Chapter 06
*Lecture Outline
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1
A Glimpse of History
 Biologists had noticed that in vats of grape juice,
alcohol and CO2 are produced while yeast cells
increase in number
• But idea not widely accepted, mocked by some
• In 1850s, Louis Pasteur set out to prove
• Simplified setup: clear solution of sugar, ammonia,
mineral salts, trace elements
• Added a few yeast cells—as they grew, sugar decreased,
alcohol level increased
• Strongly supported idea, but Pasteur failed to extract
something from inside the cells that would convert sugar
• In 1897, Eduard Buchner, a German chemist awarded
Nobel Prize in 1907 showed that crushed yeast cells
could convert sugar to ethanol and CO2;
Microbial Metabolism
 All cells need to accomplish two fundamental tasks
• Synthesize new parts
• Cell walls, membranes, ribosomes, nucleic acids
• Harvest energy to power reactions
• Sum total of these is called metabolism
• Human implications
•
•
•
•
•
Used to make biofuels
Used to produce food
Important in laboratory
Invaluable models for study
Unique pathways potential
drug targets
6.1. Principles of Metabolism
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 Can separate metabolism
into two parts
CATABOLISM
ANABOLISM
Energy source
(glucose)
• Catabolism
Cell structures
(cell wall, membrane,
ribosomes, surface
structures)
• Processes that degrade
compounds to release energy
• Energy captured to make ATP
Energy
Macromolecules
(proteins, nucleic acids,
polysaccharides, lipids)
Energy
• Anabolism
• Biosynthetic processes
• Assemble subunits of
macromolecules
• Use ATP to drive reactions
• Processes intimately linked
Subunits
(amino acids,
nucleotides, sugars,
fatty acids)
Energy
Precursor
metabolites
Waste products
Nutrients
(acids, carbon
dioxide)
(source of nitrogen,
sulfur, etc.)
Catabolic processes harvest
the energy released during the
breakdown of compounds and
use it to make ATP. The
processes also produce
precursor metabolites used in
biosynthesis.
Anabolic processes (biosynthesis)
synthesize and assemble subunits
of macromolecules that make up
the cell structures. The processes
use the ATP and precursor
metabolites produced in
catabolism.
Harvesting Energy
 Energy is the capacity to do work
 Two types of energy
• Potential: stored energy (e.g., chemical bonds, rock on
hill, water behind dam)
• Kinetic: energy of movement (e.g., moving water)
• Energy in universe cannot be
created or destroyed, but it can
be converted between forms
• This is the 1st Law of
Thermodynamics
Harvesting Energy
 Photosynthetic organisms
harvest energy in sunlight
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• Power synthesis of organic
compounds from CO2
• Convert kinetic energy of photons
to potential energy of chemical
bonds
 Chemoorganotrophs obtain
energy from organic compounds
Radiant energy
(sunlight)
Photosynthetic organisms harvest the energy
of sunlight and use it to power the synthesis
of organic compounds from CO2. This
converts radiant energy to chemical energy.
Chemical energy
(organic compounds)
Chemoorganotrophs degrade organic
compounds, harvesting chemical energy.
• Depend on activities of
photosynthetic organisms
(top): © Photodisc Vol. Series 74, photo by Robert Glusie;
(bottom): © Digital Vision/PunchStock
Harvesting Energy
 Free energy is energy available to do work
• E.g., energy released when chemical bond is broken
• Compare free energy of reactants, products
• Exergonic reactions: reactants have more free energy
• Energy is released in reaction
• Endergonic reactions: products have more free energy
• Reaction requires input of energy
• Change in free energy is same regardless of number of
steps involved (e.g., converting glucose to CO2 + H2O)
• Cells use multiple steps when degrading compounds
• Energy released from exergonic reactions powers
endergonic reactions
Components of Metabolic Pathways
 Metabolic pathways
• Series of chemical reactions that convert starting
compound end product
• May be linear, branched, cyclical
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Intermediatea
Starting compound
Intermediateb
End product
Intermediateb1
End product1
Intermediateb2
End product2
(a) Linear metabolic pathway
Starting compound
Intermediatea
(b) Branched metabolic pathway
Starting compound
Intermediated
End product
Intermediatea
Intermediatec
Intermediateb
(c) Cyclical metabolic pathway
Components of Metabolic Pathways
 Role of Enzymes
• Biological catalysts: accelerate conversion of substrate
into product by lowering activation energy
Relative energy
• Highly specific: one at each step
• Reactions would occur without, but extremely slowly
Activation
energy
with an
enzyme
Energy of
reactants
Activation
energy
without an
enzyme
Energy of
products
Progress of reaction
(a)
Starting compound
(b)
Enzyme a
Intermediatea
Enzyme b
Intermediateb
Enzyme c
End product
Components of Metabolic Pathways
 Role of ATP
• Adenosine triphospate (ATP) is energy currency
•
•
•
•
Composed of ribose, adenine, three phosphate groups
Adenosine diphospate (ADP) acceptor of free energy
Cells produce ATP by adding Pi to ADP using energy
Release energy from ATP to yield ADP and Pi
 Three processes to generate ATP
• Substrate-level phosphorylation
Unstable (high-energy) bonds
• Exergonic reaction powers
• Oxidative phosphorylation
• Proton motive force drives
• Photophosphorylation
• Sunlight used to create proton
motive force to drive
P ~ P~ P
ATP
Pi
Pi
Energy used
The energy comes
from catabolic
reactions.
Energy released
The energy drives
anabolic reactions.
P~ P
ADP
Components of Metabolic Pathways
 Role of the Chemical Energy Source and the
Terminal Electron Acceptor
 Some atoms, molecules more electronegative
than others
H2S
S0
Organic
carbon
compounds
Energy
release
Organic
carbon
compounds
CO2
SO4
FeOOH
Fe2+
NH4+
NO2– ( to form NH4+)
NO3– ( to form NH4+)
Mn2+
MnO2
Relative tendency to give up electrons
• (E.g., glucose to O2)
H2
Relative tendency to give up electrons
• Greater affinity for electrons
• Energy released when
electrons move from low
affinity molecule to high
affinity molecule
Terminal
electron
acceptors
Energy
sources
NO3– ( to form NH2)
O2
(a) Energy is released when electrons are moved from an energy source with a
low affinity for electrons to a terminal electron acceptor with a higher affinity.
Components of Metabolic Pathways
 Role of the Chemical Energy Source and the
Terminal Electron Acceptor (continued…)
 More energy released when
difference in electronegativity
is greater
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• Acceptor:
• Terminal electron acceptor
Pyruvate
NO3–
(to form
NH4–)
O2
(b) Three examples of
chemoorganotrophic
metabolism
Terminal
electron
acceptors
H2
H2S
CO2
Fe2+
Relative tendency to give up electrons
• Energy source
Glucose
Inorganic
energy
sources
Relative tendency to give up electrons
• Electron donor:
Relative tendency to give up electrons
Glucose
Terminal
as an
electron
energy source acceptors
O2
(c) Three examples of
chemolithotrophic metabolism
Components of Metabolic Pathways
 Prokaryotes remarkably diverse in using energy
sources and terminal electron acceptors
• Organic, inorganic compounds used as energy source
• O2, other molecules used as terminal electron acceptor
• Electrons removed through series of oxidation-reduction
reactions or redox reactions
• Substance that loses electrons is oxidized
• Substance that gains electrons is reduced
• Electron-proton pair, or
Transfer of electrons
hydrogen, actually moves
e
• Dehydrogenation = oxidation Compound + Compound
X
Y
• Hydrogenation = reduction
X loses electron(s).
–
Y gains electron(s).
X is the reducing agent.
Y is the oxidizing agent.
e–
Compound X + Compound Y
(reduced)
(oxidized)
X is oxidized by the reaction.
Y is reduced by the reaction.
Components of Metabolic Pathways
 Role of Electron Carriers
• Energy harvested in stepwise process
• Electrons transferred to electron carriers, which represent
reducing power (easily transfer electrons to molecules)
– Raise energy level of recipient molecule
• NAD+/NADH, NADP+/NADPH, and FAD/FADH2
Precursor Metabolites
 Precursor metabolites are intermediates of
catabolism that can be used in anabolism
• Serve as carbon skeletons for building macromolecules
• E.g., pyruvate can be converted into amino acids alanine,
leucine, or valine
Precursor Metabolites
 Recall that E. coli can grow in glucose-salts medium
• Contains just glucose, inorganic salts
• Glucose is energy source
• Glucose is starting point for all
cellular components
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• Includes proteins, lipids,
carbohydrates, nucleic acids
Glucose molecules
To:
Lipid
synthesis
To:
Amino acid
synthesis
Carbohydrate
synthesis
To:
Nucleic acid
synthesis
• Some glucose molecules
completely oxidized for energy;
others used in biosynthesis To:
CO2 molecules + energy
Overview of Catabolism
 Three central metabolic pathways
• Oxidize glucose to CO2
• Catabolic, but precursor metabolites and reducing power
can be diverted for use in biosynthesis
• Termed amphibolic to reflect dual role
• Glycolysis
• Splits glucose (6C) to two pyruvates (3C)
• Generates modest ATP, reducing power, precursors
• Pentose phosphate pathway
• Primary role is production precursor metabolites, NADPH
• Tricarboxylic acid cycle
• Oxidizes pyruvates from glycolysis
• Generates reducing power, precursor metabolites, ATP
Overview of Catabolism
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GLUCOSE
 Central metabolic
pathways
• Glycolysis
• Pentose phosphate
pathway
• Tricarboxylic acid cycle
 Key outcomes
• ATP
• Reducing power
• Precursor metabolites
 Electron Transport
Chain
2
Pentose phosphate
pathway
Starts the oxidation of glucose
Glycolysis
Oxidizes glucose to pyruvate
1
Yields
~
~
+
Reducing
power
ATP
by substrate-level
phosphorylation
Yields
Reducing
power
Biosynthesis
5
Acids, alcohols, and gases
Pyruvate
Pyruvate
3a
Fermentation
Reduces pyruvate
or a derivative
Transition step
CO2
CO2
Yields
Reducing
power
AcetylCoA
AcetylCoA
X2
CO2
CO2
3b
TCA cycle
Incorporates an acetyl
group and releases CO2
(TCA cycles twice)
Yields
ATP
by substrate-level
phosphorylation
~
~
+
Reducing
power
4
Respiration
Uses the electron transport
chain to convert reducing
power to proton motive force
Yields
~
ATP
by oxidative
phosphorylation
~
Overview of Catabolism
 Respiration transfers electrons from glucose to
electron transport chain
• Electron transport chain generates proton motive force
• Harvested
to make
ATP via oxidative phosphorylation
i.e. “Builds
a Battery
• Aerobic respiration
– O2 is terminal electron acceptor
• Anaerobic respiration
– Molecule other than O2 as terminal electron acceptor
– Also use modified version of TCA cycle
Overview of Catabolism
 Fermentation
• If cells cannot respire, will run out of carriers available to
accept electrons
• Glycolysis will stop
• Fermentation uses pyruvate or derivative as terminal
electron acceptor to regenerate NAD+
• Glycolysis can continue
6.2. Enzymes
 Enzymes are biological catalysts
• Name reflects function; ends in -ase
• Has active site to which substrate binds weakly
•
•
•
•
Causes enzyme shape to change slightly
Existing substrate bonds destabilized, new ones form
Enzymes are highly specific
Enzyme not used up
6.2. Enzymes
 Enzymes are biological catalysts
Enzyme-substrate
complex formed
Substrate
Products released
Enzyme
Active site
Enzyme
unchanged
(a)
Substrate
Substrate
Enzyme
Enzyme
(b)
(c)
(b, c): From Voet: Biochemistry, 1/e, 0471617695, 1990, John Wiley & Sons
6.2. Enzymes
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 Cofactors assist some enzymes
• Cofactors can assist different
enzymes; fewer types needed
Enzyme
• Include magnesium, zinc, copper,
other trace elements
• Coenzymes are organic cofactors
• Include electron carriers FAD,
NAD+, NADP+
Cofactor
Substrate
6.2. Enzymes
 Environmental Factors Influencing Enzyme Activity
• Enzymes have narrow range of optimal conditions
• Temperature, pH, salt concentration
• 10°C increase doubles speed of enzymatic reaction up
until maximum
• Proteins denature at higher temperatures
Enzyme activity
Optimum
temperature
Enzyme activity
• Low salt, neutral pH usually optimal
1 2 3 4 5 6 7 8 9 10 11 12 13
Acidic
Basic
Temperature
(a)
Optimum pH
(b)
6.2. Enzymes
 Allosteric Regulation
• Enzyme activity controlled by binding to allosteric site
• Distorts enzyme shape, prevents or enhances binding
• Regulatory molecule is usually end product
• Allows feedback inhibition
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Enzyme
Enzyme
Allosteric
inhibitor
Substrate
Allosteric site
Active site
(b)
(a)
Allosteric inhibitor
Starting compound
(c)
Enzyme a
Intermediatea
Enzyme b
Enzyme c
Intermediateb
End product
6.2. Enzymes
 Enzyme Inhibition
• Site to which inhibitor binds determines type
• Competitive inhibitor binds to active site of enzyme
• Chemical structure usually similar to substrate
• Concentration dependent; blocks substrate
• Example is sulfa drugs blocking folic acid synthesis
Structural
differences
PABA
(substrate)
H
HO
N
O
C
H
O
S
O
Sulfa
(inhibitor)
N
H
H
PABA
Enzyme
(a)
N
(b)
H
H
Sulfanilamide
6.2. Enzymes
 Enzyme Inhibition (continued…)
• Non-competitive inhibitor binds to a different site
• Allosteric inhibitors are one example; action is reversible
• Some non-competitive inhibitors are not reversible
– E.g., mercury oxidizes the S—H groups of amino acid
cysteine, converts to cystine
– Cystine cannot form important disulfide bond (S—S)
– Enzyme changes shape, becomes nonfunctional
6.3. The Central Metabolic Pathways
 ATP
 Reducing power: NADH, FADH2, NADPH
 Precursor metabolites
• Glucose molecules can have
different fates
• Can be completely oxidized
to CO2 for maximum ATP
• Can be siphoned off as
precursor metabolite for
use in biosynthesis
6.3. The Central Metabolic Pathways
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GLUCOSE
2
 Glycolysis
Pentose phosphate
pathway
Starts the oxidation of glucose
Yields
Glycolysis
Oxidizes glucose to pyruvate
1
P~ P~ P
+
Reducing
power
ATP
by substrate-level
phosphorylation
Yields
Reducing
power
Biosynthesis
5
Fermentation
Reduces pyruvate
or a derivative
Acids, alcohols, and gases
• Converts 1 glucose
to 2 pyruvates; yields
net 2 ATP, 2 NADH
• Investment phase:
• 2 phosphate groups
added
• Glucose split to two
3-carbon molecules
• Pay-off phase:
• 3-carbon molecules
converted to pyruvate
• Generates 4 ATP,
2 NADH total
Nets 2 ATP
3a
Glucose
Transition step
CO2
CO2
Yields
Reducing
power
Pyruvate
Pyruvate
x2
CO2
ATP
~ ~
CO2
3b
TCA cycle
Incorporates an acetyl
group and releases CO2
(TCA cycles twice)
~
ADP
Yields
P~ P ~ P +
ATP
by substrate-level
phosphorylation
1 ATP is expended to add a phosphate group.
Reducing
power
4
Respiration
Uses the electron transport
Chain to convert reducing
power to proton motive force
Yields
P~ P~ P
ATP
by oxidative
phosphorylation
Glucose
6-phosphate
2
A chemical rearrangement occurs.
Fructose
6-phosphate
~ ~
ATP
3
ATP is expended to add a phosphate group.
4
The 6-carbon molecule is split into two 3-carbon
molecules.
~
ADP
Fructose
1,6-bisphosphate
Dihydroxyacetone
phosphate
A chemical rearrangement of one of the
molecules occurs.
5
Glyceraldehyde
3-phosphate
NAD+
NADH + H+
1,3-bisphosphoglycerate
ADP
ATP
~
NAD+
6
NADH + H+
~
The addition of a phosphate
group is coupled to a redox
reaction, generating NADH and
a high-energy phosphate bond.
~
~
7
~ ~
~ ~
ATP is produced by
substrate-level
phosphorylation.
3-phosphoglycerate
8
2-phosphoglycerate
H2O
A chemical rearrangement occurs.
9
H2O
Phosphoenolpyruvate
ADP
ATP
Pyruvate
~
~ ~
~
~ ~
Water is removed, causing the
phosphate bond to become
high-energy.
10
ATP is produced by
substrate-level
phosphorylation.
6.3. The Central Metabolic Pathways
 Pentose Phosphate Pathway
• Also breaks down glucose
• Important in biosynthesis of precursor metabolites
• Ribose 5-phosphate, erythrose 4-phosphate
• Also generates reducing power: NADPH
• Yields vary depending upon alternative taken
6.3. The Central Metabolic Pathways
 Transition Step
GLUCOSE
• CO2 is removed
from pyruvate
• Electrons reduce
NAD+ to
NADH + H+
• 2-carbon acetyl
group joined to
coenzyme A to form
acetyl-CoA
• Takes place in
mitochondria in
eukaryotes
2
Pentose phosphate
pathway
Starts the oxidation of glucose
Yields
Glycolysis
Oxidizes glucose to pyruvate
1
~
~
+
Reducing
power
ATP
by substrate-level
phosphorylation
Pyruvate
Yields
CO2
Reducing
power
Biosynthesis
Pyruvate
3a
Pyruvate
NAD+
Acids, alcohols, and gases
CoA
Transition step
CO2
Yields
Transition step:
CO2 is removed, a redox reaction generates
NADH, and coenzyme A is added.
Fermentation
Reduces pyruvate
or a derivative
5
CO2
Reducing
power
AcetylCoA
AcetylCoA
NADH + H+
x2
CO2
CoA
CO2
3b
TCA cycle
Incorporates an acetyl
group and releases CO2
(TCA cycles twice)
Acetyl-CoA
1 The acetyl group is transferred
to oxaloacetate to start a new
round of the cycle.
Yields
~
~
+
Reducing
power
ATP
by substrate-level
phosphorylation
CoA
Respiration
Uses the electron transport
chain to convert reducing
power to proton motive force
4
Yields
~
~
ATP
by oxidative
phosphorylation
NADH + H+
2 A chemical
rearrangement occurs.
Oxaloacetate
Citrate
A redox reaction
generates NADH.
8
NAD+
Isocitrate
NAD+
Malate
Water is added.
7
3 A redox reaction
generates NADH
and CO2 is
removed.
NADH + H+
H2 O
CO2
Fumarate
-ketoglutarate
NAD+
4
FADH2
6
CoA
A redox reaction
generates FADH2-
NADH + H+
FAD
5 The energy released
during CoA removal is
harvested to produce ATP.
CoA
Succinyl-CoA
Succinate
CoA
~ ~
ATP
~ + Pi
ADP
CO2
A redox reaction
generates NADH,
CO2 is removed,
and coenzyme A
is added.
6.3. The Central Metabolic Pathways
 Tricarboxylic
Acid (TCA)
Cycle (Krebs)
GLUCOSE
2
Pentose phosphate
pathway
Starts the oxidation of glucose
•
•
•
•
•
~
~
+
Reducing
power
ATP
by substrate-level
phosphorylation
Pyruvate
Yields
CO2
Reducing
power
Biosynthesis
Pyruvate
3a
Pyruvate
NAD+
Acids, alcohols, and gases
CoA
Transition step
CO2
Yields
Transition step:
CO2 is removed, a redox reaction generates
NADH, and coenzyme A is added.
Fermentation
Reduces pyruvate
or a derivative
5
CO2
Reducing
power
AcetylCoA
AcetylCoA
• Completes
oxidation of
glucose
 Produces
Yields
Glycolysis
Oxidizes glucose to pyruvate
1
NADH + H+
x2
CO2
CoA
CO2
3b
TCA cycle
Incorporates an acetyl
group and releases CO2
(TCA cycles twice)
Acetyl-CoA
1 The acetyl group is transferred
to oxaloacetate to start a new
round of the cycle.
Yields
~
~
+
Reducing
power
ATP
by substrate-level
phosphorylation
CoA
Respiration
Uses the electron transport
chain to convert reducing
power to proton motive force
4
Yields
~
~
ATP
by oxidative
phosphorylation
NADH + H+
2 A chemical
rearrangement occurs.
Oxaloacetate
Citrate
A redox reaction
generates NADH.
8
NAD+
2 CO2
2 ATP
6 NADH
2 FADH2
Precursor
metabolites
Isocitrate
NAD+
Malate
Water is added.
7
3 A redox reaction
generates NADH
and CO2 is
removed.
NADH + H+
H2 O
CO2
Fumarate
-ketoglutarate
NAD+
4
FADH2
6
CoA
A redox reaction
generates FADH2-
NADH + H+
FAD
5 The energy released
during CoA removal is
harvested to produce ATP.
CoA
Succinyl-CoA
Succinate
CoA
~ ~
ATP
~ + Pi
ADP
CO2
A redox reaction
generates NADH,
CO2 is removed,
and coenzyme A
is added.
6.4. Respiration
 Uses reducing power (NADH, FADH2) generated
by glycolysis, transition step, and TCA cycle to
synthesize ATP
• Electron transport chain generates proton motive force
• Drives synthesis of ATP by ATP synthase
• Process proposed by British scientist Peter Mitchell in
1961
• Initially widely dismissed
• Mitchell conducted years of self-funded research
• Received a Nobel Prize in 1978
• Now called chemiosmotic theory
The Electron Transport Chain—Generating Proton
Motive Force
 Electron transport chain is membrane-embedded
electron carriers
•
•
•
•
•
Pass electrons sequentially, eject protons in process
Prokaryotes: in cytoplasmic membrane
Eukaryotes: in inner mitochondrial membrane
Energy gradually released
Release coupled to ejection
of protons
• Creates electrochemical
gradient (“Battery”)
• Used to synthesize ATP
• Prokaryotes can also power
transporters, flagella
Electrons from the
energy source 2 e–
Energy released is
used to generate a
proton motive force.
High energy
Low energy
Electrons donated
to the terminal
electron acceptor.
2
H+
1/
2
H2O
O2
The Electron Transport Chain—Generating Proton
Motive Force
 Components of an Electron Transport Chain
• Most carriers grouped into large protein complexes
• Serve as proton pumps
• Three general groups are notable
• Quinones
• Lipid-soluble molecules
• Move freely, can transfer electrons between complexes
• Cytochromes
• Contain heme, molecule with iron atom at center
• Several types
• Flavoproteins
• Proteins to which a flavin is attached
• FAD, other flavins synthesized from riboflavin
The Electron Transport Chain—Generating Proton
Motive Force
 General Mechanisms of Proton Ejection
• Some carriers accept only hydrogen atoms (protonelectron pairs), others only electrons
• Spatial arrangement in membrane shuttles protons to
outside of membrane
• When hydrogen carrier accepts electron from electron
carrier, it picks up proton from inside cell
– or mitochondrial matrix
• When hydrogen carrier passes electrons to electron
carrier, protons released to outside of cell
– or intermembrane space of mitochondria
• Net effect is movement of protons across membrane
• Establishes concentration gradient
• Driven by energy released during electron transfer
The Electron Transport Chain—Generating Proton
Motive Force
 Electron Transport Chain of Mitochondria
• Complex I (NADH dehydrogenase complex)
• Accepts electrons from NADH, transfers to ubiquinone
• Pumps 4 protons
• Complex II (succinate dehydrogenase complex)
• Accepts electrons from TCA cycle via FADH2, “downstream” of
those carried by NADH
• Transfers electrons to ubiquinone
• Complex III (cytochrome bc1 complex)
• Accepts electrons from ubiquinone from Complex I or II
• 4 protons pumped; electrons transferred to cytochrome c
• Complex IV (cytochrome c oxidase complex)
• Accepts electrons from cytochrome c, pumps 2 protons
• Terminal oxidoreductase, meaning transfers electrons to
terminal electron acceptor (O2)
The Electron Transport Chain of Mitochondria
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
GLUCOSE
2
Pentose phosphate
pathway
Starts the oxidation of glucose
Yields
1
Yields
Glycolysis
Oxidizes glucose to pyruvate
P ~ P ~P +
Reducing
power
ATP
by substrate-level
phosphorylation
Reducing
power
Biosynthesis
5
Pyruvate
Pyruvate
Eukaryotic cell
Fermentation
Reduces pyruvate
or a derivative
Acids, alcohols, and gases
3a Transition step
CO2
Yields
CO2
Reducing
power
AcetylCoA
AcetylCoA
x2
CO2
CO2
TCA cycle
3b
Incorporates an acetyl
group and releases CO2
(TCA cycles twice)
Yields
Inner
mitochondrial
membrane
Reducing
power
ATP
by substrate-level
phosphorylation
4
Respiration
Uses the electron transport
chain to convert reducing
power to proton motive force
Yields
P
P P
ATP
by oxidative
phosphorylation
Complex III
Complex I
4
H+
4
Ubiquinone
+
Complex II
NAD+
Complex IV
H+
2
Proton motive force
is used to drive:
H+
ATP synthase
(ATP synthesis)
10
H+
Cytochrome c
Intermembrane
space
2 e–
Path of
electrons
NADH
Use of Proton Motive Force
Electron Transport Chain
1/
2 H+
H2O
2
Mitochondrial
matrix
O2
Terminal
electron acceptor
H+
3 ATP
+ 3 Pi
3 ADP
The Electron Transport Chain—Generating Proton
Motive Force
 Electron Transport Chain of Prokaryotes
• Tremendous variation: even single species can have
several alternate carriers
• E. coli serves as example of versatility of prokaryotes
• Aerobic respiration in E. coli
• Can use 2 different NADH dehydrogenases
– Proton pump equivalent to complex I of mitochondria
• Can produce several alternatives to optimally use
different energy sources, including H2
• Lack equivalents of complex III or cytochrome c
– Quinones shuttle electrons directly to ubiquinol
oxidase, a terminal oxidoreductase
– Two versions for high or low O2 concentrations
The Electron Transport Chain—Generating Proton
Motive Force
 Electron Transport Chain of Prokaryotes (cont…)
• Anaerobic respiration in E. coli
• Harvests less energy than aerobic respiration
– Lower electron affinities of terminal electron
acceptors
• Some components different
• Can synthesize terminal oxidoreductase that uses nitrate
as terminal electron acceptor
– Produces nitrite
– E. coli converts to less toxic ammonia
• Sulfate-reducers use sulfate (SO42–) as terminal
electron acceptor
• Produce hydrogen sulfide as end product
The Electron Transport Chain—Generating Proton
Motive Force
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Prokaryotic cell
Cytoplasmic
membrane
Electron Transport Chain
NADH dehydrogenase
Uses of Proton Motive Force
Ubiquinol
veoxidase
force
rive:
H+ (2 or 4)
H+ (0 or 4)
Ubiquinone
Path of
electrons
ATP synthase
(ATP synthesis)
Active transport
(one mechanism)
10 H+
Rotation of a flagella
H+
H+
Proton motive force
is used to drive:
Transported
molecule
Outside of
cytoplasmic
membrane
2 e– –
Cytoplasm
Succinate
dehydrogenase
NADH
+
NAD+
2 H+
1/
H2O
2
O2
Terminal
electron acceptor
H+
3 ATP
+ 3 Pi
3 ADP
The Electron Transport Chain—Generating Proton
Motive Force
 ATP Synthase—Harvesting the Proton Motive
Force to Synthesize ATP
• Energy required to establish gradient
• Released when gradient is eased
• ATP synthase allows protons to flow down gradient in
controlled manner
• Uses Proton energy to add phosphate group to ADP
• 1 ATP formed from entry of ~3 protons
• Calculating yields
• Based on experiments on rat mitochondria:
~2.5 ATP made per electron pair from NADH
~1.5 ATP made per electron pair from FADH2
The Electron Transport Chain—Generating Proton
Motive Force
 Calculating theoretical maximum yields
• In prokaryotes:
•
•
•
•
Glycolysis: 2 NADH 6 ATP
Transition step: 2 NADH  6 ATP
TCA Cycle: 6 NADH  18 ATP; 2 FADH2  4 ATP
Total maximum oxidative phosphorylation yield = 34 ATP
• Slightly less in eukaryotic cells
• NADH from glycolysis in cytoplasm transported across
mitochondrial membrane to enter electron transport chain
– Requires ~1 ATP per NADH generated
The Electron Transport Chain—Generating Proton
Motive Force
 ATP Yield of Aerobic Respiration in Prokaryotes
• Substrate-level phosphorylation:
• 2 ATP (from glycolysis; net gain)
• 2 ATP (from the TCA cycle)
• 4 ATP (total)
• Oxidative phosphorylation:
•
•
•
•
6 ATP (from reducing power gained in glycolysis)
6 ATP (from reducing power gained in transition step)
22 ATP (from reducing power gained in TCA cycle)
34 (total)
• Total ATP gain (theoretical maximum) = 38
ATP Yield of Aerobic Respiration in Prokaryotes
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
GLUCOSE
2
Pentose phosphate
pathway
Starts the oxidation of glucose
Yields
Glycolysis
Oxidizes glucose to pyruvate
1
Reducing
power
GLUCOSE
ATP
by substrate-level
phosphorylation
Glycolysis
Oxidizes glucose to pyruvate
Yields
Reducing
power
Biosynthesis
5
Pyruvate
3a
Pyruvate
Fermentation
Reduces pyruvate
or a derivative
~ ~
Acids, alcohols, and gases
Transition step
CO2
Yield
CO2
2 ATP
net gain = 0
Reducing
power
AcetylCoA
AcetylCoA
x2
CO2
~ ~
CO2
3b
TCA cycle
Incorporates an acetyl
group and releases CO2
(TCA cycles twice)
2 ATP
Yields
Reducing
power
ATP
by substrate-level
phosphorylation
4
Respiration
Uses the electron transport
chain to convert reducing
power to proton motive force
Yields
ATP
by oxidative
phosphorylation
2 NADH
Oxidative
phosphorylation
~ ~
6 ATP
~ ~
Substrate-level
phosphorylation
Pyruvate
2 ATP
Pyruvate
2 NADH
AcetylCoA
~ ~
Oxidative
phosphorylation
6 ATP
Oxidative
phosphorylation
18 ATP
AcetylCoA
6 NADH
x2
CO2
2 FADH2
CO2
TCA cycle
Incorporates an acetyl
group and releases CO2
(TCA cycles twice)
~ ~
~ ~
Oxidative
phosphorylation
4 ATP
Substrate-level
phosphorylation
2 ATP
~ ~
6.5. Fermentation
 Fermentation used when respiration not an option
• E. coli is facultative anaerobe
• Aerobic respiration, anaerobic
respiration, and fermentation
GLUCOSE
Pentose phosphate
pathway
Starts the oxidation of glucose
2
Yields
Glycolysis
Oxidizes glucose to pyruvate
1
P
~
~P +
P
Reducing
power
ATP
by substrate-level
phosphorylation
Yields
Reducing
power
Biosynthesis
Fermentation
Reduces pyruvate
or a derivative
5
Pyruvate
Pyruvate
Acids, alcohols, and gases
3a
Transition step
CO2
CO2
Yields
Reducing
power
AcetylCoA
AcetylCoA
• Streptococcus pneumoniae
lacks electron transport chain
x
CO2
2
CO2
3b
TCA cycle
Incorporates an acetyl
group and releases CO2
(TCA cycles twice)
Yields
P ~
P ~
Reducing
power
P
ATP
by substrate-level
phosphorylation
+
4
• Fermentation only option
Yields
P ~
P
~
P
ATP
by oxidative
phosphorylation
• ATP-generating reactions are
only those of glycolysis
• Additional steps consume
excess reducing power
– Regenerate NAD+
Respiration
Uses the electron transport
chain to convert reducing
power to proton motive force
NADH + H+ NAD+
O
H3C
C
O
O–
C
H3C
OH
O
C
C
O–
H
Lactate
Pyruvate
(a) Lactic acid fermentation
CO2
O
H3C
C
NADH + H+
O
O
C
O–
Pyruvate
(b) Ethanol fermentation
H3C
C
H
Acetaldehyde
NAD+
OH
H3C
C
H
Ethanol
H
6.5. Fermentation
 Fermentation end products varied; helpful in
identification, commercially useful
• Ethanol
• Butyric acid
• Propionic acid
• 2,3-Butanediol
• Mixed acids
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Pyruvate
Fermentation
pathway
Microorganisms
End products
Lactic acid
Ethanol
Butyric acid
Propionic acid
Mixed acids
2,3-Butanediol
Streptococcus
Lactobacillus
Saccharomyces
Clostridium
Propionibacterium
E. coli
Enterobacter
Lactic acid
Ethanol
CO2
Butyric acid
Butanol
Acetone
Isopropanol
CO2
H2
Propionic acid
Acetic acid
CO2
Acetic acid
Lactic acid
Succinic acid
Ethanol
CO2
H2
CO2
H2
(yogurt, dairy, pickle), b (wine, beer), (acetone): © Brian Moeskau/McGraw- Hill; (cheese): © Photodisc/McGraw-Hill; (Voges-Proskauer Test), (Methyl-Red Test): © The McGraw-Hill Companies, Inc./Auburn University Photographic Services
6.6. Catabolism of Organic Compounds Other
than Glucose
 Microbes can use variety of compounds
• Excrete hydrolytic enzymes; transport subunits into cell
• Degrade further to appropriate precursor metabolites
• Polysaccharides and disaccharides
• Amylases digest starch; cellulases digest cellulose
• Disaccharides hydrolyzed by specific disaccharidases
• Lipids
• Fats hydrolyzed by lipases; glycerol converted to
dihydroxyacetone phosphate, enters glycolysis
• Fatty acids degraded by β-oxidation to enter TCA cycle
• Proteins
• Hydrolyzed by proteases; amino group deaminated
• Carbon skeletons converted into precursor molecules
6.6. Catabolism of Organic Compounds Other
than Glucose
 Microbes can
use variety of
compounds
(cont…)
• Convert to
precursor
metabolites
• Enter
appropriate
metabolic
pathways
POLYSACCHARIDES
Starch
Cellulose
amylases
lipases
glycerol
cellulases disaccharidases
monosaccharides
(simple sugars)
Pentose phosphate
pathway
LIPIDS (fats)
DISACCHARIDES
Lactose Maltose
Sucrose
+
GLUCOSE
PROTEINS
proteases
Amino acids
deamination
fatty acids
NH3
Glycolysis
Applies to
both branches
In glycolysis
Pyruvate
Pyruvate
AcetylCoA
AcetylCoA
X2
TCA cycle
ß-oxidation
removes
2-carbon units.
6.7. Chemolithotrophs
 Prokaryotes unique in ability to use reduced
inorganic compounds as sources of energy
• E.g., hydrogen sulfide (H2S), ammonia (NH3)
• Produced by anaerobic respiration from inorganic molecules
(sulfate, nitrate) serving as terminal electron acceptors
• Important example of nutrient cycling
• Four general groups
6.8. Photosynthesis
 Photosynthesis
• Plants, algae, several groups of bacteria
• General reaction is
Light Energy
6 CO2 + 12 H2X
C6H12O6 + 12 X + 6 H2O
where X indicates element such as oxygen or sulfur
• Can be considered in two distinct stages
• Light reactions (light-dependent reactions)
– Capture energy and convert it to ATP
• Light-independent reactions (dark reactions)
– Use ATP to synthesize organic compounds
– Involves carbon fixation
6.8. Photosynthesis
 Photosynthesis (continued…)
• Many variations found in approaches
• Oxygenic and anoxygenic
6.8. Photosynthesis
 Capturing Radiant Energy
• Colors observed are those of wavelength reflected
• Pigments are located in photosystems within membranes
• Chlorophylls (plants, algae, cyanobacteria)
• Bacteriochlorophylls (anoxygenic bacteria)
– Absorb different wavelengths than chlorophylls
• Accessory pigments absorb at additional wavelengths
– Carotenoids (many photosynthetic prokaryotes and
eukaryotes)
– Phycobilins (cyanobacteria, red algae)
• Antennae pigments form complex
• Funnel energy to reaction-center pigment
6.8. Photosynthesis
 Reaction-center pigments
• Donate excited electrons to electron transport chain
– Chlorophyll a (plants, algae, cyanobacteria)
– Bacteriochlorophylls (anoxygenic bacteria)
• Cyanobacteria: photosystems in membranes of stacked
structures inside cell termed thylakoids
• Plants, algae: thylakoids in stroma of chloroplast
• Endosymbiotic theory explains
• Purple bacteria: in cytoplasmic
membrane, extensive infoldings
• Green bacteria: specialized
chlorosomes attached to
cytoplasmic membrane
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Photosystem
Radiant
energy
Electron
transport chain
Reaction-center
chlorophyll
e–
Chlorophyll
molecule
Photosynthetic membrane
Converting Radiant Energy into Chemical Energy
 Light-dependent reactions in cyanobacteria and
photosynthetic eukaryotes
• Two distinct photosystems (I and II)
• Cyclic photophosphorylation
– Photosystem I alone produces ATP
– Reaction-center chlorophyll is terminal electron acceptor
• Non-cyclic photophosphorylation
– Used when cells need both ATP and reducing power
• Electrons from photosystem II drive photophosphorylation
– Are then donated to photosystem I
– Photosystem II replenishes electrons by splitting water
– Generates oxygen (process is oxygenic)
• Electrons from photosystem I reduce NADP+ to NADPH
Converting Radiant Energy into Chemical Energy
Excited
chlorophyll
Proton gradient
formed for ATP
synthesis
Excited
chlorophyll
H+
Electron
carrier
e–
NADP
reductase
e–
e–
Energy of electrons
NADPH
NADP+
Proton
pump
Reactioncenter
chlorophyll
Radiant
energy
Electron
carrier
Electron
carrier
Reactioncenter
chlorophyll
e-
Radiant
energy
Water-splitting
enzyme
Z
2 H2O
e–
4 H
Photosystem II
+ O2
Proton pump
Photosystem I
NADP reductase
Converting Radiant Energy into Chemical Energy
 Light-dependent reactions in anoxygenic
photosynthetic bacteria
• Each has single photosystem
• Cannot use water as electron donor, so anoxygenic
• Use electron donors such as hydrogen gas (H2), hydrogen
sulfide (H2S), organic compounds
• Purple bacteria: photosystem similar to photosystem II
• Energy of electrons insufficient to reduce NAD+
– Instead expend ATP to use reversed electron transport
• Green bacteria: photosystem similar to photosystem I
• Electrons can generate proton motive force or reduce NAD+
6.9. Carbon Fixation
 Chemolithoautotrophs, photoautotrophs use CO2 to
synthesize organic compounds: carbon fixation
• In photosynthetic organisms: light-independent reactions
• Consumes lots of ATP, reducing power
• Reverse process of oxidizing compounds to CO2 liberates a
lot of energy!
• Calvin cycle most commonly used
• Three essential stages
• Incorporation of CO2 into organic compounds
• Reduction of resulting molecule
• Regeneration of starting compound
• Six “turns” of cycle: net gain of one fructose-6-phosphate
• Consumes 18 ATP, 12 NADPH per fructose molecule
6.9. Carbon Fixation
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1
6
Carbon dioxide is added to ribulose
1,5-bisphosphate to start a new
round of the cycle.
CO2
12 molecules
3-phosphoglycerate
~ ~
6 molecules
ribulose 1,5-bisphosphate
12 ATP
STAGE 1
~
12 ADP
12 molecules
1,3-bisphosphoglycerate
~
STAGE 3
6 ADP
STAGE 2
~ ~
6 ATP
3
6 molecules
ribulose 5-phosphate
Ribulose 1,5-bisphosphate is
regenerated so that the cycle
Can continue.
12 NADPH + H+
12 molecules
glyceraldehyde
3-phosphate
12 NADP+
12 Pi
Series
of complex
reactions
2
1 molecule
fructose 6-phosphate
Cell components
ATP and NADPH are used to reduce
the product of stage1, producing
glyceraldehyde 3-phosphate, which
can be used in biosynthesis.
6.10. Anabolic Pathways—Synthesizing Subunits from
Precursor Molecules
 Prokaryotes remarkably similar in biosynthesis
• Synthesize subunits using central metabolic pathways
• If enzymes lacking, end product must be supplied
• Fastidious bacteria require many growth factors
• Lipid synthesis requires fatty acids, glycerol
• Fatty acids: 2-carbon units added to acetyl group from
acetyl-CoA
• Glycerol: dihydroxyacetone phosphate from glycolysis
• Nucleotide synthesis
•
•
•
•
DNA, RNA initially synthesized as ribonucleotides
Purines: atoms added to ribose 5-phosphate to form ring
Pyrimidines: ring made, then attached to ribose 5-phosphate
Can be converted to other nucleobases of same type
6.10. Anabolic Pathways—Synthesizing Subunits from
Precursor Molecules
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Glycolysis
Pentose phosphate
pathway
Glucose 6-phosphate
Fructose 6-phosphate
Lipopolysaccharide
(polysaccharide)
Ribose 5-phosphate
Erythrose 5-phosphate
Nucleotides
amino acids
(histidine)
Amino acids
(phenylalanine,
tryptophan,
tyrosine)
Peptidoglycan
Dihydroxyacetone
phosphate
Lipids
(glycerol
component)
3-phosphoglycerate
Amino acids
(cysteine,
glycine, serine)
Phosphoenolpyruvate
Amino acids
(phenylalanine,
tryptophan, tyrosine)
Pyruvate
Pyruvate
Acetyl-CoA
Acetyl-CoA
Amino acids
(alanine,
leucine, valine)
Lipids
(fatty acids)
Oxaloacetate
Amino acids
(aspartate, asparagine,
isoleucine, lysine,
methionine, threonine)
X2
- ketoglutarate
TCA cycle
Amino acids
(arginine, glutamate,
glutamine, proline)
6.10. Anabolic Pathways—Synthesizing Subunits from
Precursor Molecules
 Amino Acid Synthesis
• Synthesis of glutamate provides mechanism for
incorporation of nitrogen into organic material
• Ammonium (NH4+) commonly used via glutamate synthesis
• Transamination can generate other amino acids
NH2
α-ketoglutarate
Glutamate is
synthesized
by adding ammonia
to the precursor
metabolite
α-ketoglutarate.
Aspartate
Oxaloacetate
NH3 (ammonia)
NH2
Glutamate
The amino group (NH2) of glutamate can be
transferred to other carbon compounds to
produce other amino acids.
6.10. Anabolic Pathways—Synthesizing Subunits from
Precursor Molecules
 Amino Acid Synthesis (continued…)
• Aromatic amino acids: carefully regulated branch points
• Tryptophan is feedback inhibitor of enzyme that directs
branch to its synthesis
– Pathway instead leads to tyrosine, phenylalanine
– Tyrosine, phenylalanine likewise inhibit first enzyme of
branch leading to their synthesis
• The three amino acids also regulate formation of original
7-carbon compound (three different enzymes catalyze)
From glycolysis
Phenylalanine
Compound
a
3-C
+
4-C
From pentose
phosphate pathway
7-C
compound
Branch
point II
Branch
point I
Tyrosine
Compound
b
Tryptophan