BIO 302 Exam 3

Cellular Respiration

A aerobic process where organisms use oxygen to break down food molecules to obtain chemical energy (i.e, ATP and high energy electron carriers like NADH and FADH2) for cell functions

Animals, plants, fungi, algae

Cellular respiration pathways in cells

1) glycolysis

• in the cytosol

2) the citric acid cycle

• mitochondrial matrix

3) oxidative phosphorylation

• mitochondrial matrix

2 pyruvate, 2 ATP, 2 NADH

The net products of glycolysis are:

**The oxidation of sugars begins with glycolysis

Pyruvate Dehydrogenase

What oxidizes pyruvate to acetyl CoA and CO2?

Contains 3 enzymes and some 60 polypeptides

NADH is produced in this reaction

Mitochondrial Matrix

Where pyruvate is converted to acetyl CoA (pyruvate oxidation)

NADH is produced in this reaction

In the mitochondria

Where fatty acids are converted to acetyl CoA

Each fatty acid in the form of fatty acyl CoA is broken down by a cycle of reactions that trims two carbons at a time from its carboxyl end

1 acetyl CoA, 1 NADH, 1 FADH2

Each turn of the cycle generates (fatty acids cycle)

Oxidation

Most of the cells _____ reactions occur in the mitochondria

Most of its ATP is generated here too

Citric Acid Cycle

The _________ then converts the acetyl group in acetyl CoA to CO2 and H 2O

Much of the energy released in these oxidation reactions is stored as high-energy electrons in the activated carriers NADH and FADH2.

The high energy electrons are used to produce ATP through a process called oxidative phosphorylation

One cycle with three names

Citric Acid Cycle

Krebs Cycle

Tricarboxylic acid (TCA) cycle

3 NADH, 1 FADH2, 1 GTP, 2 CO2

The products of the citric acid cycle

Essential requirements for harnessing energy in ATP

• a membrane containing a series of electron carriers (electron-transport chain), a pump protein, and an ATP synthase

• source of high energy electrons (e-) derived from the oxidation of glucose

• protons (H+)

Proton Pump

Harnesses the energy of electron transfer to pump protons derived from water, creating a proton gradient across the membrane

Proton Gradient

Serves as an energy store and is used to drive the synthesis of ATP by the ATP synthase

Chemiosmotic Coupling

The linkage of electron transport, protein pumping, and ATP synthesis was formerly called the chemiosmotic hypothesis

Allows cells to harness the energy of electron transfers (in the same way that energy is stored in a battery)

Characteristics of mitochondria

• contains own DNA and RNA’

• constantly change shape and position in a cell

• position varies between cell types depending on where the majority of ATP is needed

4 compartments of mitochondria

1. Matrix: highly concentrated mixture of enzymes

2. Inner membrane: Folded into numerous cristae, contains proteins that carry out oxidative phosphorylation

3. Outer membrane: Large channel-forming proteins (called porins)

4. Inter membrane space

Inner mitochondrial membrane

Protons are pumped across

oxidative phosphorylation

NADH donates

a hydride ion to the electron-transport chain

High energy electrons

Generated during glycolysis and the citric acid cycle, power the production of ATP

Oxidative Phosphorylation in Mitochondria

The combined efforts of the ETC and ATP synthesis via ATP synthase is referred to as _______

Involved both the consumption of O2 and the addition of a phosphate group to ADP to form ATP

Four respiratory enzyme complexes

NADH dehydrogenase complex

cytochrome c reductase complex

succinate dehydrogenase

cytochrome c oxidase complex

**inner mitochondrial membrane

Complexes I, III, IV

Directly pump protons from the matrix into the intermembrane space

Complex II does not directly pump proton, but it does promote proton pumping in complexes III and IV

Proton Pumping

Requires energy

The four protein complexes get this energy by transferring electrons through a series of coupled reaction (unidirectional)***

1) NADH donates its two electrons directly to NADH dehydrogenase

2) FADH2 donates its two electrons to succinate dehydrogenase

Accept electrons from NADH or FADH2

Complexes I and III

Accept these in the form of a hydride ion (H-)

Redox Centers

Clusters of atoms that have different affinities for electrons based on their unique atomic configurations

Electrons move mainly between metal atoms that are tightly bound to the proteins***

Each subsequent redox center

Has a higher affinity for the electrons than the previous one in the sequence, allowing for a unidirectional flow of electrons through the chain

When an electron is passed through redox centers

A small amount of energy is released, allowing some of the complexes to harness this energy and use it to pump protons through the complexes, across the membrane

Ubiquinone (coenzyme Q) and Cytochrome C

Electron “shuttling” molecules in ETC

These molecules assist in transferring the electrons through the complexes

Coenzyme Q

The last redox center in Complex I (NADH dehydrogenase) donates two electrons to _____ molecule

Can accept two electrons from EITHER Complex I (NADH dehydrogenase) or Complex II (succinate dehydrogenase) and shuttle them to Complex III (cytochrome c reductase)

Complex II

Does NOT pump protons across the membrane

High energy electrons that enter Comlex II are first donated from FADH2

Complex II also transfers electrons between several redox centers before donating them to Coenzyme Q

Goal of electron transport chain

Establish a proton gradient

Without it, ATP synthesis cannot work

Coenzyme Q - hydrophobic

Coenzyme Q is highly hydrophobic, and rests in the hydrophobic core of the inner mitochondrial membrane

Cytochrome C

A small protein that tethers itself to the intermembrane face of the inner mitochondrial membrane

Carries electrons from Complex III (cytochrome c reductase) to Complex IV (cytochrome c oxidase)

• Because of its small size, cytochrome c is only able to shuttle one electron at a time into complex IV***

When 4 electrons build up in Complex IV

When this happens, they undergo a series of reactions that convert a molecule of oxygen to two molecules of water via interactions with other protons in the mitochondrial matrix

**The reason we breathe oxygen is so that it can serve as the final electron acceptor at the end of the electron transport chain

ETC unidirectional flow

Electrons can flow through the respiratory chain in the following directions:

1) Electrons donated from NADH: Complex I » Coenzyme Q » Complex III » Cytochrome C » Complex IV

2) Electrons donated from FADH2: Complex II » Coenzyme Q » Complex III » Cytochrome C » Complex IV

Proton-motive force

The role the membrane potential and pH that adds to the driving force pulling H+ across the membrane

The total electrochemical gradient of H+ across the inner mitochondrial membrane:

- a large force due to the membrane potential (∆V)

- a smaller force due to the H+ concentration gradient (∆pH)

Electrochemical gradient of protons

One of the key roles of the inner mitochondrial membrane is to act as a barrier to positively charged protons

This allows a concentration gradient to be maintained where the concentration of protons is vastly different on either side of this membrane

**Concentration of protons in the intermembrane is MUCH GREATER than the concentration of protons in the matrix

ATP production

The electrochemical proton gradient across the inner mitochondrial membrane allows ATP synthase to generate ATP from ADP and Pi

ATP synthesis, via the ATP synthase enzyme, is the chemiosmotic component of oxidative phosphorylation

Lollipop

ATP synthase is shaped like a…

The enzyme is composed of a head porton, called the F1 ATPase, and a transmembrane H+ carrier called the F0

Both are formed from multiple subunits

ATP synthase enzyme

Synthesizes ATP by using proton flow from one side of the inner mitochondrial membrane to the other

This binding of protons triggers the protein to rotate

Chemical energy (in the form of protons) is converted to rotational energy, allowing synthesis of ATP to ADP +Pi to occur

If there is no proton gradient

ATP synthase subunits stops rotating and the cell can quickly become starved of the energy and die

Reversible Coupling Device

ATP synthase

Can convert the energy of the electrochemical proton gradient into chemical-bond energy or vice versa

Can either synthesize ATP by harnessing the H+ gradient (A) or pump

protons against their electrochemical gradient by hydrolyzing ATP (B).

Protein complexes together

Very densely packed on the inner mitochondrial membrane

Together, they make the entire surface of the inner mitochondrial membrane a giant cellular power plant

Electron transport + chemiosmosis = Oxidative phosphorylation

Symport

Electrochemical gradient of H+ drives the import of pyruvate and Pi

Antiport

Pumps out ATP and pump in ADP (ADP-ATP exchange) that depends a voltage gradient across the membrane (membrane potential) and move one negative charge out of the mitochondrion

30 ATP

One glucose oxidation produces

In cytosol

1 NADH » 1.5 ATP

Transport of NADH across the inner membrane requires energy

Inside mitochondrial matrix

1 NADH » 2.5 ATP

1 FADH2 » 1.5 ATP

Mitochondria maintains a High ATP/ADP ratio in cells (ATP is

10 times higher than ADP)

Blocking electron transport

In the inner mitochondrial membrane by the poison cyanide will cause cell death due to the stop of energetically unfavorable reactions

ATP is used

As an energy source to drive energetically unfavorable reactions since ATP hydrolysis is energetically favorable

Uncoupling Agents

H+ carriers that can insert into the mitochondrial inner membrane

Prevent ATP from being made (DNP)

DNP

Uncoupling agent

Causes the inner membrane to be permeable to protons, allowing H+ to flow into the mitochondrion without passing through ATP synthase, so that ATP can no longer be made

Such uncoupling occurs naturally in some specialized fat cells called brown fat cells, most of the energy from oxidation is dissipated as heat rather than converted into ATP

Bacteriorhodopsin

How can you demonstrate that proton gradients can power ATP production?

When added to artificial vesicles, the protein generates a proton gradient in response to light

In artificial vesicles containing both bacteriorhodopsin and an ATP synthase, the proton gradient drives the formation of ATP

• Uncoupling agents that abolish the gradient eliminate the ATP synthesis.

membrane-enclosed organelles

In eukaryotic cells, membrane-enclosed compartments are called

Internal membranes create enclosed compartments and
organelles in which different metabolic processes are segregated

Nucleus

The most prominent organelle, which is surrounded by a double membrane known as the nuclear envelope and communicated with the cytosol via nuclear pores that perforate the envelope

Endoplasmic Reticulum (ER)

The major site for synthesis of new membranes

• Rough ER: having ribosomes attached to its cytosolic surface

• Smooth ER: Lacking ribosomes

ER functions

• Adrenal gland cells: steroid hormone synthesis

• Liver cells: detoxify alcohol

• Smooth ER: sequester Ca 2+ from the cytosol and the release of Ca 2+ to cytosol triggers secretion of signal molecules and the contraction of muscle cells

Golgi Apparatus

Receives proteins and lipids from the ER, modifies them, and then dispatches them to other destinations in the cells

Lysosomes

Small sacs of digestive enzymes degrade worn-out organelles, as well as macromolecules and particles taken into the cell by endocytosis

Endosomes

Compartments containing endocytosed materials

Peroxisomes

Small organelles with a single membrane contain enzymes used in a variety of oxidative reactions that break down lipids and destroy toxic molecules

Mitochondria

Pyruvate oxidation, citric acid cycle, and oxidative phosphorylation

Chloroplasts

Photosynthesis

54

Relative volume of cytosol

22

Relative volume of mitochondria

Evolution of membrane-enclosed organelles

Likely began with an expansion of the plasma-membrane

An ancient archaeon cell possibly enlarged its plasma membrane, allowing it to interact with aerobic bacteria

Protrusions invaginated giving rise to various membrane-enclosed organelles by enclosing the archaeon’s genetic material, engulfing aerobic bacteria, and fusing to form the components of the endomembrane system (ER and Golgi apparatus)

Evolution of organelles

This can explain why the nucleus in present-day eukaryotes is:

surrounded by a double-layered membrane, and why mitochondria have two membranes, their own genomes, and do not participate in vesicular transport of the endomembrane system

Ribosomes in the cytosol

The synthesis of virtually all proteins in the cell begins here

Import of proteins

***one of three mechanisms

1) Transport across membranes

2) Transport through nuclear pores

3) Transport by vesicles

Sorting Signal

Directs the protein to the organelle in which it is required

***Proteins that lack such signals remain as permanent residents in the cytosol

Transport through nuclear pores

Importing proteins (1/3)

Proteins moving from the cytosol into the nucleus that are transported through the nuclear pores that penetrate the inner and outer nuclear membranes

Transport across membranes

Importing proteins (2/3)

Proteins moving from the cytosol into the ER (resident vs non-resident proteins) mitochondria, or chloroplast are transported across the organelle membrane by protein translocators located in the membrane

**Unfolding is required for protein transport across the membrane

Transport by vesicles

Importing proteins (3/3)

Transport vesicles are loaded with a cargo of proteins from the lumen of one compartment of the endomembrane system (ER, Golgi apparatus, endosomes and lysosomes) and discharge their cargo into a second compartment by membrane fusion

H3N

Signals import into ER

N-terminus of protein

COO-

Retention in lumen of ER

Indicates the C-terminus of a protein

N-terminal signal sequence

Proteins destined for the ER ***necessary

Those destined to remain in the cytosol lack this sequence

if the signal sequence is removed from the ER protein and attached to the cytosolic protein, the proteins end up in an abnormal location in the cell

Nuclear envelope

Double membrane is penetrated by nuclear pores

Encloses the DNA and defines the nuclear compartment

Traffic occurs in both directions through the pores

• Newly made proteins destined for the nucleus enter from the cytosol

• RNA Molecules, which are synthesized in the nucleus, and ribosomal subunits, which are assembled in the nucleus, are exported

Nuclear Lamina

The inner nuclear membrane contains proteins that act as binding sites for the chromosomes and provide anchorage for the ______

Nuclear pore

Composed of about 30 different proteins

Each one contains water-filled passages through which small water-soluble molecules can pass freely

Many proteins that line the nuclear pore contain extensive and unstructured regions, preventing the passage of large molecules

Nuclear Localization Signal

The signal sequence that directs a protein from the cytosol into the nucleus typically consists of one or two short sequences containing several positively charged lysines or argines

Nuclear transport receptors

Bind to the nuclear localization signal on newly synthesized proteins destined for the nucleus

Help direct the new protein through the nuclear pore into the nucleus

• Once the protein has been delivered, the nuclear transport receptor is returned to the cytosol through the nuclear pore for reuse

GTP hydrolysis

Drives nuclear transport

Ran-GTP

Binds to nuclear transport receptor, causing it to release cargo in the nucleus

In the cytosol an accessory protein triggers Ran to hydrolyze its bound GTP to GDP (releases it from the receptor)

Steps of GTP hydrolysis

1) A nuclear transport receptor picks up its cargo protein in the cytosol and enters the nucleus

2) In the nucleus, Ran-GTP binds to the nuclear transport receptor, causing it to release its cargo

3) The nuclear transport receptor - still carrying the Ran-GTP is transported back through the pore to the cytosol

4) In the cytosol, an accessory protein triggers Ran to hydrolyze its bound GTP to GDP. Ran-GDP falls off the nuclear transport receptor, which is then free to bind another cargo protein destined for the nucleus

similar cycle for mRNAs and other large molecules

Unfolded form

In what form are proteins imported into mitochondria?

Receptor on the outer mitochondrial membrane

**Import into mitochondria

What recognizes the mitochondrial signal sequence?

Import receptor protein***

Contact site in membrane

**Import into mitochondria

Where does the receptor-protein complex move after binding?

Translocators across inner and outer membrane

How does the protein enter the mitochondria?

**Diffuses laterally

(TOM and TIM - outer and inner)

The two translocators MUST be aligned

Signal Peptidase

Is what cleaves off the signal sequence in the mitochondria

**Import into mitochondria

Chaperone Proteins

Help to pull the protein across the membrane and help it to refold

**Import into mitochondria

The ER is….

the most extensive membrane network in cells

Membrane-bound ribosomes

Attached to the cytosolic side of the ER membrane and are making proteins that are being translocated into ER

Making and importing the proteins into the ER at the same time (translation and translocation)

Free Ribosomes

Unattached to any membrane and are making all of the other proteins encoded by the nuclear DNA

Many ribosomes bind to each mRNA molecule, forming a polyribosome

Signal-Recognition Particle (SRP)

Binds to the exposed ER signal sequence (coming out of the bottom of ribosome) and to the ribosome, thereby slowing protein synthesis by the ribosome

• slows down translation because it has to carry the entire complex closer towards the ER membrane

SRP-ribosome complex

SRP binds to an SRP receptor in the ER membrane

The SRP is released, passing the ribosome to a translocation channel in the ER membrane

Finally, the translocation channel inserts the polypeptide chain into the membrane and starts to transfer it across the lipid bilayer

Translocation Channel

Binds the signal sequence and actively transfers the rest of the polypeptide across the lipid bilayer as a loop

During the translocation process, the signal peptide is cleaved from the growing protein by a signal peptidase

Cleaved signal sequence

Done by signal peptidase

Is ejected into the bilayer, where it is degraded, and the translocated polypeptide is released as soluble protein into the ER lumen

Channel closes once the protein has been released