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· A molecular assembly in the __ carries out the synthesis of ATP
o This enzyme complex was originally called the __ because it was discovered through its catalysis of the reverse reaction, the __
§ __ emphasizes its actual role in the mitochondrion. It is also called __
- inner mitochondrial membrane
- mitochondrial ATPase or F1F0 ATPase
- hydrolysis of ATP
- ATP synthase
- Complex V
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· How is the oxidation of NADH coupled to the phosphorylation of ADP?
o A method was suggested to be of the __
§ Proposal: __ and __ are couple by a __ across the inner mitochondrial membrane
· The transfer of electrons through the __ leads to the __
· The __ concentration becomes lower in the matrix, and an electric field with the __ __ is generated
- chemiosmotic hypothesis
- electron transport and ATP synthesis
- proton gradient
- respirator chain
- pumping of protons from the matrix to the cytoplasmic side of the inner mitochondrial membrane
- H+
- matrix side negative
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· Protons then do what?
o This flow of protons drives __ by __
§ The energy-rich unequal distribution of protons is called the __, which can be thought of as being composed of two components: __ and __
flow back into the matrix to equalize the distribution
- the synthesis of ATP by ATP synthase
- proton-moive force
a chemical gradient and a charge gradient
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o A chemical gradient for protons can be represented as a __
o The charge gradient is created by __
- pH gradient
- the positive charge on the unequally distributed protons forming the chemical gradient
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o Mitchell proposed htat
§ __
· Electron transport does generate a __ across the inner mitochondrial membrane
o The pH outside is__ units lower than inside, and themembrane potential is __, the outside being __
- Proton motive force= chemical gradient and charge gradient
- proton gradient
- 1.4
- 0.14 V
- positive
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· The role of the respiratory chain was played by bacteriorhodopsin, a membrane protein from halobacteria that pumps proteins when illuminated
o Synthetic vesicles containing bacteriorhodopsin and __ purified from beef heart were created
§ When the vesicles were __, ATP was formed
· The __ and __ are biochemically separate systems, linked only by a __
- mitochondrial ATP synthase
- exposed to light
- respiratory chain and ATP synthase
- proton-motive force
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· Electron transport generates a __; and, __ by __ can be powered by a __
· ATP synthase is a __. Much of the “stick” part, called the __, is embedded in the __. The 85-Angstrom-diameter ball, called the __, protrudes into the mitochondrial matrix
o The F1 subunit contains the __
§ In fact, the isolated F1 subunits display __
- proton-motive force
- ATP synthesis by ATP synthase
- proton-motive force
- large, complex enzyme that looks like a ball on a stick
- F0 subunit
- inner mitochondrial membrane
- F1 subunit
- catalytic activity of the synthase
- ATPase activity
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· The F1 subunit consists of __
o The __ and __subunits, which makes up the bulk of the F1, are arranged alternately in a __; they are homologous to one another and are members of the P-loop NTPase family
§ Both bind __but only the __subunits participate directly in catalysis.
- five types of polypeptide chains with the indicated stoichiometry
- alpha and Beta
- hexameric ring
- nucleotides
- Beta
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· Beginning ust below the __ and __ is a __.
o The __subunit includes a long __ that extends into the center of the __
§ The __subunit breaks the symmetry of the __: each of the beta subunits is distinct by virtue of its interaction with a different face of gama
- alpha and Beta subunits
- central stalk consisting of the γ and ε proteins
- gama
- helical coiled coil
- alpha3-beta3 hexamer
- gamma
- alpha3beta3 hexamer
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· The __ is a hydrophobic segment that spans the __
o F0 contains the __; this channel consists of a ring comprising from __subunits that are embedded in the membrane
§ A single __ binds to the outside of the ring
- F0 subunit
- inner mitochondrial membrane
- proton channel fo the complex
- 10 to 14 c
- a subunit
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· The F0 and F1 subunits are connected in two ways:
- o By the central γε stalk and by an exterior column
- The exterior column consists of one a subunit, two b subunits, and the δ subunit
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· ATP synthase catalyze the formation of ATP from __ and __
o The actual substrates are __
§ A __ of ADP attacks the __ of Pi to form a __intermediate, which then dissociates into ATP and H2O
o Enzyme bound ATP forms reaily in the __
§ When ADP and Pi were added to ATP synthase in H2O, O became incorporated into Pi through the synthesis of ATP and its subsequent hydrolysis
- ADP and orthophosphate
- ADP and ATP complexed with Mg2terminal oxygen atom
- phosphorus atom
- pentacovalent
- absence of a proton-motive force
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· The rate of incorporation of oxygen into Pi showed that about equal amounts of bound ATP an ADP are in equilibrium at the catalytic site, even in the absence of a proton gradient
o ATP does not leave the catalytic site unless __
§ Thus, the role of the proton gradient is not to __ but to __
- protons flow through the enzyme
- form ATP but to release it from the synthase
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o The fact that __ are components of the F1 moiety of the ATPase means that there are __, each performing one of three different functions at any instant. The proton-motive force causes the three active sites to do what as protons flow through the membrane-embedded component of the enzyme
three beta subunits
- three active sites on the enzyme
- sequentially change functions
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o The enzyme can be thought of as having a moving part and a stationary part:
§ The moving unit (rotor) consisting of the __
§ The stationary unit (stator) is composed of the remainder fo the molecule
o The mechanism is a __
§ A __subunit can perform each of three sequential steps in the synthesis of ATP by changing conformation
What are they?
§ Interactions with the __ make the three __ subunits unequivalent
- c ring and the γε stalk
- binding-change mechanism
- beta
- · ADP and P bind
- · ATP synthesis
- · ATP release
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o At any given moment, one beta subunit will be in the__
§ This conformation does what?
§ A second subunit will be in the __
· This conformation does what?
- L, or loose, conformation
- binds ADP and Pi
- T, or tight, conformation
- binds ATP with great avidity, so much so that it will convert bound ADP and Pi into ATP
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o Both the T and L conformations are sufficiently __that they cannot __
§ The final subunit will be in the __
· This form has a more __ and can __
o The rotation of the gamma subunit does what?
§ ADP and Pi bound in the subunit in the __ are doing what?
- constrained
- release bound nucleotides
- O, or open, form
- open conformation
- bind or release adenine nucleotides
- drives the interconversion of these three forms
- T form
- transiently combining to form ATP
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o Suppose:
§ The gamma subunit is rotated by 120 degrees in a counterclockwise direction
· This rotation does what?
§ The L form site is __, enabling the transformation of __
· The __in the __ can now depart from the enzyme to be replaced by ADP and Pi
converts the T form site into an O-form site with the nucleotide bound as ATP
converted into a T form site
an additional ADP and Pi into ATP
ATP
O form site
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· An additional 120-degree rotation converts this __ into __, trapping these substrates
o Each subunit progresses from the __ to the __ to the __ with no two subunits ever present in the same conformational form
§ This mechanism suggests what?
- O-form site into an L-form site
- T to the O to the L form
- that ATP can be synthesized and released by driving the rotation of the gamma subunit in the appropriate direction
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· Rotation was determined through __, which allowed the __assembly to be immobilized on a glass surface that had been coated with nickel ions
o This property of the tags allowed the a3b3 assembly to be immobilized on a glass surface that had been coated with nickel ions
§ The gamma subunit was linked to a fluorescently labeled __ to provide a long segment that could be observed under a microscope
o The addition of ATP caused the actin filament to do what?
§ the gamma subunit was __, driven by __
- tagging
- a3B3
- actin filament
- rotate unidirectionally in a counterclockwise direction
- rotating, driven by the hydrolysis of ATP
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o the gamma subunit rotates in __, each increment corresponding to the __
o the enzyme appears to operate near __; that is, essentially all of the energy released by ATP hydrolysis is converted into __
- 120 degree increments
- hydrolysis of a single ATP molecule
- 100% efficiency
- rotational motion
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· How does proton flow through F0 drive the rotation of the gamma subunit?
o The mechanism depends on the __
§ The stationary a subunit directly __
· Although the structure of the a subunit has not yet been experimentally determined, a variety of evidence is consistent with a structure that includes two __
- structures of the a and c subunits of F0
- abrupts the membrane-spaning ring formed by 10 to 14 c subunits
- two hydrophilic half-channels that do not span the membrane
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o Thus, protons can pass into either of these channels, but they cannot __
§ The a subunit is positioned such that __
- move completely across the membrane
- each half-channel directly interactions with one c subunit
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· Structure of the c subunit: eaech poypeptide chain forms a pair of __ that __. An __ is in the middle of one of the helices
o The key to proton movement across the membrane is that, in a proton-rich environment, a __ and will __
o The c subunit with the bound proton then does what?
- alpha helices that span the membrane
- aspartic acid residue
- p proton will enter a channel and bind the aspartate residue
- rotates through the membrane until the aspartic acid is in a proton-poor environment of the other half-channel, where the proton is released
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· 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 __
o Its rotation is favored by the __
§ Thus, the c subunit with the __ moves from __ into the __; and, the other c subunits move in unison
- rotation of the c ring
- ability of the newly protonated aspartic acid residue to occupy the hydrophobic environment of the membrane
- newly protonated aspartic acid
- contact with the cytoplasmic half-channel
- membrane
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o The a unit does what as the c ring rotates. Each proton that enters the cytoplasmic half-channel of the a unit moves through the membrane by doing what?
- remains stationary
- riding around on the rotating c ring to exit through the matrix half-channel into the proton-poor environment of the matrix
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· How does the rotation of the c ring lead to the synthesis of ATP?
o The c ring is tightly linked to the __
§ Thus, as the c ring turns, the __ in turn promotes the __through the binding-change mechanism
§ The rotation of the gamma subunit in turn promotes the __ through the __
· The exterior column formed by the __ prevents the __ unit from rotating
- gamma and ε subunits
- gamma and ε subunits
- synthesis of ATP
- synthesis of ATP through the binding-change mechanism
- two b chains and the delta subunit
- alpha3beta3 hexamer
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o The number of c subunits in the c ring appears to range between __, determining the number of protons that must be transported to generate a molecule of ATP
§ Each 360-degree rotation of the gamma subunit leads to the __
· Thus, if there are 10 c subunits in the ring, each ATP generated requires the transport of __
o __ must flow in for each ATP
o Electrons from NADH pump enough protons to generate __, whereas those from FADH2 yield __
- 10 and 14
- synthesis and release of three molecules of ATP
- 10/3= 3.33 protons
- 3 protons
- 2.5 molecules of ATP
- 1.5 molecules of ATP
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· The __ and __ of ATP synthase are members of the P-loop NTPase family of proteins
o The P-loop regions of the B subunits will bind either__ or __, depending on __. The conformational changes take place in an orderly way, driven by the __
- alpha and beta subunits
- ADP or ATP
- which of three different faces of the gamma subunit they interact with
- rotation of the gamma subunit
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