Oxidative Phosphorylation

Proton-motive force can be calculated if the ______________ and ______________ between matrix and outside matrix is known.

charge difference pH difference

The purpose of the electron transport chain is to generate a proton motive force, that is used by the mitochondrial ATPase or ATP synthase to generate ATP. The relationship between the electron transport chain and ATP synthase is explained by the ______________ hypothesis.

chemiosmotic.

Which complexes have iron-sulfur clusters?

complexes 1-3.

CN-, N3- and CO block the electron transfer in _________________.

cytochrome c oxidase.

__________________ is a small water-soluble carrier that shuttles electrons from Q-cytochrome c oxidoreductase (complex III) to cytochrome c oxidase (complex IV).

cytochrome c.

The transfer of electrons from NADH or FADH2 to molecular oxygen is mediated by protein complexes that are part of the ________________ and are located in the inner mitochondrial membrane. The electron transport through these protein complexes leads to pumping of protons out of the matrix.

electron transport chain.

Inhibition of ATP synthase by inhibiting proton flow prevents ________________.

electron transport.

_____________ takes place in mitochondria (enzymes needed are here), is the major source of energy for aerobic organisms.

oxidative phosphorylation.

________________ is the major pathway for generation of ATP and consists of the electron transport chain and ATP synthase.

oxidative phosphorylation.

Partial reduction of O2 generates highly reactive oxygen derivatives, called _______________.

reactive oxygen species (ROS). **implicated in many pathological conditions.

The citric acid cycle enzyme succinate dehydrogenase which generates FADH2 is part of the _______________ complex.

succinate-Q reductase complex (Complex II).

ROS include _______________.

superoxide ion, peroxide ion, and hydroxyl radical.

Electron-transport chain generates a proton gradient, which is used to _____________.

synthesize ATP.

Disruption of Complex I is the most common cause of mitochondrial disease - causes ______________.

blindness.

Uncoupling occurs in mitochondria in _____________.

brown fat.

2,4-Dinitrophenol (DNP) uncouples electron transport from phosphorylation in mitochondria by carrying the protons across the inner mitochondrial membrane. It stops ATP synthesis without affecting electron transport from NADH to O2.

True.

FADH2 generated in the citric acid cycle reduces Q to QH2, which then enters the Q pool.

True.

Total 30 ATP per glucose and 26 are generated during oxidative phosphorylation from NADH and FADH2 electrons.

True.

Electrons from FADH2 are transferred to Q. Succinate-Q reductase transfers only electrons and NOT protons. It is NOT a proton pump.

True. That is the reason why the electrons of FADH2 produce less ATP compared to NADH.

Inhibition of the ATP-ADP translocase prevents __________________.

oxidative phosphorylation.

Cytoplasmic NADH cannot enter the mitochondria. Electrons from cytoplasmic NADH are carried across the mitochondrial membrane by two major mitochondrial shuttles: 1. 2.

1. Glycerol 3-phosphate shuttle (muscle). 2. Malate-aspartate shuttle (heart and liver).

What are the 2 electron carriers of the respiratory chain?

1. ubiquinone (coenzyme Q or Q). 2. cytochrome c.

Electrons of cytosolic NADH transported by the glycerol 3-phosphate shuttle, yield ______ ATP instead of _______ ATP

1.5 instead of 2.5

-It takes two electrons to reduce a single atom of oxygen. ________ protons are translocated per NADH and _________ protons per FADH2. - Since 4 protons are required for each ATP synthesized, the P/O ratio is 10:4 = ________ ATP for NADH - Electrons from FADH2 do not pass through complex I and contribute to pumping of 6 protons and 6:4 =________ ATP per FADH2.

10 6 2.5 1.5

In the electron transport chain, NADH contributes to pumping of ________ protons and FADH2 to pumping of _________ protons from the matrix. Electrons from FADH2 bypass the first proton pump (complex I).

10 6.

Electrons of cytosolic NADH transported by malate-aspartate shuttle yield ________ ATP.

2.5

Complete oxidation of glucose to CO2 and H2O yields 30 molecules of ATP, _________ of which are generated by oxidative phosphorylation.

26.

the respiratory chain consists of 4 complexes. what are they?

3 proton pumps and a physical link to the citric acid cycle.

_____________ protons must flow into the matrix for each ATP formed.

3.

the complete oxidation of glucose yields about ___________ moleculues of ATP.

30 (26 are from oxidative phosphorylation).

The synthesis of 1 ATP requires _________ H+.

4 (3 in ATP synthesis and release, and 1 for ATP transport).

What is the best description of respiratory control or acceptor control? A. ADP is the most important factor in determining the rate of oxidative phosphorylation. B. ATP synthesis by the ATP synthase requires the flow of oxygen via the F0 subunit. C. Electron transport during oxidative phosphorylation is tightly coupled to glucose production. D. ATP-ADP translocase requires oxygen to transport ATP into the mitochondria. E. The transfer of electrons from cytochrome c to oxygen via cytochrome c oxidase is controlled by carbon monoxide (CO).

A. ADP is the most important factor in determining the rate of oxidative phosphorylation.

Which of the following enzymes are found in the glyoxylate cycle, but not in the citric acid cycle? A. malate synthase and isocitrate dehydrogenase. B. glyoxylate synthase and malate synthase. C. citrate lyase and malate dehydrogenase. D. malate synthase and isocitrate lyase. E. succinyl CoA synthetase and glyoxylate synthase.

A. malate synthase and isocitrate dehydrogenase.

Electrons do not flow through the electron-transport chain to O2 unless _______________.

ADP is simultaneously phosphorylated to ATP.

ATP and ADP cannot diffuse freely across the inner mitochondrial membrane and are transported by the ATP-ADP translocase. The flow of ATP and ADP are coupled, meaning ______________.

ADP must enter the mitochondria for ATP to leave.

The level of ____________ is the most important factor in determining the rate of oxidative phosphorylation. This regulation is called respiratory control or acceptor control.

ADP. **ADP levels increases when ATP is consumed.

Uncouplers carry protons across the inner mitochondrial membrane. The electron-transport chain functions, but _______________ does not occur because the proton gradient can never form.

ATP synthesis.

The inner mitochondrial membrane is impermeable to metabolites and ions. To generate ATP, ADP and Pi have to be transported into the mitochondrial matrix. The ADP transporter is also an ATP transporter. It is called the ________________. For each ADP transported into the mitochondria, an ATP exits the mitochondria.

ATP-ADP translocase. **ATP-ADP translocase is an antiporter and makes up 15% of the inner mitochondrial membrane proteins.

Defenses against oxidative damage include ______________.

Antioxidant vitamins (vitamin E & vitamin C) and exercise.

________________: changes in the properties of the three b subunits of F1 allows sequential ADP and Pi binding, ATP synthesis and release.

Binding charge mechanism.

ATP-ADP translocase is a unidirectional exchange of ATP for ADP (antiport). Pi also needed in matrix - Phosphate transporter is a symporter of Pi and H+, electro-neutral, but does effect chemical component (pH).

Both transporters use up some of the proton motive force. Transport of ATP changes the charge gradient and transport of Pi changes the pH gradient. The proton motive force used for both transporters is equivalent of 1 proton. These transporters are associated with the ATP synthase complex.

Proton motive force has 2 components: 1. _______________ is generated by the difference in pH inside and outside the matrix (outside has more protons and is more acidic than matrix). 2. ______________ is generated by the charge difference inside and outside the matrix (outside has more protons, which is more positive and matrix has less protons and is more negative).

Chemical gradient (pH). Charge gradient (protons).

_________________ catalyzes the reduction of O2 to two molecules of H2O.

Cytochrome c oxidase (Complex IV).

_______________ pumps four protons from matrix to intermembrane space. Oxidative phosphorylation occurs under aerobic conditions, because this reaction requires oxygen. Why does it require oxygen?

Cytochrome c oxidase. Oxygen is the final electron acceptor.

The glycerol 3-phosphate shuttle:

Electrons from NADH can enter the mitochondrial electron-transport chain by reducing dihydroxyacetone phosphate to glycerol 3-phosphate. Electrons are transferred to an FAD prosthetic group in a membrane-bound glycerol 3-phosphate dehydrogenase, which reoxidizes glycerol 3-phosphate. Subsequent electron transfer to Q to form QH2 allows these electrons to enter the electron-transport chain. The electrons are transferred from NADH to FADH2 and subsequently to Q to form QH2.

___________ subunit spans the inner mitochondrial membrane and contains the proton channel (10 to 14 c subunits).

F0.

___________ subunit protrudes into the mitochondrial matrix and contains the catalytic activity (a3, b3, g, d and e).

F1.

succinate = ___________.

FADH2.

_______________ are electron carriers. They accept and donate electrons.

Iron-sulfur proteins.

Structure of ATP synthase: the part of the enzyme complex (the F0 subunit) is embedded in the inner mitochondrial membrane, whereas the remainder (the F1 subunit) resides in the matrix.

It consists of 2 units: F0= proton-conducting unit -contains the proton channel. F1= ATP-synthesizing unit - contains the catalytic activity (beta-subunit).

In what conformation are nucleotides trapped in the b subunit?

L/loose.

_______________ generate small amounts of superoxide anions and hydrogen peroxide, called reactive oxygen species or ROS, that are harmful for cells.

Mitochondria.

________________ are semiautonomous organelles and contain their own DNA and encode a variety of proteins.

Mitochondria.

The malate-aspartate shuttle consists of two membrane transporters and four enzymes. The electrons of cytoplasmic NADH are transferred to ______________ NADH. Thus, there is no energy loss and 2.5 ATP are generated per cytoplasmic NADH.

Mitochondrial.

Oxidative phosphorylation regenerates ___________ for use in glycolysis.

NAD+.

Defects in electron-transport chain not only reduce ATP synthesis, but increase the amount of ____________, leading to increased mitochondrial damage.

ROS.

Oxidative phosphorylation can be characterized as a process in which electrons from _______________ are transferred to molecular oxygen. The transfer of electrons generates a proton gradient that is used to generate ______________.

NADH and FADH2. ATP by ATP synthase.

In oxidative phosphorylation, ATP is formed as a result of the transfer of electrons from ____________ to __________ by a series of electron carriers.

NADH or FADH2 O2

The driving force for oxidative phosphorylation is the electron-transfer potential of _____________ relative to that of O2. The electrons from NADH are not directly transferred to O2. They are carried by electron carriers and the reduction of each electron carrier releases energy that contributes to the proton gradient.

NADH or FADH2.

The electrons from NADH enter the chain at ________________.

NADH-Q oxidoreductase (Complex I). **Four protons are simultaneously pumped out of the mitochondria by Complex I.

Electron flow from NADH to O2 is mediated through three large protein complexes. These complexes pump protons out of the mitochondria, generating a proton gradient. What are the 3 complexes?

NADH-Q oxidoreductase (Complex I). Q-cytochrome c oxidoreductase (Complex III). Cytochrome c oxidase (Complex IV).

Summary of Oxidative Phosphorylation:

NADH-Q oxidoreductase pumps 4 protons from the matrix; Q-cytochrome c oxidoreductase pumps 2 protons and cytochrome c oxidase pumps 4 protons from the matrix. The 2 electrons from NADH contribute to the removal of 10 protons from matrix, while the 2 electrons from FADH2 contribute to the removal of 6 protons (first proton pump is skipped). Per NADH 2.5 ATP and per FADH2 1.5 ATP are generated.

Rotenone (fish/insect poison) and amytal (barbiturate sedative) block the electron transfer in _______________.

NADH-Q oxidoreductase.

Electrons from NADH are transferred to Q by _________________ and electrons from FADH2 are transferred to ubiquinone or Q by _________________. Q is hydrophobic and diffuses through the membrane and docks to the next complex (III) - Q-cytochrome c oxidoreductase - which transfers electrons from ubiquinol (QH2) to cytochrome c - The transfer of electrons is coupled to pumping of two protons.

NADH-Q oxidoreductase. succinate-Q reductase.

Electron transfer starts with complex I or the ___________________. It is L-shaped and consists of two parts - hydrophobic horizontal arm in the membrane - a vertical arm facing the matrix. It catalyzes the transfer of electrons from NADH to ________________. The transfer of the electrons from NADH to Q is associated with the pumping of 4 protons out of the matrix.

NADH-Q oxidoreductase. ubiquinone Q.

•A strong reducing agent (___________) readily donates electrons and has a negative E0′. •A strong oxidizing agent (__________) readily accepts electrons and has a positive E0′.

NADH. O2.

Is succinate Q-reductase a proton pump?

No.

In what conformation can nucleotides bind or be released from the b subunit?

O/open.

QH2 carries two electrons while cytochrome c carries only one electron. The mechanism for coupling electron transfer from QH2 to cytochrome c is called the _____________.

Q cycle.

___________________ is the second proton pump in the respiratory chain and catalyzes the transfer of electrons from QH2 to cytochrome c.

Q-cytochrome c oxidoreductase (Complex III). **This electron transfer is coupled to the pumping of two protons from the matrix.

Antimycin A blocks the electron flow in ________________.

Q-cytochrome c oxidoreductase.

________________, is a measure of a molecule's tendency to donate or accept electrons.

Reduction potential E0′ or redox potential.

Superoxide dismutase and catalase help protect against ROS damage. ________________ converts superoxide radicals to hydrogen peroxide and oxygen. ___________ converts hydrogen peroxide into water and oxygen.

Superoxide dismutase. Catalase.

The 3 beta subunits are identical but in complex with the gamma subunit they have different conformations: _________________.

T tight confirmation - has a high affinity for ADP, Pi and ATP. The beta subunit in T confirmation forms the ATP, but ATP cannot be released. The L confirmation is the loose conformation that binds ADP and Pi but cannot release them. O stands for open form - nucleotides can be released.

In what conformation is ATP synthesized from ADP and Pi, but ATP cannot be released?

T/tight.

Summary of oxidative phosphorylation.

The flow of electrons from NADH or FADH2 to molecular oxygen via the respiratory or electron-transport chain leads to pumping of protons from the matrix to the intermembrane space, generating a proton motive force, which is then used to synthesize ATP via the ATP synthase. Electron transport is coupled to ATP synthesis via a proton motive force.

Mitochondria are thought to have developed by an _________________ event. It is assumed that mitochondria were originally independent free-living organisms and at some point were engulfed by another organism and became part of that organism.

endosymbiotic. **The evidence for this endosymbiotic event comes from the fact that the mitochondria have their own DNA and have the double membrane system and can generate energy (ATP) by oxidative phosphorylation.

The interconversion of these three forms is driven by rotation of the _________ subunit, which requires proton flow. (Proton flow releases the newly synthesized ATP).

gamma. **counterclockwise rotation.

In muscle, electrons from cytoplasmic NADH enter the electron-transport chain via the _____________ shuttle.

glycerol 3-phosphate shuttle.

The ATP balance is shown for muscle, which uses the _____________ shuttle and makes _________ ATP per cytosolic NADH from glycolysis.

glycerol 3-phosphate. 1.5

Energy is released when ______________ are transferred to oxygen. The energy is used to establish a ______________.

high-energy electrons. proton gradient.

All of the enzymes of oxidative phosphorylation are localized within the ________________.

inner mitochondrial membrane.

Where does oxidative phosphorylation occur?

inner mitochondrial membrane.

______________ is the site of electron transport and ATP synthesis.

inner mitochondrial membrane.

A deficiency of ________________ results in Friedreich's ataxia, a disease that affects the nervous, heart and skeletal system.

iron-sulfur clusters. **cannot produce enough iron-sulfur clusters, slows down the electron transport chain, systems do not get enough ATP.

What is the function of cytochrome c in the electron transport chain?

it transports electrons from complex III to complex IV.

In heart and liver, electrons from cytoplasmic NADH are used to generate mitochondrial NADH in the _____________ shuttle.

malate-aspartate shuttle.

Liver uses the ______________ shuttle and produces 32 ATP per glucose because cytosolic NADH from glycolysis generates 2.5 ATP in liver and heart.

malate-aspartate.

A ______________ redox potential means that a molecule has lower affinity for electrons compared to H2. A ______________ redox potential means, the molecule has a higher affinity for electrons than H2.

negative. positive.

Uncoupling of electron transport from ATP synthesis, generates heat, a process called _______________. Such uncoupling is facilitated by uncoupling protein 1 (UCP-1), also called thermogenin, an integral protein of the inner mitochondrial membrane.

non-shivering thermogenesis. **use proton gradient to generate heat instead of ATP.

Mitochondria: --Outer membrane is permeable to small molecules and ions because of channel protein ___________. --Inner membrane is _____________.

porin. impermeable.

Electrons are transferred in the direction of ______________ redox potential.

positive.

___________ help with electron transfer and sit in catalytic site complexes.

prosthetic groups.

What 2 subunits comprise ATP synthase?

proton-conducting unit (F0) and catalytic/ATP-synthesizing unit (F1).

ATP synthesis is powered by the _____________.

proton-motive force.

Electron transport chain and ATP synthase are separate systems, linked only by a _____________.

proton-motive force.

Electrons from NADH and FADH2 are transferred to O2 via protein complexes in the inner mitochondrial membrane. This leads to pumping of protons out of the mitochondrial matrix, generating a ________________. ATP synthesis occurs when the protons flow back.

proton-motive force.

Inhibition of electron-transport chain prevents oxidative phosphorylation by inhibiting the formation of the ________________.

proton-motive force.

Oxidation of fuels and phosphorylation of ADP are coupled by a _____________ gradient across the inner mitochondrial membrane.

proton.

Enzyme-bound ATP forms readily in the absence of a proton-motive force. However, ATP does not leave the catalytic site unless ____________ flow through the enzyme.

protons. **The binding change mechanism accounts for the synthesis and release of ATP in response to proton flow.

P/O ratios are maximum values and the efficiency is not always 100%, because __________________.

some of the proton gradient can be used to make heat instead of generating ATP.

What are the 2 major ways to generate ATP?

substrate-level phosphorylation and oxidative phosphorylation (majority).

An additional complex, __________________, delivers electrons from FADH2 to Q-cytochrome c oxidoreductase.

succinate Q-reductase (Complex II).

In the citric acid cycle, FAD is reduced to FADH2 by the _________________ by conversion of succinate to fumarate.

succinate dehydrogenase. **Succinate dehydrogenase is part of the Complex II (succinate-Q reductase).

Electron transfer or respiratory chain contains 3 proton pumps and 2 electron carriers. Electrons from FADH2 enter the electron transport chain via _________________ and are transferred onto ubiquinone Q.

succinate-Q dehydrogenase (complex II).

ATP synthase phosphorylates ADP to ATP. The interesting finding about the ATP synthase is that ATP can be formed by the ATP synthase in the absence of the proton motive force. The proton motive force (flow of protons though the ATP synthase) is required to release the synthesized ATP from the ATP synthase. This requires the binding change mechanisms and depends on the properties of _______________.

the 3 beta subunits of F1.

the entry of ADP into mitochondria is coupled to _____________.

the exit of ATP by the ATP-ADP translocase.

Oligomycin and dicyclohexylcarbodiimide (DCCD, antifungal agent) inhibits the ATP synthase by preventing ________________.

the influx of protons.

the rate of oxidative phosphorylation is determined by ___________.

the need for ATP.

This last complex pumps 4 protons from the matrix. Cytochrome c oxidase accepts four electrons from four molecules of cytochrome c in order to catalyze _______________.

the reduction of O2 to two molecules of H2O.

__________________ is a hydrophobic electron carrier that is located in the inner mitochondrial membrane and transports electrons from NADH-Q oxidoreductase (complex I) and succinate-Q reductase to Q-cytochrome c oxidoreductase (complex III).

ubiquinone (coenzyme Q or Q).

What is the P/O ratio?

used to express the relationship between oxygen consumption (respiration) and ATP synthesis (phosphorylation) ADP phosphorylated/oxygen reduced to water.

The _____________ subunit connects the F1 and F0 components of ATP synthase.

γ (gamma).