***12. Oxidative Phosphorylation

What is Oxidative Phosphorylation?

The metabolic process where cells use enzymes to oxidize nutrients, releasing energy to produce ATP

What is the primary energy currency of the cell?

ATP.

What are the two phases of oxidative phosphorylation?

1. Production of reduced NADH and FADH2

2. Use of generates energy to produce ATP = oxidative phosphorylation.

Energy produced during the transfer of electrons in the ETC is used to:

Pump protons into the intermembrane space.

The energy produced when those proteins reenter the mitochondrial matrix is used to:

Synthesize ATP.

Oxidative Phosphorylation is:

Electron transport chain (ETC) coupled with ATP synthesis.

How do electrons flow in oxidative phosphorylation?

From NADH and FADH2 through a series of carriers to reach O2.

Why can’t IMM enter the mitochondira?

Because it lacks an NADH transporter.

What does the Glycerol-3-P shuttle do?

Transfers electrons from cytosolic NADH into the mitochondria for oxidative phosphorylation.

Glycerol-3-P Shuttle:

NADH → FADH2

Glycerol-3-P becomes oxidized by the mitochondrial isoenzyme, which causes ____.

FAD to be reduced to FADH2.

What does the malate shuttle do?

Moves electrons from NADH produced during glycolysis in the cytoplasm across the inner mitochondrial membrane for oxidative phosphorylation.

Malate Shuttle:

Transfers electrons from cytosolic NADH → mitochondrial NADH.

How is oxaloacetate reduced to malate?

With NADH.

IMM special carriers:

• Adenine nucleotide antiporter

• Phosphate transporter

What does the adenine nucleotide antiporter do?

• Imports 1 ADP from cytosol.

• Exports 1 ATP into the cytosol.

What does the phosphate transporter do?

Carries phosphate from the cytosol into the matrix.

Where is the Electron transport chain located?

Inner mitochondrial matrix.

ETC Structure:

• 4 large multiprotein complexes (I-IV)

• 2 small carriers: coenzyme Q (CoQ) and cytochrome C

• Prosthetic groups

What do ETC carriers do?

Transfer electrons between complexes, to finally combine with O2 and H+ → H20.

What is the first and largest protein enzyme in the ETC?

Complex I (NADH: CoQ oxidoreductase)

Complex I Energy use:

Energy is lost with each passing and is used to pump 4H+ from the matrix into the inner membrane space.

What is Complex II?

Complex II (succinate dehydrogenase) oxidizes succinate to fumarate (TCA cycle) with production of FADH2.

Is energy lost in Complex II?

No. No energy is lost.

*NO Protons are pumped at this stage.

Which complex serves as the parallel entry point into the ETC?

Complex II.

What is Coenzyme Q known as?

Ubiquinone.

What kind of derivative is CoQ?

A quinone derivative from cholesterol.

What is the ONLY lipid-soluble and non-protein bound component of ETC?

CoQ.

What does CoQ do?

Is a mobile carrier of electrons from Complexes I and II → Complex III.

How many electrons does CoQ carry at a time?

2 electrons.

What is Complex III also known as?

Coenzyme Q-cytochrome c reductase OR the cytochrome bc1 complex.

What is Cytochrome c?

A mobile electron carrier that brings electrons to complex IV.

How many electrons does Cytochrome c carry?

1 electron at a time.

What happens during Complex III?

High drop in energy with electron movement → 4H+ are pumped into the IMS.

How many H+ are pumped into the IMS during complex III?

4H+.

What is Complex IV?

Cytochrome a+a3 (cytochrome oxidase).

What happens during Complex IV?

Transfers electrons to oxygen to form water and helps build the proton gradient needed to produce cellular energy (ATP).

(When 4 electrons are available, 4 protons are used to reduce and split O2 to form 2 molecules of H2O.

How many H+ molecules are pumped into the IMS during Complex IV?

2H+ per 1 H20 from the matrix are pumped into the IMS.

What is a reactive oxygen species?

Partially reduced oxygen is very unstable and avid for electrons.

In reactive oxygen species, how many electrons can O2 accept?

4 electrons.

What is a superoxide?

When CoQ accidentally interacts with O2.

What is Oxidative stress?

A cellular imbalance that happens when unstable molecules called free radicals outnumber the body’s protective antioxidants.

*(Free-radical mediated damage)

What causes Oxidative stress?

• Lipid peroxidation

• Proteins oxidation and degradation or aggregation

• DNA damage (base oxidation or double strand breaks)

What are cellular defenses against oxygen toxicitiy?

• Enzymes

• Antioxidants

What are the two entry points for the ETC Chain? ****

1. NADH → Complex I

2. FADH → Complex II

What is the final acceptor of the ETC? ****

Oxygen.

(in complex IV)

How many electrons are needed to reduce oxygen to convert to water? ****

4 electrons → 2 sets of 2 electrons since there are 2 oxygen atoms that need to be reduced to make H20.

How many protons are pumped out of complex IV per atom of oxygen? ****

2 protons / 1 atom of oxygen.

What do ETC inhibitors do?

Block the flow of electrons to oxygen and inhibit ATP synthesis.

What are ETC inhibitors for Complex I?

• Rotenone

• Barbiturates

What are ETC inhibitors for Complex III?

• Antimycin A

What are ETC inhibitors for Complex IV?

• Cyanide (CN-)

• Carbon monoxide (CO)

What is the Chemiosmotic Theory?

The energy needed to phosphorylate ADP → ATP is produced by a flow of protons against an electrochemical gradient.

How is the proton gradient established in the Chemiosmotic theory?

The proton gradient is established by H+ pumped from the Matrix → IMS using the energy released by the ETC through complexes I, III and IV.

What enzyme helps with ATP synthesis?

ATP Synthase.

Components of ATP Synthase:

*Multisubunit enzyme

• Membrane domain (F0; embedded in the IMM)

• Extramembraneous domain (F1; sphere that protrudes into the matrix)

Which part of the ATP synthase subunit is mobile? ****

Membrane domain; IMM ****

(rotates)

Which part of the ATP synthase subunit is fixed?

Extramembraneous domain (matrix) ***

(does not move)

One complete c ring rotation produces ___ of ATP. ****

3 molecules of ATP.

How are NADH and FADH2 oxidized? ****

Via the mitochondrial electron transport chain.

Where is the electrochemical proton gradient established? ****

Across the inner mitochondrial membrane.

What does the proton gradient do? ****

Drives ATP synthesis.

What do the inhibitors of electron transport do?

Block ATP synthesis.

Oxidative phosphorylation donors:

NADH & FADH2.

Oxidative phosphorylation electron acceptor:

O2.

What does Oxidative Phosphorylation require to proceed? ****

1. Electron donors (NADH & FADH2)

2. Electron acceptor (O2)

3. Intact mitochondrial membrane

4. Functional ETC components

5. ATP synthase

ATP synthesis and ETC are ____ in normally functioning mitochondria.

Coupled.

What happens if ATP synthase is inhibited or has inadequate supply of ADP?

• ATP synthesis is inhibited

• O2 will not be consumed

• ETC components accumulate in reduced states.

What is Oligomycin?

Binds to the F0 domain, closing the H-channels and preventing the reentry of H+ into the matrix → causes inhibited ATP synthesis and blocked oxidative phosphorylation.

What do uncoupling proteins do?

(In the IMM)

Form channels that allow H+ to reenter the matrix without synthesis of ATP.

What happens after uncoupling?

• ATP production decreases

• O2 consumption and ETC rate increase.

• Energy is released as heat in non-shivering thermogenesis.

• UPC1 / thermogenin is responsible for heat production in the mitochondria-rich brown adipose tissue.

What does UPC1 / thermogenin do?

Generates heat via non-shivering thermogenesis.

• Found primarily in brown adipose tissue

• Is a mitochondrial IMM.

What is Dinitrophenol?

A lipophilic H+ carrier that disrupts the proton gradient by carrying protons across the IMM.

Who has a large amount of brown adipose tissue? ***

Babies.

(maintain their body through non-shivering thermogenesis)

What is an uncoupler that can disrupt the proton gradient?

Dinitrophenol.

(Adverse effects: Heart failure and myocardial infarction)

How many proteins are involved in oxidative phosphorylation disorders?

13.

How are the proteins involved in OP disorders encoded and where are they synetheiszed?

• Encoded by mtDNA

• Synthesized in : Matrix

What is the mutation rate of mtDNA vs nuclear DNA in OP? ***

mtDNA mutation rate is 10x greater than nuclear DNA → genetic defects in OP enzymes.

T/F: Hereditary defects are common in OP. ***

False. They are rare.

Why is mutation rate so high in OP? ****

1. We lack proofreading capacity for mtDNA

2. We have a lot of reactive oxidative species in the mitochondria → induces DNA damage.

If you have a hereditary defect in OP, what occurs?

• Lactic acidosis

• Muscle and nerve pathology (tissues with high ATP requirements)

What is Lebers’s hereditary optic neuropathy? ***

A Complex I defect;

Bilateral neuroretinal degeneration with optic nerve damage.

What is Leigh syndrome? ***

F0 defect (ATP synthase)!!!! (complex V)

Optic nerve atrophy, hypotonia, ataxia, respiratory abnormality.