Oxidative phosphorylation II Flashcards
Protons are pumped across what membrane as they flow through the Respiratory chain
the inner mitochondrial membrane
What is the chemiosmotic hypothesis
- Purposed by Peter Mitchell in 1961
- ETC is accompanied by transport of protons from matrix to cytoplasmic side of inner membrane
- This generates a pH gradient and membrane potential
- Constitutes a proton motive force
- composed of two comments:
- chemical gradient (pH gradient)
- charge gradient - used to dive ATP synthesis (via ATP synthase)
- composed of two comments:
what was used to supply evidence for the chemiosmotic hypothesis
- Synthetic phospholipid vesicles containing Bacteriorhodopsin and mitochondrial ATP synthase purified form beef heart were created
- purified ATP synthase and bacteriorhodopsin reconstituted into the vesicles (bacteriorhodopsin pumps protons when illuminated)
- Incubated with ADP+Pi
- Exposed to light and ATP generated
- — experiment clearly showed that the respiratory chain and ATP synthase are biochemically separate systems, linked only by a proton-motive force
The F0 unit of the ATP synthase is located
embedded in the inner mitochondrial membrane
The F1 unit of ATP synthase is located
protrudes into the mitochondrial matrix
The F1 subunit of ATP synthase consists of ____ types of polypeptide chains
5 (alpha3, beta3, gamma, delta, and epsilon)
The alpha and beta components of subunit F1 in ATP synthase are arranged alternately in a
hexameric ring
Which of the ATP synthase subunits contain catalytic domains
F1
What is complex V of the ETC
ATP synthase
ATP synthase is a ____ and ____ structure
ball and stick
What is the stick component of the ATP synthase
F0
what is the ball component of the ATP synthase
F1
Which of the subunits in ATP synthase have proton channels
F0
does Isolated F1 subunit displays ATPase activity
Yes
Both Alpha and Beta on F1 bind nucleotides but only the ____ are catalytically active
Beta
Above the alpha and beta is a stalk made of ____ and ____ proteins
gamma and epsilon
The gamma subunit has a long helical coil that extends into the
center of the alpha3 and beta3 hexamer
What breaks the symmetry of the beta3alpha hexamer: each of the Beta subunits is distinct by virtue of its interaction with a different face of the _____
The gamma subunit, gamma subunit
The F0 has a proton channel complex. This channel consists of a ring comprising from ____ c subunits that are embedded in the matrix
8-14 c subunits
F0 and F1 are subunits of ATP synthase are connected by what two ways
- by the central gamma/epsilon stalk
- by an exterior column consists of one alpha subunit, two beta subunits, and the delta subunit
ATP synthase molecules associate with each other to form
dimers
ATP synthase dimers come together to form
oligomers
What is the purpose of ATP synthase molecules associating with one another to form dimers and then oligomers
- association stabilizes the individual enzymes to the rotational forces required for catalysis and facilitates the curvature of the inner mitochondrial membrane
- maintains curvature in inner membrane
The ATP synthases are located
at the tips of the crust, thereby enhancing the efficiency of ATP synthesis
-Cristae allow the proton gradient to be in ____ proximity to the ATP synthase
close
What is the role of the proton gradient in ATP synthase
ATP can be formed in the absence of proton gradient but it can’t leave the catalytic site unless protons flow through the enzyme. Thus the role of the proton gradient is not to form ATP but to release it form the synthase
ATP synthase when incubated with ADP and Pi forms
ATP in the absence of the proton gradient
How many active sites are there on the ATP synthase
3 (the beta3 units of the F1 subunit)
What is the moving unit of the ATP synthase
The moving unit, or rotor, consists of the c ring and the gamma epsilon stalk
The three beta subunits can each perform three sequential steps in the synthesis of ATP by
Changing conformations
What are the three sequential steps in ATP synthesis
- ) ADP and Pi binding
- ) ATP synthesis
- ) ATP release
What is the O conformation in the beta subunit
it is the open conformation. This allows for the release of ATP and for ADP and Pi to enter the subunit
What is the L conformation in the beta subunit
Loose, Has a strong binding affinity for ADP and Pi thus locking them in the subunit
What is the T conformation in the beta subunit
Tight, has a strong affinity for the ATP and actually drives ADP and Pi together to form ATP
How much does the gamma subunit rotate to move conformation states in the beta subunits
counterclockwise 120 degrees
each beta subunit progresses from ___ to ___ to ___ conformation
T,O,L
Are any two beta subunits ever in the same conformation state
No
What powers the rotation of the c ring on ATP synthase F0 subunit
The movement of protons through the half channels from the high proton concentration of the cytoplasm to the low proton concentration of the matrix powers the rotation of the c ring.
The c subunit of ATP synthase is composed of
- 2 alpha helices that span the membrane.
- Aspartic acid (glutamic acid) residue in one of the helices lies on the center of the membrane (these bind to the protons)
how many a subunits bind to the outside of the ring (8-14 c subunits)
a single a subunit
When glutamate is in a high proton environment it will ____
bind the proton
if glutamate is charged (unprotonated) the c subunit will
not move into the membrane
Does the a unit rotate with the c ring
no
The a subunit is composed of
2 half channels, that allow protons to enter and pass partway but not completely
The alpha and beta subunits of ATP synthase are members of the ______ family of proteins
P-loop NTPase family
Is the regeneration of NAD+ for use in glycolysis one function of the ETC
Yes
The major function of oxidative phosphorylation is to generate
ATP from ADP
Do ATP and ADP readily move across the inner mitochondrial membrane into the cytoplasm
No (ATP-ADP translocase is used)
What enables ATP and ADP to transverse the permeability barrier (inner mitochondrial membrane)
ATP-ADP translocase
Mechanism of mitochondrial ATP-ADP translocase
- Catalyzes the coupled entry of ADP into the matrix and the exit of ATP from it
- The binding of ADP form the cytoplasm favors eversion of the transporter to release ADP into the matrix.
- Subsequent binding of ATP from the matrix to the everted from favors eversion back to the original conformation, releasing ATP into the cytoplasm
The flow of ATP and ADP are
coupled, ADP enters the mitochondrial matrix only if ATP exits, and vice versa.
ATP-ADP translocase is what kind of transporter
antiporter
Inhibition of translocase leads to
the subsequent inhibition of cellular respiration
about a quarter of the proton-motive force generated by the respiratory chain is consumed by
ATP-ADP translocase’s exchange process
The regulation of cellular respiration is governed primarily by the need for
ATP
The complete oxidation of glucose yields about ____ molecules of ATP
30
glucose is completely oxidized to
CO2
Regulation of cellular respiration
- levels of ATP regulate respiration
- electrons flow through ETC only when ADP phosphorylated to ATP
- Regulation by ADP levels called respiratory control
The regulation of the rate of oxidative phosphorylation by the ADP level is called
respiratory control or acceptor control
electrons are transferred to O2 only if ADP is
concomitantly phosphorylated to ATP
Regulation of ATP synthase
- inhibitory factor I (IF1) -inhibits hydrolytic activity of ATP synthase
- prevents the reverse reaction, i.e. ATP breakdown - is useful to prevent the hydrolysis of ATP when oxygen not available to make protein gradient
Warburg effect
- the switch form oxidative phosphorylation to aerobic glycolysis as the principle means for ATP synthesis
- caused by the over-expression of IF1 (inhibitory factor 1), which is common in many types of cancers
Some organisms can uncouple oxphos from ATP synthesis. Why?
Used to generate heat and maintain body temperature (important in hibernating animals)
in animals the uncoupling of oxidative phosphorylation from ATP synthesis to generate heat is found in
brown adipose tissue (BAT)
why is brown adipose tissue reddish brown
due to cytochromes in the numerous mitochondria and hemoglobin present in extensive blood supply
Brown adipose tissue is rich in
mitochondria
The inner mitochondrial membrane of brown adipose tissue mitochondria contain large amounts of
uncoupling protein (UCP-1) or thermogenin
what is the function of uncoupling protein 1 (thermogenin)
Transfers protons form cytoplasm to matrix side without producing ATP, thus converting engr. to heat instead of ATP
what are the two other uncoupling proteins
UCP-2 (found in a variety of tissues)
UCP-3 (found in skeletal muscle and brown fate)
-may play a role in homeostasis
The family of uncoupling proteins may play a role in
homeostasis
_____ inhibits ATP synthase by preventing the influx of protons by binding to the carboxylate group of the c subunits required for proton binding
Oligomycin (Antibiotic and anti fungal agent)
Uncouples electron transport from ATP synthase
2,4-dinitrophenol (dissipates the proton gradient)
____ and ____ inhibit ATP-ADP translocate thus inhibiting ATP export
Atractyloside and bongkrekic acid
How does Oligomycin inhibit Oxidative phosphorylation
Inhibits ATP synthase by preventing the influx of protons through ATP synthase by bidding to the carboxylate group of the c subunits
How does 2,4 dinitrophenol inhibit oxidative phosphorylation
uncouples electron transport from ATP synthase
In the presence of 2,4 dinitrophenol (DNP) electron transport from NADH to O2 proceeds in a normal fashion, but ATP is not formed by mitochondrial ATP synthase, because the proton-motive force across the inner mitochondrial membrane is continuously dissipated.
- Leads to increase in oxygen consumption and release of Heat
Inhibit electron transfer in NADH-Q oxidoreductase
Rotenone and Amytal
Inhibits the electron flow form cytochrome bH in Q-cytochrome c oxidoreductase
Antimycin A
Cyanide (CN-), Azide (N3-), and Carbon monoxide (CO) inhibit
Cytochrome c Oxidase
cyanide and Azide react with the ferric form of heme a3 and carbon monoxide inhibits the ferrous form
what inhibits cytochrome c oxidase
Cyanide, Axide, and carbon monoxide
Atractyloside and Bonkrekic inhibit
ATP export by inhibiting ATP-ADP translocase
