspartate shuttle is reversible
Which shuttle is irreversible: malate-aspartate OR glycerol phosphate shuttle?
glycerol phosphate shuttle is irreversible
How many ATP result when the Malate-Aspartate Shuttle is used?
Leads to 3 ATP produced
1 NADH –> 3 ATP
How many ATP result when the Malate-Aspartate Shuttle is used?
Leads to 2 ATP produced
1 NADH –> 2 ATP
Where does the malate-aspertate shuttle function?
heart, liver, kidneys
What does the malate-aspertate shuttle do?
generates NADH in the mito matrix, which then enters complex I
(cytocol??)
-during gluconeogenesis, it releases oxaoacetate (in the form of malate) into cyto for later rxns in gluconeogenic path
What does the glycerophosphate shuttle do?
converts FAD to FADH2 in the inner mito membrane –> which donates its electrons to ETC at CoQ
Atractyloside (state the mechanism and effects on ETC)
prevents transport and availability of ADP for ATP synthesis by inhibiting ADP/ATP antiporter
Amytal and Rotenone
state the mechanism and effects on ETC
blocks complex I;
allows SOME production of ATP from FADH2 via complex II (bypass); note: amytal is reversible
Antimycin (state the mechanism and effects on ETC)
anti-fungal; blocks complex III;
NO ATP made:
Fully reduced: Complex I, CoQ and Complex II;
Fully Oxidized: cyt c; complex IV
Carbon Monoxide (state the mechanism and effects on ETC)
blocks complex IV;
NO ATP production;
competes for O2 for biding to reduced heme-a3 (Fe2+) in complex IV
Cyanide (state the mechanism and effects on ETC)
blocks complex IV;
NO ATP production;
bind to oxidized form of heme-a3 (Fe3+) –> prevent O2 reduction
Treatment for CN poisoning
1) Nitrate (trap CN in MetHb)
2) Thiosulfate to make it soluble to urinate
Oligomycin (state the mechanism and effects on ETC)
blocks complex V: NO ATP production;
binds F0 portion of ATP synthase, blocking proton path, prevents re-entry of H+ into mito matrix, stops ETC and prevents redox reactions (everything will remain in oxidized form once H+ movement stops)
