Oxidative phosphotylation Flashcards
What are the three types of electron transfer that occur in oxidative phosphorylation?
1) Direct transfer (no H+ move, only electrons)
-Fe3+ –> Fe2+
2) As a hydrogen atom
-reduction of coQ to FAD
3) As a hydride ion
-NADH to FMN in complex 1
why are the complexes in the ETC needed?
Because when groups are in contact the electrons transfer very fast
-the rate of transfer drops 10 fold for every 1.4 nanometer difference
what is the name of complex 1?
NADH-Ubiquinone oxidoreductase
what is the name of complex 2?
Succinate-ubiquinone oxidoreductase
what is the name of complex 3?
Ubiquinol-cytochrome C oxidoreductase
what is the name of complex 4?
Cytochrome C oxidase
what complex is also in the CAC?
complex 2 (succinate DH)
Succinate-ubiquinol oxidoreductase
how do FMN and FAD differ? how are they similar? where are they present in the ETC?
FMN is a riboflavin attached to a phosphate group
-in complex I
FAD contains FMN as a part of its structure, in addition to an AMP attached to the phosphate
- in complex II
What are flavoproteins? what nutrient are they derived from?
proteins that contain a nucleotide derivative of riboflavins as a prosthetic group
derived from Vit B2
how many electrons can flavins accept and pass on?
can accept 2 at a time and pass on 1
This is important as NADH and FADH2 release 2 electrons at complex I and II
how would you explain the reduction activity of Fe-S clusters? How does it differ from a Rieske Fe-S cluster?
the reduction potentials vary, with lower reduction potential at the beginning of the complex and higher potential towards the end
Rieske Fe-S clusters are attached to His groups rather than Cys groups and they have a higher affinity for electron pulling
How many oxidation and reductions can Fe-S clusters undergo (electron movement)?
they can only move ONE electron
how many classes of cytochromes are present in the mitochonria?
3
what peaks would you expect to observe in the reduced vs oxidized form of cytochrome C? what does the subscript beside the “Cty c” tell you?
1 major peak in oxidized form
1 major peak and 2 smaller peaks in the reduced form
the number in a subscript indicates the absorption peak
what factors contribute to the difference in reduction potential of cytochromes?
1) difference in structure of apoprotein
2) Difference in heme groups
3) difference in location within the complex
what are the majority cofactors in complex 1? how does this impact movement of electrons?
Fe-S clusters
-they can only pick up and move one electron at a time
why is it so important that FMN is the first cofactor in complex 1?
it can pick up 2 electrons but pass on 1
-minimizing electron leakage is essential to prevent formation of ROS
what complex are there no H+ pumped across? why?
Succinate-ubiquinol oxidoreductase (complex 2)
-there is not enough energy produced as electrons move through complex 2 for H+ to be moved across the membrane
how many electrons can cytochromes pick up and move?
pick up one, move one
when given delta G values for each complex, what does this tell us?
the delta G of each complex indicates the amount of energy released from the movement of electrons to pump protons across the membrane
when an inhibitor is present in the ETC, what will be reduced and what will be oxidized when?
prior to the inhibitor everything is reduced and after the inhibitor everything will be oxidized as oxygen pulls all the electrons towards it
where does Antimycin inhibit the ETC?
complex 3
where does CN- or CO inhibit the ETC?
the last point where electrons leave, inhibiting oxygen from accepting the electrons
what is the source of electrons for complex 2?
succinate
why is FADH2 not an electron donor in the ETC?
FADH₂ is generated within Complex II and immediately transfers its electrons to ubiquinone (CoQ) as part of the ETC. It does not serve as an independent electron donor for Complex II because it is produced and used within the complex itself
where does movement of electrons vs protons occur in NADH ubiquinone oxidoreductase?
movement of electrons mainly in the hydrophilic part and movement of H+ mainly in the hydrophobic part
are there heme cofactors present in NADH ubiquinone oxidoreductase?
no, there are only Fe-S clusters
it is a non-heme iron protein
how many H+ move for every pair of electrons at the NADH ubiquinone oxidoreductase?
4
explain the movement of electrons in the NADH ubiquinone oxidoreductase
electrons move from NADH to FAD which get picked up by each Fe-S cluster
the last Fe-S cluster passes the electron to N2 which causes a conformational change that is transmitted from the Aq region to helixes along the membrane to the hydrophobic region
changes in conformation of the helix regions cause it to think the residues are being charged which will change the pKa to pick up H+ and pass it on
what structure must cross a membrane?
alpha helix
why coQ able to move very easily within the inner mitochondrial membrane?
it is very hydrophobic due to the 50 isoprene side chains within Q10
how many electrons can coQ accept?
very flexible in its ability to pick up electrons, can pick up 1 or 2
what makes coQ a good antioxidant?
the flexibility for it to pick up electrons
-1 or 2 electrons and doesn’t need to pick up a H+ with it
is coQ a protein?
no, it is the only non-protein component of the ETC
where in complex 3 does antimycin bind?
at Qi, completely blocking the transfer of e- through complex 3
why does QH2 donate electrons to pick them back up again?
in order to pump protons across the IMS
explain the Q cycle in the ubiquinol cyt C oxidoreductase:
1) QH2 is reduced to Q and 2 e- are released
-one e- goes to Fes then C1 where Cyt C is reduced and the e- is taken to complex 4
-the other e- is transferred from heme b (low affinity) to heme b (high affinity)
2) 2H+ are pumped from the matrix to the IMS
3) when Qi accepts the e-, Q is partially reduced
4) another QH2 is reduced and the e- follow the same path
5) the second e- that is taken to Qi converts the partially reduced Q to QH2 with the uptake of 2H+
6) the oxidation of the second QH2 pumps 2 more H+ from the matrix to IMS, causing a total of 4H+ to be pumped
what is the overall rxn for the Q cycle?
what is the main function of cyt C? what is another main function it has in the body?
1) transfer e- from complex 3 to 4
2) apoptosis (programmed cell death)
therefore, it is highly conserved
how many e- and H+ are needed to reduce an O2 molecule? how many H2O does this create?
4 e- and 4H+
produces 2 water molecules
how many H+ are pumped through Cyt C oxidase?
2
how many H+ are pumped for every 2 e- going through complex 4?
2H+ for every 2 e-
what is the purpose of heme b in succinate ubiquinone oxidoreductase?
prevents leakage of e- as they move to form QH2
how are electrons transferred in succinate ubiquinone oxidoreductase?
1) succinate is oxidized to fumarate and the e- are passed to FAD which is then reduced to FADH2
2) the e- in FADH2 are passed to the Fe-S one at a time and is picked up by Q which is reuduced to QH2
what enzyme in the NADH shuttle system oxidizes NADH coming from glycolysis?
cytosolic 3-phosphoglycerol dehydrogenase
what enzyme in the NADH shuttle system reduces FADH, odizing 3-phosphoglycerol into DHAP?
mitochondrial 3-phosphoglycerol dehydrogenase
what is Q a substrate for in the NADH shuttle system into the mitochondria?
substrate for the mitochondrial 3-phosphoglycerol dehydrogenase
what are the pros and cons of the malate-apartate shuttle?
pros: yields more energy because NADH enters at complex 1
cons: takes longer due to many step conversions
explain how NADH is produced with the malate-aspartate shuttle?
1) OAA is reduced to malate in the cytosol with cytosolic malate dehydrogenase
2) malate is able to cross the membrane and enter the matrix through transporters
3) malate is oxidized by NAD+ using matrix malate dehydrogenase to yield OAA and NADH
4) NADH is taken to ETC and OAA then goes through a transamination and is converted to aspartate to be able to cross back over the membrane and continue the shuttle cycle
5) once in the matrix, aspartate is deaminated and converted back to OAA
what components are present in the ATP synthase? what are they responsible for? and how do they function?
F0: crosses the membrane as alpha helices and is the proton channel
-rotates as protons move through, transferring kinetic energy to F1
F1:Catalytic component that extends into matrix. ATP synthesis occurs here. composed of alternating α,β units and a γ subunit
-γ unit rotates in response to F0 movement, causing a conformation change in αβ.
-ADP and Pi are bound and converted into ATP
what does rotational catalysis refer to? explain this process
the binding mechanism for ATP synthesis
In the L conformation ADP and Pi bind loosely and is catalytically inactive
In the T conformation ADP and Pi are tightly bound and ATP is made (catalytically active)
In the O (open) confirmation, there is a low affinity for ligands and ATP is released. (catalytically inactive)
what causes the change in confirmation in rotational catalysis?
The rotation of γ, causing changes in the conformation of the α,β dimers
What types of transporters are seen in the ETC? what do they transport?
Adenine nucleotide translocase (antiporter)
-brings ADP into matrix and ships ATP out
H+ phosphate symporter
-brings Pi into the matrix with H+ to be used as a substrate for ATP synthesis
what part of the gradient does the adenine nucleotide translocase utilize?
electrical
True or False: the electron transport is what drives the synthesis of ATP
False, the electrochemical gradient created from the H+ movement is what is responsible for the generation of ATP
-as long as there is a H+ gradient, ATP can be synthesized (racker experiment)
what is the effect of an uncoupler? what is an example of an uncoupling protein?
uncoupling allows H+ to bypass ATP synthase into the matrix without making ATP (produces heat)
-thermeogenin
What are the metabolic results of uncoupling?
1) decreased ATP synthesis
2) increased O2 consumption
3) increased heat production
what is the effect of norepinephrine on brown fat? why is this?
Nep will increase heat generation through uncoupling
- it is very sensitive to signals from the nervous system
what is the mechanism for uncoupling of mitochondria in brown fat? what effect does fat have on uncoupling protein?
It is a G-coupled mechanism and is stimulated by norepinephrine
the phosphorylation of Hormone-sensitive triacylglycerol is units active form and will break down TAGs to release free fatty acids
-free FA bind directly to uncoupler proteins to open it
-free FA have electrons that supply energy to the ETC
what signals inhibit uncoupler protein (thermogenin) in non-shivering thermogenesis?
ADP and GDP
what is the effect of DNP on oxidative phosphorylation?
It uncouples oxidative phosphorylation without disrupting mitochondrial structure
the structure allows it to diffuse back and forth between the membrane and dissipate the gradient by displacing the [H+]
what do you expect to be added to a suspension of mitochondria at each point?
1: ADP + Pi
2: ATP synthase inhibitor
3: uncoupler
**10 mark Q on final
How does the ATP generation differ in skeletal muscle vs the liver given the complete oxidation of glucose? why?
30 in skeletal muscle and 32 in the liver
the skeletal and brain use the phosphate glycerol shuttle system which yields less energy due to it bypassing complex 1 in the ETC
**10 mark Q on final
explain the ATP yield from the complete oxidation of glucose in muscle vs liver tissues:
From 1Glucose / 2 pyruvate
LIVER
Glycolysis: (7 ATP)
-2 NADH (x2.5 = 5 ATP)
-2ATP
Pyruvate dehydrogenase: (5 ATP)
-2 NADH (x2.5 =5 ATP)
CAC : (20 ATP)
-6NADH (x2.5 = 15 ATP)
-2FADH (x1.5 = 3 ATP)
-2GTP
MUSCLE
From 1Glucose / 2 pyruvate
Glycolysis: (5 ATP)
-2 NADH (x1.5 = 3ATP)*
-2ATP
Pyruvate dehydrogenase: (5 ATP)
-2 NADH (x2.5 =5 ATP)
CAC : (20 ATP)
-6NADH (x2.5 = 15 ATP)
-2FADH (x1.5 = 3 ATP)
-2GTP
what is thermogenin also reffered to? what does it do?
Uncoupling protien 1 (UCP1)
-natural uncoupler that facilitates protons across the inner mitochondrial membrane without the use of the ATP synthase (dissapating the H+ gradient)
-found in brown adipose tissue and regulated by FA and bodily needs
