Dr Agnieszka Adamczewska

L6 – Cellular respiration

Summer School2015

Images from Wikimedia Commons

Major Concepts

1. Identify the three end-product options for glycolysis, and under what conditions these end-products form

2. State in words (not chemical formulae) the overall reaction of the glycolytic pathway, understand parts that are common and different

3. Understand how the overall balance sheet for glycolysis is obtained, and show the methods of reaction “coupling” that the cell uses

4. Say where the enzymes of the glycolytic pathway are to be found in eukaryotic cells and in prokaryotic cells

5. Explain what oxidation reduction reactions are and the special role of the coenzyme NAD+/NADH

Major Concepts

6. Give the names of, and recognise the equations for, the overall reactions of, the two major parts of the cellular respiration sequence - glycolysis and tricarboxylic acid (TCA) cycle.

7. Describe how a H+ pumping mechanism is coupled to a proton-driven ATP synthase.

8. State how many ATP molecules are produced per glucose molecule in the glycolytic pathway and in the whole respiratory pathway.

9. Describe where in the respiratory pathway CO2 is released, and where O2 is consumed.

Macromolecules store energy

Energy is stored in carbon-carbon bonds

e.g. glycogen or starch, fats and oils

Get energy out of food through catabolic reactions

Image from Campbell Biology 8e Australian Version © Pearson Education Inc.

Metabolism: chemical reactions that occur within cells• Catabolism – breaking down organic matter to release

energy• Anabolism – using energy to produce cellular components

CatabolismComplex molecules Simple molecules

Anabolism

ADP ATPPi

Metabolism (L3)

ATP: energy carrierMade through metabolism of energy rich molecules.

- carbohydrates are converted into glucose

- lipids are processed by β-oxidation

CatabolismComplex molecules Simple molecules

ADP ATPPi

ATP: Adenosine triphosphate

Hydrolysis

ADP Adenosine diphosphate

Energy conversions in cells

Energy from macromolecules is released by cellular respiration. Initial breakdown of macromolecules produces simple sugars, fatty acids, glycerol, and amino acids. Subsequent gradual oxidation of the fuel molecules by removal of electrons from C-C and C-H bonds releases energy:

Energy conversions located in the cytosol 1. Glycolysis converts glucose to pyruvate2. Fermentation to lactate and alcohol

Energy conversions located in mitochondria in the presence of O2

3. -oxidation of lipids produces acetyl CoA4. Citric acid cycle converts pyruvate to acetyl CoA and finally CO2

5. Electron transport chain (NADH, FADH2 > O2 > H2O) drives proton pumps, proton gradient is coupled to synthesis of ATP

Energy conversionpathwaysCellular respiration

12

3

4

5

CYTOSOL

MITOCHONDRION

Pathway described in 1930's- a major biochemical triumph- involves a number of steps

one glucose (C6)

two pyruvate (2 x C3)

Glycolysis

GlycolysisGlucose (6C)

2 ATP

2 ADP

3 steps

Fructose 1,6-bisphosphate (unstable)

G3P (3C)G3P (3C)NAD+

NADH

2 ATP

2 ADP

Pyruvate (3C)

NAD+

NADH

2 ATP2 ADP

Pyruvate (3C)

5 steps

Mitochondria image from Wikimedia Commons

glyceraldehyde 3 phosphate

Cytosol

Glycolysis Energy Conversions

Net yield

2 NADH2 ATP

Glycolysis“Splitting glucose”

Glucose• from hydrolysis of polysaccharides (L2)• enters cell via facilitated diffusion - Glucose-Na+

symport (L5)

Cytosol – 10 enzymes

Glucose (6C) + 2 ATP + 2 NAD+ + 2 ADP + 2 Pi 2 Pyruvate (3C) + 4 ATP + 2 NADH

Net yield: 2 ATP

NAD+ Nicotinamide Adenine Dinucleotide

Electron carrier (coenzyme )

Imag

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edia

Com

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NAD+ reduced to NADH by transfer of H+ from food

1. Glycolysis: Processing of glucose to pyruvate

a) Glucose (6C) is phosphorylated using 2 ATP and split into two molecules of glyceraldehyde 3-phosphate (3C). Total 5 steps, consuming 2 ATP

b) Oxidation in another 5 steps to 2 molecules of pyruvate (3C) and production of 4 ATP (net yield =2 ATP/glucose)

• Pyruvate can be converted in the absence of O2 by alcoholic fermentation to ethanol (in yeast and bacteria) or by lactate fermentation to lactate (muscle tissue)

• In the presence of O2 pyruvate enters mitochondria, is converted (decarboxylated) to a 2C compound acetyl CoA (a substrate for citric acid cycle) CO2, and NADH;

2. FermentationAnaerobic conversion of pyruvate

to alcohol or lactic acid

Alcohol fermentation

REDUCTION

By yeast and many bacteria.

Used by humans for thousands of years in brewing, winemaking, and baking (CO2 bubbles from baker’s yeast)

Lactic acid fermentation

By certain fungi and bacteria

Used in dairy industry to make cheese and yogurt

A product of muscle exercise, may enhance muscle performance (pain from K+ ions) REDUCTION

FermentationNo oxygen

Glycolysis then fermentation

Fungi, bacteria, animals

lactic acid

Yeast, plants

ethanol + CO2

Glycolysis1×

Glucose

2ATP2ADP + 2Pi

2NAD+ 2NADH + 2H+

2× Pyruvate

2×CO22×

Ethanol

Glycolysis1×

Glucose

2ATP2ADP + 2Pi

2NAD+ 2NADH + 2H+

2× Pyruvate

2×Lactic acid

Pyruvate A key juncture in catabolism

Food Chloroplast

• Site of cellular respiration (energy production)

• Double membrane: permeable outer membrane and impermeable, folded inner membrane (cristae) containing enzymes of respiration

Images from Wikimedia Commons

Outer membraneInner membrane

MatrixCrista

Mitochondria (L4)

Mitochondria: number, shape, and subcellular location are highly variable

Glucose + oxygen carbon dioxide + water + energy

Cytosol

MitochondriaATP ATP ATP

Matrix

Intermembranespace

Crista

NADH

Glycolysis

NADHFADH2

Krebs cycle Oxidative phosphorylation

Mitochondria image from Wikimedia Commons

Fuel in, energy out

Intermediate reaction Pyruvate to Acetyl CoA

MatrixInner mitochondrial membrane

Outer mitochondrial membrane

Pyruvate

Pyruvate dehydrogenase

complex

CoA

CoA

NAD+NADH + H+

CO2

Acetyl-CoA

Mitochondria image from Wikimedia Commons

Other fuel molecules

• Other carbohydrates (apart from glucose)

• Fats and proteins can also be broken down and enter pathways

b-oxidation

Image from Campbell Biology 8e Australian Version © Pearson Education Inc.

3. -Oxidation of lipids

-oxidation degrades long-chain fatty acids by 2C atoms at a time

Last reaction splits off acetyl CoA (energy in C-C bond) and enters Citric acid cycle

NADH, FADH2, energy (electron) carriers

h

NAD+ and FAD reduced to NADH and FADH2

ATP is formed

8 steps, each catalysed by different enzyme

Krebs cycleFADH2

NADH

CO2 evolved

AcetylCoA (2C)

Oxaloacetate (4C) Citrate (6C)

NADH CO2

5CNADH

CO24C

4C

4C

4C

6C

TCA cycle Citric acid cycle

4. Krebs cycle

accept e- from intermediates

ATP

ATP ATP

2× NADH

Glycolysis

6× NADH

2× FADH2

Krebs cycle

Mitochondria image from Wikimedia Commons

C6H12O6 + 0O2 6CO2 + 0H2O + energy

Chemical bonds to electrons

glucose

CO2 CO2

Chemical bonds to electrons

1 glucose:

Glycolysis 2 pyruvate + 2 ATP + 2 NADH

2 pyruvate 2 acetyl CoA + 2 NADH + 2 CO2

TCA cycle: 2 acetyl CoA 6 NADH + 2 FADH2 +

4 CO2 + 2 ATP

1 glucose 4 ATP + 8 NADH + 2 FADH2 + 6 CO2

5. Electron transport chain (ETC)

(Proton-motive force i.e. the power in movement of protons)

NADH and FADH2

Inner membrane of mitochondria

Four protein complexes of acceptors

Oxygen needed

Inner membrane

MatrixCrista

Mitochondria image from Wikimedia Commons

Intermembrane space

Mitochondrial matrix

Innermitochondrial

membrane

NADHdehydrogenase

bc1 complexCytochrome

oxidase complex

H+

C

H+

H+

NAD+

Q

e–

H+

e–

5. Electron transport chain

FAD

FADH2

NADH

Start with NADH (or FADH2) as primary electron donor

H2O2H+ + 1/2O2

Finish with O2 as terminal electron acceptor

H+H+

H+

H+

H+

H+

H+H+

H+H+

H+

H+

H+

H+

H+

Chain of redox reactionsInner mitochondrial membraneElectrons move to higher redox potentials, towards oxygen

with highest electron affinityEnergy released is used to pump H+ from matrix to inter-

membrane space

2e

NADH + H+

High energy

Low energy

Cyt a (Fe3+)

½ O2 + 2H+

H2O

Cyt a (Fe2+)

Cyt c (Fe3+)

Cyt c (Fe2+)

Cyt b (Fe3+)2H+ + NAD+

Cyt b (Fe2+)

Energy released

Electrons “falling” from

NADH to oxygen

Intermembrane space

Mitochondrial matrix

Innermitochondrial

membrane

NADHdehydrogenase

bc1 complexCytochrome

oxidase complex

H+

C

H+

H+

NAD+

Q

e–

H+

e–

Generating proton gradient

FAD

FADH2

NADH

Start with NADH (or FADH2) as primary electron donor

H2O2H+ + 1/2O2

Finish with O2 as terminal electron acceptor

H+H+

H+

H+

H+

H+

H+H+

H+H+

H+

H+

H+

H+

H+

Chemiosmosis couples the ETC to ATP synthesis!

IMS

IMM

OMM

FADFADH2

NAD+NADH + H+ H2O H+ + OH-

½ O2 + 2H+ H2O

AT

P syn

tha

se

ADP + Pi ATP

Electron transport chain

complexity of 1o, 2o, 3o and 4o structure

embedded in inner membrane of mitochondria

highly conserved in nature

ATP synthase

Matrix

Intermembrane space

Inner membrane

Image from http://www.rcsb.org/pdb/101/motm.do?momID=72

ATP synthase

Matrix

Inner membrane

Image from http://www.rcsb.org/pdb/101/motm.do?momID=72

RotorH+ bindingH+ ions flow down gradient and enter half channel in stator

H+ ions enter binding sites in rotor, changing shape of subunits so rotor spins

Each H+ ion makes one complete turn before being released to matrix

Spinning of rotor causes internal rod to spin

Turning rod activates catalytic sites in knob

Catalytic knob

Intermembrane space

H+

H+

H+

H+

ADP + Pi ATP

Glucose + oxygen carbon dioxide + water + energy

Cytosol

MitochondriaATP ATP ATP

Matrix

Intermembranespace

Crista

NADH

Glycolysis

NADHFADH2

Krebs cycle Oxidative phosphorylation

Mitochondria image from Wikimedia Commons

Fuel in, energy out

2x 2x ~34x

2

Aerobic respiration = lots of energy

C6H12O6 + 6 O2 6 CO2 + 6 H2O + ~38 ATP

Where are these ATPs from?

Glycolysis = 2 ATP

TCA = 2 ATP

Oxidative phosphorylation (ETC + chemiosmosis)

= ~34 ATP

• Infoldings of the inner mitochondrial membrane (cristae) greatly increase the number of electron transport chain proteins and ATP synthase proteins

Cristae increases surface area

Lack of oxygen?

Need oxygen to accept final electrons

If no oxygen, complex IV keeps electrons

Þ All protein complexes keep electronsÞ No pumping of H+ into intermembrane spaceÞ No H+ gradientÞ No energy for ATP synthase

No ATP made in ETC - ATP from glycolysis and TCA not enough

Most cells cannot survive long without oxygen

Prokaryotic cell – no mitochondria

Still need ATP!

Nucleoid region

PilusFlagellum

Ribosome

Cell wall

Plasma membrane

Capsule

Image from Wikimedia Commons

Aerobic Bacteria do it too!

Cell membrane

Electron carrier

ATP synthase complex ATP

ADP + Pi

H+

NADH

NAD+

O2

H2O

H+

Enzymes embedded in bacterial cell membrane

Anaerobic respiration

Prokaryotes living in anaerobic environment

- no oxygen e.g. waterlogged soils, intestines

Nitrate (NO3-) or sulfate (SO4

2-) are the terminal electron acceptors

Products include CO2, inorganic substance, ATP

e.g. C6H12O6 + 12 KNO3 (potassium nitrate)

6 CO2 + 6 H2O + 12 KNO3 (potassium nitrite) + ATP

Summary

Cytoplasm and mitochondria are sites of cellular respiration

Glucose + oxygen carbon dioxide + water + energy

Aerobic respiration has four stages

For one glucose molecule:

Glycolysis = 2 ATP, TCA = 2 ATP, Oxidative phosphorylation (ETC, chemiosmosis) = ~34 ATP

Chemiosmosis - electrons in ETC used to pump protons into inter-membrane space, this establishes a proton gradient across inner membrane, protons accumulate, lowers pH

Redox reactions integral to ETC

Fermentation and anaerobic respiration in absence of O2

Aerobic respiration

Stage Where Main starting materials

Main end products

Glycolysis Cytoplasm Glucose Pyruvate, ATP, NADH

forming Acetyl CoA

Matrix of mitochondria Pyruvate

Acetyl CoA, CO2, NADH

Citric acid cycle Matrix of mitochondria

Acetyl CoA, H2O

CO2, NADH, FADH2, ATP

Oxidative phosphorylation

Inner membrane mitochondria

O2, NADH, FADH2

ATP, H2O

• Read:

• Knox B, et al. (2010) Biology: an Australian perspective.

- Chapter 6 Harvesting energy