Glucose OxygenCarbon dioxide

Water

ADP + Pi ATP

This equation is a summary of a complex metabolic pathway consisting of 4 main stages:

1. Glycolysis

2. The Link Reaction

3. The Krebs Cycle

4. Oxidative phosphorylation

Before we look at these stages in detail, we need to look at the structure of a mitochondrion, and see how structure relates to function:

Draw this diagram:

How many labels can you remember?

1-2μm long

Outer mitochondrial membrane - OMM

Inner mitochondrial membrane - IMM

Inter membranal space - IMS

CristaeMitochondrial DNA

Matrix

Ribosomes

Stalked particles – ATP synthase molecules

Increases surface area for membrane proteins involved in oxidative phosphorylation –increasing ATP production

Contains the genes for mitochondrial proteins – eg ATP synthase

Compartmentalises the link reaction, Krebs cycle and OP from the cytoplasm Provides a space for the

build up of a H+ ion (proton) electrochemical gradient

Separates IMS from matrix. Contains membrane proteins involved in OP. H+

ions move across by facilitated diffusion through ATP synthase molecules.

Allow the translation of mitochondrial proteins

H+ ions move by facilitated diffusion through ATP synthase molecules, allowing the synthesis of ATP.

The ‘background material’ of the mitochondrion. Contains the enzymes that catalyse the link reaction and the reactions of the Krebs cycle.

How does the structure of a mitochondrion allow it to carry out its functions?

For each of these labels, try to relate the structural feature to its function.

1. GlycolysisHappens in the cytoplasm – not in mitochondria.

Glucose

Hexose bisphosphate

Triose phosphate

Triose phosphate

Pyruvate Pyruvate

2 ATP

2 ADP + Pi

2 ATP

2 ATP

2 ATP

2 ADP + Pi

oxNAD

redNAD

oxNAD

redNAD

The first stage uses ATP.Glucose is activated in a phosphorylation reaction.

Triose phosphate is oxidised to pyruvate

1. How many carbon atoms have all these molecules got?2. What’s the net gain of ATP from glycolysis?3. What happens to the 2 Pi from the 2ATP in stage 1?

Triose phosphate

Pyruvate

oxNAD

redNAD

Oxidation, reduction and coenzymes.

• NAD is a coenzyme that acts as a hydrogen carrier.• It takes part in reactions catalysed by dehydrogenases.• NAD – nicotinamide adenine dinucleotide .• It can be oxidised – oxNAD, or reduced – redNAD.

• When oxNAD becomes reduced, it gains 2 hydrogen atoms.

• A hydrogen atom is a proton H+ and en electron e-

The simplified reaction is this:

oxNAD redNAD

The ‘proper’ reaction is this:

NAD+ + 2H+ + 2e- NADH + H+

2H 2 hydrogen atoms

OIL RIGOxidation is loss. Reduction is gain

(of electrons)When a substance loses electrons,

it becomes oxidised.When a substance gains electrons,

it becomes reduced.

Using this information, explain what’s happening in the above reaction in terms of oxidation, reduction, electron gain and loss, and hydrogen atoms.

2. The link reactionHappens in the matrixLinks glycolysis and the Krebs cycle

Pyruvate

Acetate

Acetyl coenzyme A Coenzyme A

Acetate

CO2

oxNAD

redNAD

Acetate combines with CoA in order for it to be ‘fed into’ the Krebs cycle.

This is a decarboxylation reaction… …and a dehydrogenation.

How many carbon atoms does each molecule have?How many times does the link reaction have to happen per glucose molecule?

Acetate

Oxaloacetate Citrate

3. The Krebs cycleHappens in the matrix

CO2

CO2

The ATP so far in glycolysis and the Krebs cycle has been produced in substrate level phosphorylation.This contrasts with oxidative phosphorylation, the next stage.

Stage ATP redNAD redFAD

Glycolysis

Link reaction

Krebs cycle

Total

Fill in this table per glucose molecule:

Note where the following reactions occur:DecarboxylationDehydrogenationOxidationReductionPhosphorylation

4. Oxidative phosphorylationHappens in the IMM, matrix and IMS

red

NA

D

oxN

AD

red

FAD

oxF

AD

2H+ 2H+2H+

2H+

½O2 + 2e- + 2H+ H2O

2e-2e-2e-

Using this information, can you work out the maximum theoretical yield of ATP from one glucose molecule?

Oxygen is ‘the final electron acceptor’

The maximum theoretical yield of ATP per glucose is 38.In reality this is not achieved.Can you suggest why not?

• Pyruvate has to be actively transported from the cytoplasm into the matrix.• ADP and Pi has to be actively transported from the cytoplasm into the matrix.• Some H+ ions ‘leak’ across the IMM, back into the matrix.

This means that instead of 3 ATPs per redNAD, it’s 2.5, and instead of 2ATPs per redFAD, it’s 1.5.

What’s the yield of ATP per glucose now?

Chemiosmosis.

The movement of high energy electrons down an electron transport chain……energy from the electrons used to pump H+ ions / protons across a membrane……the formation of a H+ ion / proton / electrochemical gradient……the facilitated diffusion of ions through an ATP synthase complex……the production of ATP using the energy from the movement of the H+ ions.

We have seen this process in:• Oxidative phosphorylation• The light dependent reactions of photosynthesis

Electron transport chain

e- e-

H+

H+

But what experimental evidence do we have for chemiosmosis?

Evidence for chemiosmosis.For each piece of evidence, summarise it, and explain HOW it provides evidence for chemiosmosis.

1.. pH gradientLow pH in IMS

Higher pH in matrix

The same for chloroplasts –but where would the low and high pHs be? Draw a diagram to explain.

2. Stopping the build up of the H+ ion gradient.Dinitrophenol causes the IMM to be very ‘leaky’ to H+ ions.Mitochondria treated with DNP do not produce ATP.

H+

H+

H+

Extension question:DNP was used as a diet pill in the 1930s –explain how it makes you thin.(Don’t try it. It causes cataracts to form, and damages the nervous system!)

3. Isolated thylakoids can be made to produce ATP.

Anaerobic respirationLooking back at all the stages of respiration, try to work out what the consequences would be if there was no oxygen – the final electron acceptor.

No oxygen oxidative phosphorylation stops / ‘backs up’ no oxNAD regenerated Krebs cycle / link reaction stops

But cells still need ATP! How does ATP production continue without oxygen?

In mammals:Glycolysis continues, but something different happens to pyruvate:

Glucose

Hexose bisphosphate

2 Triose phosphate

2 Pyruvate

2 ATP

2 ATP

4 ATP

4 ADP + Pi oxNAD

redNAD

2 LactateLactate dehydrogenase

Pyruvate is reduced to lactate.This regenerates oxNAD so glycolysis can continue.What are the problems here?1. ATP yield is low – only 2 per glucose – but better than none!2. Lactate is toxic causing nausea, pain and eventually stopping

muscle contraction.

What happens to the lactate?• Lactate is converted back to pyruvate in hepatocytes.• Pyruvate enters the link reaction / Krebs cycle as normal.• Pyruvate can also be converted to glycogen for storage.• Removal of lactate requires oxygen – this is the oxygen debt.

Anaerobic respiration in yeast.

Yeast, a unicellular fungus – Saccharomyces cerevisiae – has been responsible for the staple diet of students for hundreds of years…

…this is because of 2 substances produced by yeast cells when they respire anaerobically:

ethanol and carbon dioxide

Why are each important in brewing and baking……and why don’t you get drunk eating bread?

Glucose

Hexose bisphosphate

2 Triose phosphate

2 Pyruvate

2 ATP

2 ATP

4 ATP

4 ADP + Pi oxNAD

redNAD

The process is similar to that in mammals, but pyruvate has a different fate:

2 Ethanal 2 EthanolPyruvate

decarboxylaseEthanol

dehydrogenase

CO2

Pyruvate is decarboxylated to ethanal. This produces CO2 - bubbles!Ethanal is reduced to ethanol.This regenerates oxNAD so glycolysis can continue.What are the problems here?1. ATP yield is low – only 2 per glucose – but better than none!2. Ethanol is toxic, eventually killing the population of yeast cells that

produced it.

Now construct a table to compare and contrast anaerobic respiration in mammals and yeast.

This is a simplified (!) version of a famous wallchart published by Roche Applied Science. You can get one for free if you ask them nicely!

Amongst other things, you should be able to spot the following:

GlycolysisLink reactionKrebs cycle

Oxidative phosphorylationThe Z scheme

RuBPChemiosmosis x 2

The ornithine cycleCAMP

DopamineHaemoglobin

CelluloseLactoseLignin

Histamine

Many other molecules can be used as respiratory substrate – not just glucose:

Glucose

Hexose bisphosphate

Triose phosphate

Pyruvate

CoAAcetyl CoA

Krebs cycle

Amino acids

LipidsGlycerol

Fatty acids

Proteins

This diagram shows where the molecules and their ‘fragments’ enter the pathways of respiration.

What’s a respiratory substrate?‘A substance used to produce ATP in a

cell by respiration’

Different respiratory substrates have different energy contents:

Carbohydrate: 17 kJ g-1

Protein: 17 kJ g-1

Fat: 39 kJ g-1

Why?

GlucoseCysteine – an amino acid

Palmitic acid – a fatty acid

What proportion, in %, of hydrogen atoms does each molecule have?

It’s all about hydrogen.

Energy is obtained from respiratory substrates by oxidising them, forming reduced coenzyme – redNAD.

Oxidation involves dehydrogenation.

If a molecule has proportionately more hydrogen atoms, then more redNADmolecules can be formed, and more ATP molecules can be synthesised during oxidative phosphorylation.

Answers:Glucose 12/24 = 50%Cysteine: 7/14 = 50%Palmitic acid: 32/50 = 64%

Some extension slides follow:

ETC animation

ATP synthase animation