Electron Transport and Oxidative Phosphorylation

• Final stages of aerobic Mitochondrial Electron Transport

oxidation of biomolecules in • How did we get here?

eukaryotes occur in the
mitochondrion • Summary of glycolysis

glucose + 2 NAD+ + 2Pi + 2ADP  2 pyruvate + 2ATP + 2NADH + 2H+

• Reduced coenzymes NADH
and FADH2 from: • Summary of the citric acid cycle (including pyruvate dehydrogenase)

(1) Aerobic oxidation of pyruvate + 4 NAD+ + FAD + GDP + 2 H 20  3 CO2 + 4NADH + 4H+ + Pi + GTP + FADH 2

pyruvate by the citric

acid cycle
(2) Oxidation of fatty acids
and amino acids
Electron Transport Chain is the
process by which NADH and
FADH2 are oxidized and a proton
Electron Transport and Oxidative Phosphorylation
gradient is formed.

Oxidative phosphorylation is the process of

making ATP by using the proton gradient generated
by the ETC.
Respiration by mitochondria
• Oxidation of substrates is coupled to the
phosphorylation of ADP
• Respiration (consumption of oxygen)
proceeds only when ADP is present
• The amount of O2 consumed depends upon
the amount of ADP added

Location of mitochondrial complexes

• Inner mitochondrial membrane:
a. Electron transport chain: oxidizes
reduced coenzymes
b. ATP synthase: machinery to synthesize
ATP
 Electron transport and oxidative phosphorylation
capture the energy in the redox potential of NADH and
FADH2 – 2 separate processes that are COUPLED to
result in ATP production
Extensive folding of IMM provides a large surface area on
the matrix side to form lots of assemblies of proteins to
maximize ATP production

(1) Respiratory electron-transport chain (ETC) Series of enzyme complexes embedded in the inner
mitochondrial membrane, which oxidize NADH and FADH2. Oxidation energy is used to transport
protons creating a proton gradient – protons pumped from matrix to intermembrane space across IMM

(2) ATP synthase uses the proton gradient energy to produce ATP; It is the release of the energy in the
gradient back through the membrane through the protein ATP Synthase that drives ATP synthesis
Overview of electron transport chain and
oxidative phosphorylation
Electron transport chain (ETC)

– Series of sequential
oxidation/reduction (redox)
reactions
– Finally see role of oxygen

– Passes electrons from

NADH or FADH2 to O2
producing H2O through a
series of protein complexes
(source of metabolic water!)

– Since NAD+ and FAD are in

limited supply, they must be
recycled.
- FOUR protein complexes in the IMM make up the ETC
- Complexes I, II, III, IV
- Work together in succession to catalyze redox reactions
- Electrons are transferred to
molecular oxygen that is Recycling is accomplished by oxidation and
then reduced to water transfer of electrons to oxygen.
- Electrons move through the ADP + Pi ATP
complexes in order
NADH + H+ + 1/2 O2 NAD+ + H2O
• Electrons from ADP + Pi ATP
NADH enter at
Complex I FADH2 + 1/2 O2 FAD + H2O
• Electrons from
NAD+ and FAD are then available for additional
FADH2 enter at
oxidative metabolism. The energy released during
Complex II
electron transport is coupled to ATP synthesis.
• Flow of electrons is

spontaneous and thermodynamically favorable because the next carrier has greater affinity for
electrons than the previous
• In each reaction, an electron donor is oxidized and an electron acceptor is reduced

– Areduced + Boxidized  Aoxidized + Breduced

• Compounds differ from one another in how readily they will be oxidized or reduced
– can be compared using Eo’ (volts)
– starting with 1 M “A” and 1 M “B”, the component with most positive (low) redox potential
will be reduced and the component with the most negative (high) reduction potential will be
oxidized

– Electrons flow downhill – spontaneously moving from molecules that are strong electron
DONORS to strong electron ACCEPTORS = move from high energy state to low energy state
–
 • NADH = strongest donor
• O2 = strongest acceptor
• The redox potential energy of
NADH is released stepwise via
the electron transport chain
– The flow of electrons results in energy
that is released in increments through
the ETC

– Energy is used to pump protons (H+)

across the inner mitochondrial
membrane (IMM) and set up the pH
gradient
– It is the release of the energy in the
gradient back through the membrane
through the intergral membrane protein
ATP Synthase that drives ATP
synthesis

Co-factors in Electron Transport

• Complexes contain enzymes with electron carrying groups or oxidation – reduction components
• Protein components use metal-
containing prosthetic groups or
flavins to carry electrons
• Metal-containing groups such as
iron-sulfur clusters, copper
ions, hemes
• Flavins:
• (Complex I) FMN - FMNH2
• (Complex II) FAD - FADH2
Mobile electron carriers – serve as links between ETC complexes
1. Ubiquinone (Q)
• Also called coenzyme Q
• A membrane-soluble low molecular weight compound
• Long hydrophobic tail keeps Q anchored in the mitochondrial inner membrane
• Q is a lipid soluble molecule that diffuses within the lipid bilayer, and shuttles electrons from
Complexes I and II and pass them to III
• Not a part of any complex

2. Cytochrome c
• A peripheral membrane protein associated with
the outer face of the membrane, transports
electrons from III to IV
• Cytochromes are heme-containing proteins –
contains Fe
• Not a part of any complex
• Shuttles electrons and protons from Complex III
to Complex IV Structure of cytochrome c heme group.
Overview of Electron Transport
• The electron transport chain is associated with the mitochondrial inner membrane
• Complexes I-IV contain multiple cofactors, and are involved in electron transport
• OVERALL TRANSFER OF 2 ELECTRONS FROM NADH THROUGH ETC TO
MOLECULAR OXYGEN:

NADH + H+ + ½ O2  NAD+ +H2O

• Complexes I – where NADH electrons enter the chain

• Complex II – Where FADH2 electrons enter the chain
• Electrons passed from Complex I or II to Coenzyme Q
• Coenzyme Q shuttles electrons to complex III
• Complex III shuttles electrons to cytochrome C
• Cytochrome C shuttles electrons to Complex IV
• Complex IV transfers electrons to O2 which is then reduced to water
• Flow through Complexes I, III and IV release energy which is used to pump protons across the
IMM and form a “proton gradient”
• Proton gradient has lots of potential energy
• When the energy is released (protons flow back into matrix through ATP synthase), the energy
drives ATP synthesis
Electron transfer and proton flow in Complex I
• Also called NADH-ubiquinone oxidoreductase
• Complex I includes a flavoprotein (contains FMN – related to FAD) and proteins with Fe-S
centers (iron-sulfur clusters)
• These proteins provide two centers for oxidation reduction reactions
• Transfers electrons from NADH to Coenzyme Q via FMN and iron-sulfur proteins
• NADH transfers a two electrons as a hydride ion (H:-) to FMN
• Reduction of Q to QH2 requires 2 e-
• About 4 H+ translocated per 2 e- transferred

Electron transfer in Complex II

• Succinate-ubiquinone oxidoreductase
• Same as succinate dehydrogenase, a component of the TCA cycle
• Succinate dehydrogenase
• Directs transfer of electrons from succinate to CoQ via FADH2.
• Catalyzes the reduction of Q to QH2
• Acyl-CoA dehydrogenase
• From -oxidation of fatty acids. It also transfers electrons to CoQ via FADH2.
 • Complex II proteins provide two centers for oxidation reduction reactions
• FAD  FADH2
• Fe3+  Fe2+ (iron-sulfur cluster)
• FAD of Complex II is reduced in a 2-electron transfer of a hydride ion from succinate
• Complex II does NOT contribute to proton gradient, but supplies electrons from succinate
**Note that all electrons from FADH2 and NADH must pass through CoQ.**

Electron transfer and proton flow in Complex III

• Ubiquinol-cytochrome c oxidoreductase
• Transfers electrons to cytochrome c
 • Complex III contains several cytochromes (heme prosthetic group) and Fe-S center proteins
which provide several centers for oxidation reduction reactions
• Oxidation of one QH2 is accompanied by the translocation of 4 H+ across the inner
mitochondrial membrane
• Two H+ are from the matrix, two from QH2
• Regenerates Q for next round

lectron transfer and proton flow in Complex IV

• Cytochrome c oxidase - Combination of cytochromes
• A complex of 10 protein subunits that contains 2 cytochromes (a and a3) and proteins with
copper centers that provide multiple centers for oxidation-reduction
• Consists of, 2 types of prosthetic groups - 2 heme and 2 Cu.
• Fe3+  Fe2+
• Cu2+  Cu1+
• Source of electrons is cytochrome c (links Complexes III and IV)
• Catalyzes a four-electron reduction of molecular oxygen (O2) to water (H2O)
• Cytochromes a and a3 are the only species capable of direct transfer of electrons to
oxygen.
• Translocates H+ into the intermembrane space and contributes to the proton gradient
Complex IV contributes to the proton gradient

Net effect is transfer of four H+ for each pair of e-

O2 + 4 e- + 4H+ 2 H2O
1. Proton translocation of 2 H+ for each pair of
electrons transferred (each O atom reduced)
HOWEVER, FOR EACH PAIR OF ELECTRONS (e.g. NADH), ONLY GET 2H+
TRANSFERRED TO INTERMEMBRANE SPACE IN COMPLEX IV

SUMMARY OF ELECTRON TRANSPORT CHAIN

Mitochondrial Electron Transport

NADH is oxidized by Complex I Succinate is oxidized by Complex II

Complex I is reduced Complex II is reduced
Complex I is oxidized by Ubiquinone Complex II is oxidized by Ubiquinone
Ubiquinone is reduced Ubiquinone is reduced

Ubiquinone is free to diffuse through the mitochondrial inner membrane

Ubiquinone is oxidized by Complex III

Complex III is reduced
Complex III is oxidized by Cytochrome C
Cytochrome C is reduced

Cytochrome C is free to diffuse through the mitochondrial inter-membrane space

Cytochrome C is oxidized by Complex IV

Complex IV is reduced
Complex IV is oxidized by oxygen
Oxygen is reduced

Electron Transport and Oxidative Phosphorylation

ENERGETICS:
• Complex I, Complex III and Complex IV pump protons across the inner mitochondrial
membrane
– pumping uses the energy liberated from the oxidation of NADH and FADH2
– pumping generates a membrane potential because it generates an electrochemical
gradient
• negative inside, positive outside
• alkaline inside, acidic outside
The Chemiosmotic Hypothesis
• Proposed by Peter Mitchell in the 1960’s (Nobel Prize in 1978)
• A proton concentration gradient serves as the energy reservoir for driving ATP formation
• Electron transport through the ETC generates a proton gradient (pumps H+ from the matrix to
the intermembrane space)
• Protonmotive force (p) is the energy of the proton concentration gradient
• Protons that are translocated into the intermembrane space by electron transport, flow back into
the matrix via ATP synthase
– H+ flow forms a circuit (similar to an electrical circuit)
• The transmembrane protein, ATP synthase, catalyzes the phosphorylation of ADP in a reaction
driven by movement of H+ across the inner membrane into the matrix
• As protons move back into the matrix through ATP Synthase, the energy stored in the
electrochemical proton gradient is used to make ATP

Coupling of electron transport

with ATP synt hase

H+ H+ H+ H+ H+ H+
H+
H+ H+ H+ H+

Inner mitochondrial membrane

H + H+ H+
Electron
EElectron
Elleeccttrroonn FF1 --ATP
ATP
F 11-ATP
Transport
TTrraannssppoorrt
Transport H+
ssynthase
syF
yn1n-ATP
tthhaassee
Chain
CChain
Chhaaiinn ADP + Pi ATP
csynthase
coom
complexmpplleexx
ANIMATION complex

http://www.science.smith.edu/departments/Biology/Bio111/etc.html
http://www.wiley.com/college/fob/anim/
Chapter 17
 Fig. 17-8 -- The Mitochondrial Electron Transport Chain
ATP Synthase
• F0F1 ATP Synthase uses the proton
gradient energy for the synthesis of
ATP
• Large transmembrane protein complex
• Faces into the mitochondrial matrix –
spans the IMM
• Composed of a “knob-and-stalk”
structure
• F0 (stalk) has a proton channel
which spans the membrane.
• Forms a proton pore
• Membrane-spanning portion –
integral membrane protein
• Made up of 4 different subunits
• Fo subunit composition: a1b2c9-12
(c subunits form cylindrical,
membrane-bound base)

• F1 (knob) contains the catalytic subunits (ATP-synthesizing subunits)

• Where ATP synthesis takes place
• F1 knobs: inner face of the inner mitochondrial membrane
• (subunit composition:   )


•   oligomer of F1 is connected to c subunits by a multisubunit stalk of  and 

 

chains
• Passage of protons through the Fo (stalk) into the matrix is coupled to ATP formation
• Estimated passage of 3 H+ / ATP synthesized
• Fo is sensitive to oligomycin, an antibiotic that binds in the channel and blocks H+ passage,
thereby inhibiting ATP synthesis
Mechanism of ATP Synthase
• F1-F0 complex serves as the molecular apparatus for coupling H+ movement to ATP synthase.
• There are 3 active sites, one in each  subunit
• Passage of protons through the Fo channel causes the rotor to spin in one direction and the stator
to spin in the opposite direction

Proton flow  C unit rotates   rotates  conformation change  ATP synthesized

Animation:
http://www.stolaf.edu/people/giannini/biological%20anamations.html
mitochondrial electron transport
ATP synthase
ATP synthase mechanism
Binding-change mechanism of ATP synthase (This page is NOT
for the Exam)
1. ADP, Pi bind to an open site
2. Inward passage of protons, cause a conformational change, and
ATP is synthesized from ADP and Pi
3. ATP released from open site, ADP and Pi form ATP in the tight
site

 subunits are asymmetric:

T is catalytically active, binds to ADP tight;
O has low affinity for substrate
L is also inactive, but can bind ADP

ADP+Pi first binds to L state; rotation of

 subunits converts L to T, T to O
and O to L states, one ATP is made.

The binding change mechanism. F1 has three chemically identical but conformationally
distinct interacting  protomers: O, the open conformation, has very low affinity for ligands
and is catalytically inactive; L binds ligands loosely and is catalytically inactive; T binds ligands
tightly and is catalytically active. ATP synthesis occurs in three steps: (1) ADP and Pi bind to
site L. (2) An energy-dependent conformational change converts binding site L to T, T to O,
and O to L. (3) ATP is synthesized at site T and ATP is released from site O. The enzyme
returns to its initial state after two more passes of this reaction sequence. The energy that
drives the conformational change is apparently transmitted to the catalytic   assembly via
 

the rotation of the  assembly, here represented by the centrally located asymmetric object
(green). [After Cross, R.L. Annu. Rev. Biochem. 50, 687 (1980).]

Animation:
http://www.wiley.com/college/fob/anim/
Chapter 17: Fig. 17-21 -- The Binding Change Mechanism for ATP Synthesis
Regulation:
• Electrons do not flow unless ADP is present for phosphorylation
• Increased ADP levels cause an increase in catabolic reactions of various enzymes including:
a. glycogen phosphorylase
b. phosphofructokinase
c. citrate synthase

Active Transport of ATP, ADP and Pi Across the Inner Mitochondrial Membrane
• ATP is synthesized in the mitochondrial matrix
• ATP must be transported to the cytosol, and ADP and Pi must enter the matrix
• ADP/ATP carrier exchanges mitochondrial ATP4- for cytosolic ADP3-
• The exchange causes a net loss of -1 in the matrix (draws some energy from the H+ gradient)
• Adenine nucleotide translocase: unidirectional exchange of ATP for ADP (antiport)
• Symport of Pi and H+ is electroneutral
The P:O Ratio

molecules of ADP phosphorylated

P:O ratio = -----------------------------------------
atoms of oxygen reduced
• Translocation of 3H+ required by ATP synthase for
each ATP produced
• 1 H+ needed for transport of Pi, ADP and ATP
• Net: 4 H+ transported for each ATP synthesized

Calculation of the P:O ratio

Complex I III IV
#H+ translocated/2e- 4 4 2
Since 4 H+ are required for each ATP synthesized:
For NADH: 10 H+ translocated / O (2e-)
P/O = (10 H+/ 4 H+) = 2.5 ATP/O
For succinate substrate = 6 H+/ O (2e-)
P/O = (6 H+/ 4 H+) = 1.5 ATP/O
RESPIRATORY INHIBITORS & UNCOUPLERS:
Inhibitors are chemicals that can block electron transfer
through specific complexes in the ETC
• Complex I: blocked by rotenone, barbiturates
• Complex III: blocked by antimycin A
• Complex IV: blocked by cyanide, azide, carbon monoxide

Uncouplers
• In some special cases, the coupling of the two processes
can be disrupted.
• Uncouplers stimulate the oxidation of substrates in the
absence of ADP
• Large amounts of O2 are consumed but no ATP is
produced.
• Uncouplers are lipid-soluble weak acids
• Both acidic and basic forms can cross the inner mitochondrial membrane
• Uncouplers deplete any proton gradient by transporting protons across the membrane
• Do NOT affect electron transport
• Allow protons back into the matrix without making ATP
• Stimulate oxygen consumption

2,4-Dinitrophenol: an uncoupler
• Used as a diet/weight loss drug
• Hydrophobic low molecular weight
substance that can diffuse through
the mitochondrial inner membrane
• Shuttles protons across the
membrane and dissipates proton
gradient
• ATP synthesis goes down
– ADP concentration in cells goes up and acts as a stimulator
– Signals to turn on pathways to make ATP
– Therefore, electron transport and O2 consumption turned on fully and is NOT regulated
 • Energy produced by electron transport released as HEAT rather than harnessed into ATP
synthesis
• Fuels (carbs and fats) are consumed at great rates and get quick weight loss BUT
– Get heavy breathing – using lots of oxygen
– Excessive fever (heat generation)
– BIG problem – no control over uncoupling
– Brain, heart and muscles are affected as well
• 2,4-dinitrophenol is extremely toxic and pulled from the market

NATURAL UNCOUPLERS
• In newborn and hibernating animals, brown fat oxidizes large amounts of substrate (mostly fatty
acids) to generate heat
• ‘Brown fat’- brown because of the large number of mitochondria and their associated
cytochromes
• In brown fat mitochondria oxidation of NADH and FADH2 is uncoupled from ATP synthesis
– Mitochondria contain thermogenin (uncoupling protein).
– Thermogenin allows the release of energy as heat instead of ATP.
– Thermogenin dissipates proton
electrochemical gradient

• By providing another channel for return of

protons - bypasses ATP synthase
• Also called uncoupling protein (UCP)
• In brown fat mitochondria, the energy
that would have been used to make ATP is
liberated as heat
Electron Transport Chain and Oxidative Phosphorylation Animation Websites:
1. http://www.cat.cc.md.us/biotutorials/cellresp/etsar.html

2. http://wunmr.wustl.edu/EduDev/LabTutorials/Cytochromes/etc_movie.html

3. http://faculty.nl.edu/jste/electron_transport_system.htm

4. http://cwx.prenhall.com/horton/chapter14/deluxe.html
Live Figures:
Figure 14.10 Mitochondrial electron transport.
Figure 14.19 Demonstration of the rotation of a single molecule of
ATP synthase

5. http://www.wiley.com/college/fob/anim/
Chapter 17
 Fig. 17-8 -- The Mitochondrial Electron Transport Chain
 Fig. 17-18 -- The Coupling of Electron Transport and ATP Synthesis
 Fig. 17-21 -- The Binding Change Mechanism for ATP Synthesis

6. Great site with lots of information:

http://www.brookscole.com/chemistry_d/templates/student_resources/shared_resources/animations/oxi
dative/oxidativephosphorylation.html

7. http://www.science.smith.edu/departments/Biology/Bio111/etc.html **

8. http://www.stolaf.edu/people/giannini/biological%20anamations.html **

mitochondrial electron transport

ATP synthase
ATP synthase mechanism
Glycogen Metabolism
 CH2OH CH2OH
H O O
glycogen
H H H
H H
OH H OH H 1
O
OH
O
H OH H OH

CH2OH CH2OH 6 CH2 CH2OH CH2OH

H O H H O H H 5 O H H O H H O H
H H H H H
OH H OH H OH H 1 4 OH H OH H
4 O O
O O OH
OH
3 2
H OH H OH H OH H OH H OH

Glycogen is a polymer of glucose residues linked by

 (14) glycosidic bonds, mainly
 (16) glycosidic bonds, at branch points.
Glycogen chains & branches are longer than shown.
Glucose is stored as glycogen predominantly in liver and
muscle cells.
 CH2OH
H O H
H
OH H
OPO32
Glycogen catabolism OH
H OH
(breakdown): glucose-1-phosphate

Glycogen Phosphorylase catalyzes phosphorolytic

cleavage of the (14) glycosidic linkages of glycogen,
releasing glucose‐1‐phosphate as reaction product.
glycogen(n residues) + Pi 
glycogen (n–1 residues) + glucose‐1‐phosphate
This phosphorolysis may be compared to hydrolysis:
Hydrolysis: R-O-R' + HOH  R-OH + R'-OH
Phosphorolysis: R-O-R' + HO-PO32-  R-OH + R'-O-PO32-
 H O
Pyridoxal phosphate (PLP), C
O
O
a derivative of vitamin B6, H2
P C OH
serves as prosthetic group O
O
for Glycogen 
Phosphorylase. N
H
CH3

pyridoxal phosphate (PLP)

 lysine Enz
H
(CH2)4
H3N+ C COO
N+
CH2 O HC H
O H2
CH2 P C O
O
CH2
O

CH2 N CH3
H
 NH3 Enzyme (Lys)-PLP Schiff base

Pyridoxal phosphate (PLP) is held at the active site by a

Schiff base linkage, formed by reaction of the aldehyde of
PLP with the ‐amino group of a lysine residue.
In contrast to its role in other enzymes, the phosphate of
PLP is involved in acid/base catalysis by Phosphorylase.
 Enz

(CH2)4

N+
O HC H
O
The Pi substrate binds H2
P C O
between the phosphate of O
O
PLP and the glycosidic O

linking the terminal glucose N CH3
residue of the glycogen. H
Enzyme (Lys)-PLP Schiff base

After the phosphate substrate donates H+ during

cleavage of the glycosidic bond, it receives H+ from the
phosphate moiety of PLP.
PLP then takes back the H+ as the phosphate O attacks C1
of the cleaved glucose to yield glucose‐1‐phosphate.
Glycogen
Phosphorylase: GlcNAc
PLP
a homodimeric
enzyme, subject to
allosteric control.
inhibitor
It transitions between
“relaxed” (active) &
“tense” (inhibited) PLP
GlcNAc
conformations.
Diagram comparing
Human Liver
relaxed and tense Glycogen Phosphorylase PDB 1EM6
conformations.
A glucose analog, N‐acetylglucosamine (GlcNAc), is
adjacent to pyridoxal phosphate at the active site in the
crystal structure shown.
A class of drugs
developed for treating PLP GlcNAc
the hyperglycemia of
diabetes
(chloroindole‐ inhibitor
carboxamides), inhibit
liver Phosphorylase
allosterically. GlcNAc PLP
These inhibitors bind
at the dimer interface, Human Liver
stabilizing the inactive Glycogen Phosphorylase PDB 1EM6
(tense) conformation.
Question: Why would an inhibitor of Glycogen
Phosphorylase be a suitable treatment for diabetes?
A glycogen storage site on the surface of the
Phosphorylase enzyme binds the glycogen particle.
Given the distance between storage & active sites,
Phosphorylase can cleave (14) linkages only to
within 4 residues of an (16) branch point.
This is called a "limit branch".
Explore the structure of muscle Glycogen
Phosphorylase with Chime.
Debranching enzyme has 2 independent active sites,
consisting of residues in different segments of a single
polypeptide chain:
 The transferase of the debranching enzyme
transfers 3 glucose residues from a 4‐residue limit
branch to the end of another branch, diminishing
the limit branch to a single glucose residue.
 The (16) glucosidase moiety of the debranching
enzyme then catalyzes hydrolysis of the (16)
linkage, yielding free glucose. This is a minor
fraction of glucose released from glycogen.
The major product of glycogen breakdown is
glucose‐1‐phosphate, from Phosphorylase activity.
 Enzyme-Ser-OPO32 Enzyme-Ser-OH Enzyme-Ser-OPO32

CH2OH CH2OPO32 CH2OPO32

H O H H O H H O H
H H H
OH H OH H OH H
OH OPO32 OH OPO3
2
OH OH

H OH H OH H OH
glucose-1-phosphate glucose-6-phosphate

Phosphoglucomutase catalyzes the reversible reaction:

glucose‐1‐phosphate  glucose‐6‐phosphate
A serine OH at the active site donates & accepts Pi.
The bisphosphate is not released.
Phosphoglycerate Mutase has a similar mechanism, but
instead uses His for Pi transfer.
 Glycogen Glucose
Hexokinase or Glucokinase
Glucose-6-Pase
Glucose-1-P Glucose-6-P Glucose + Pi
Glycolysis
Pathway
Pyruvate
Glucose metabolism in liver.

Glucose‐6‐phosphate may enter Glycolysis or (mainly in

liver) be dephosphorylated for release to the blood.
Liver Glucose‐6‐phosphatase catalyzes the following,
essential to the liver's role in maintaining blood glucose:
glucose‐6‐phosphate + H2O  glucose + Pi
Most other tissues lack this enzyme.
 O

CH2OH HN

H O H
Glycogen H O N
OH H O O
synthesis
OH O P O P O CH2
O
 
H OH O O H H
H H
UDP-glucose OH OH

Uridine diphosphate glucose (UDP‐glucose) is the immediate

precursor for glycogen synthesis.
As glucose residues are added to glycogen, UDP‐glucose is
the substrate and UDP is released as a reaction product.
Nucleotide diphosphate sugars are precursors also for
synthesis of other complex carbohydrates, including
oligosaccharide chains of glycoproteins, etc.
 O
UDP-Glucose Pyrophosphorylase
CH2OH HN

H O H
H O N
OH H O O O O

OH O P O + 
O P O P O P O CH2
O
H OH O O O O H H

glucose-1-phosphate UTP H
OH OH
H

PPi O

CH2OH HN

H O H
H O N
OH H O O

OH O P O P O CH2
O
 
H OH O O H H
H H
UDP-glucose OH OH
UDP‐glucose is formed from glucose‐1‐phosphate:
 glucose‐1‐phosphate + UTP  UDP‐glucose + PPi
 PPi + H2O  2 Pi
Overall:
 glucose‐1‐phosphate + UTP  UDP‐glucose + 2 Pi
Spontaneous hydrolysis of the ~P bond in PPi (P~P) drives
the overall reaction.
Cleavage of PPi is the only energy cost for glycogen
synthesis (one ~P bond per glucose residue).
Glycogenin initiates glycogen synthesis.
Glycogenin is an enzyme that catalyzes attachment of
a glucose molecule to one of its own tyrosine
residues.
Glycogenin is a dimer, and evidence indicates that the
2 copies of the enzyme glucosylate one another.

Tyr active site

active site Tyr

Glycogenin dimer
 6 CH
2OH tyrosine residue
UDP-glucose
H
5 O H of Glycogenin
H O O C O
4 OH H 1
OH O P O P O Uridine HO C CH
3 2 H2
H OH O O NH

6 CH
2OH
O-linked 5 O
glucose H H
H
C O
residue 4 OH H 1
OH O C CH + UDP
3 2 H2
H OH NH

A glycosidic bond is formed between the anomeric C1 of

the glucose moiety derived from UDP‐glucose and the
hydroxyl oxygen of a tyrosine side‐chain of Glycogenin.
UDP is released as a product.
 6 CH
2OH
O-linked 5 O
glucose H H
H
C O
residue 4 OH H 1
OH O C CH + UDP
3 2 H2
H OH NH
UDP-glucose
CH2OH CH2OH

H O H H O H
H H C O
OH H OH H
OH O O C CH + UDP
H2
H OH (1 4) H OH NH
linkage

Glycogenin then catalyzes glucosylation at C4 of the

attached glucose (UDP‐glucose again the donor), to yield an
O‐linked disaccharide with (14) glycosidic linkage.
This is repeated until a short linear glucose polymer with
(14) glycosidic linkages is built up on Glycogenin.
Glycogen Synthase then catalyzes elongation of
glycogen chains initiated by Glycogenin.

Question: Where would you expect to find

Glycogenin within a cell?

Answer: Most of the Glycogenin is found associated

with glycogen particles (branched glycogen chains)
in the cytoplasm.
Glycogen Synthase catalyzes transfer of the glucose
moiety of UDP‐glucose to the hydroxyl at C4 of the
terminal residue of a glycogen chain to form an
(1 4) glycosidic linkage:
glycogen(n residues) + UDP‐glucose 
glycogen(n +1 residues) + UDP

A branching enzyme transfers a segment from the end of

a glycogen chain to the C6 hydroxyl of a glucose residue
of glycogen to yield a branch with an (16) linkage.
 Glycogen Synthesis
UTP UDP + 2 Pi
glycogen(n) + glucose-1-P glycogen(n + 1)

Glycogen Phosphorylase Pi

Both synthesis & breakdown of glycogen are spontaneous.

If both pathways were active simultaneously in a cell,
there would be a "futile cycle" with cleavage of one ~P
bond per cycle (in forming UDP‐glucose).
To prevent such a futile cycle, Glycogen Synthase and
Glycogen Phosphorylase are reciprocally regulated, by
allosteric effectors and by phosphorylation.
Glycogen Phosphorylase in muscle is subject to allosteric
regulation by AMP, ATP, and glucose‐6‐phosphate.
A separate isozyme of Phosphorylase expressed in liver is
less sensitive to these allosteric controls.
 AMP (present significantly when ATP is depleted)
activates Phosphorylase, promoting the relaxed
conformation.
 ATP & glucose‐6‐phosphate, which both have binding
sites that overlap that of AMP, inhibit Phosphorylase,
promoting the tense conformation.
 Thus glycogen breakdown is inhibited when ATP and
glucose‐6‐phosphate are plentiful.
 Glycogen Glucose
Hexokinase or Glucokinase
Glucose-6-Pase
Glucose-1-P Glucose-6-P Glucose + Pi
Glycolysis
Pathway
Pyruvate
Glucose metabolism in liver.
Glycogen Synthase is allosterically activated by
glucose‐6‐P (opposite of effect on Phosphorylase).
Thus Glycogen Synthase is active when high blood
glucose leads to elevated intracellular glucose‐6‐P.
It is useful to a cell to store glucose as glycogen when the
input to Glycolysis (glucose‐6‐P), and the main product of
Glycolysis (ATP), are adequate.
Regulation by covalent modification
(phosphorylation):
The hormones glucagon and epinephrine activate
G‐protein coupled receptors to trigger cAMP cascades.
 Both hormones are produced in response to low
blood sugar.
 Glucagon, which is synthesized by ‐cells of the
pancreas, activates cAMP formation in liver.
 Epinephrine activates cAMP formation in muscle.
The cAMP cascade results in phosphorylation of a serine
hydroxyl of Glycogen Phosphorylase, which promotes
transition to the active (relaxed) state.
The phosphorylated enzyme is less sensitive to allosteric
inhibitors.
Thus, even if cellular ATP & glucose‐6‐phosphate are
high, Phosphorylase will be active.
The glucose‐1‐phosphate produced from glycogen in liver
may be converted to free glucose for release to the
blood.
With this hormone‐activated regulation, the needs of the
organism take precedence over needs of the cell.
Commonly used terminology:
 "a" is the form of the enzyme that tends to be active,
and independent of allosteric regulators (in the case
of Glycogen Phosphorylase, when phosphorylated).
 "b" is the form of the enzyme that is dependent on
local allosteric controls (in the case of Glycogen
Phosphorylase when dephosphorylated).
 Hormone (epinephrine or glucagon)
via G Protein (G-GTP)

Adenylate cyclase Adenylate cyclase

(inactive) (active)
catalysis
ATP cyclic AMP + PPi
Activation Phosphodiesterase
Signal cascade AMP
by which Protein kinase A Protein kinase A
(inactive) (active)
Glycogen ATP
Phosphorylase ADP

is activated. Phosphorylase kinase Phosphorylase kinase (P)

(b-inactive) (a-active)
Phosphatase ATP
Pi ADP

Phosphorylase Phosphorylase (P)

(b-allosteric) (a-active)
Phosphatase
Pi
The cAMP cascade induced in liver by glucagon or
epinephrine has the opposite effect on glycogen
synthesis.
Glycogen Synthase is phosphorylated by Protein
Kinase A as well as by Phosphorylase Kinase.
Phosphorylation of Glycogen Synthase promotes the
"b" (less active) conformation.
The cAMP cascade thus inhibits glycogen synthesis.
Instead of being converted to glycogen, glucose‐1‐P
in liver may be converted to glucose‐6‐P, and
dephosphorylated for release to the blood.
 Glycogen Glucose
Hexokinase or Glucokinase
Glucose-6-Pase
Glucose-1-P Glucose-6-P Glucose + Pi
Glycolysis
Pathway
Pyruvate
Glucose metabolism in liver.

High cytosolic glucose‐6‐phosphate, which would result

when blood glucose is high, turns off the signal with
regard to glycogen synthesis.
The conformation of Glycogen Synthase induced by the
allosteric activator glucose‐6‐phosphate is susceptible to
dephosphorylation by Protein Phosphatase.
Insulin, produced in response to high blood glucose,
triggers a separate signal cascade that leads to
activation of Phosphoprotein Phosphatase.
This phosphatase catalyzes removal of regulatory
phosphate residues from Phosphorylase,
Phosphorylase Kinase, & Glycogen Synthase enzymes.
Thus insulin antagonizes effects of the cAMP cascade
induced by glucagon & epinephrine.
 Phosphorylase Kinase inactive

Phosphorylase Kinase-Ca++ partly active

P-Phosphorylase Kinase-Ca++ fully active

Ca++ also regulates glycogen breakdown in muscle.

During activation of contraction in skeletal muscle, Ca++ is
released from the sarcoplasmic reticulum to promote
actin/myosin interactions.
The released Ca++ also activates Phosphorylase Kinase,
which in muscle includes calmodulin as its  subunit.
Phosphorylase Kinase is partly activated by binding of
Ca++ to this subunit.
 Phosphorylase Kinase inactive

Phosphorylase Kinase-Ca++ partly active

P-Phosphorylase Kinase-Ca++ fully active

Phosphorylation of the enzyme, via a cAMP cascade

induced by epinephrine, results in further activation.
These regulatory processes ensure release of
phosphorylated glucose from glycogen, for entry into
Glycolysis to provide ATP needed for muscle contraction.
During extended exercise, as glycogen stores become
depleted, muscle cells rely more on glucose uptake from
the blood, and on fatty acid catabolism as a source of ATP.
A genetic defect in the isoform of an enzyme expressed in
liver causes the following symptoms:
 After eating a CHO meal, elevated blood levels of
glucose, lactate, & lipids.
 During fasting, low blood glucose & high ketone bodies.
Which liver enzyme is defective? Glycogen Synthase
Explain Symptoms:
 After eating, blood glucose is high because liver cannot
store it as glycogen. Some excess glucose is processed
via Glycolysis to produce lactate & fatty acid precursors.
 During fasting, glucose is low because the liver lacks
glycogen stores for generation of glucose.
Ketone bodies are produced as an alternative fuel.
Question: How would you nutritionally treat
deficiency of liver Glycogen Synthase?
 Frequent meals of complex carbohydrates
(avoiding simple sugars that would lead to a rapid
rise in blood glucose)
 Meals high in protein to provide substrates for
gluconeogenesis.
Glycogen Storage
Diseases are genetic glycogen
enzyme deficiencies
associated with excessive
glucose-1-P
glycogen accumulation
Glucose-6-Phosphatase
within cells.
glucose-6-P glucose + Pi
Some enzymes whose
deficiency leads to fructose-6-P
glycogen accumulation Phosphofructokinase
are part of the inter‐ fructose-1,6-bisP
connected pathways
Glycolysis continued
shown here.
Symptoms in addition to excess glycogen storage:
 When a genetic defect affects mainly an isoform of an
enzyme expressed in liver, a common symptom is
hypoglycemia, relating to impaired mobilization of
glucose for release to the blood during fasting.
 When the defect is in muscle tissue, weakness &
difficulty with exercise result from inability to
increase glucose entry into Glycolysis during exercise.
 Additional symptoms depend on the particular
enzyme that is deficient.
 Symptoms, in addition to
Glycogen Storage Disease
glycogen accumulation
Type I, liver deficiency of hypoglycemia (low blood
Glucose-6-phosphatase (von glucose) when fasting, liver
Gierke's disease) enlargement.
Type IV, deficiency of liver dysfunction and early
branching enzyme in various death.
organs, including liver
(Andersen's disease)
Type V, muscle deficiency of muscle cramps with exercise.
Glycogen Phosphorylase
(McArdle's disease)
Type VII, muscle deficiency of inability to exercise.
Phosphofructokinase.
GLYCOGEN METABOLISM

Glycogen Breakdown Is Catalyzed by Glycogen Phosphorylase

In skeletal muscle and liver, the glucose units of the outer branches of glycogen enter the
glycolytic pathway through the action of three enzymes: glycogen phosphorylase, glycogen
debranching enzyme, and phosphoglucomutase. Glycogen phosphorylase catalyzes the
reaction in which an (α1,4) glycosidic linkage between two glucose residues at a nonreducing
end of glycogen undergoes attack by inorganic phosphate (Pi), removing the terminal glucose
residue as glucose 1-phosphate. This phosphorolysis reaction is different from the hydrolysis
of glycosidic bonds by amylase during intestinal degradation of dietary glycogen and starch.
In phosphorolysis, some of the energy of the glycosidic bond is preserved in the formation of
the phosphate ester, glucose 1-phosphate. Pyridoxal phosphate is an essential cofactor in the
glycogen phosphorylase reaction; its phosphate group acts as a general acid catalyst, promoting
attack by Pi on the glycosidic bond. (This is an unusual role for pyridoxal phosphate; its more
typical role is as a cofactor in amino acid metabolism

Glycogen phosphorylase acts repetitively on the non-reducing ends of glycogen branches until
it reaches a point four glucose residues away from an (α1,6) branch point, where its action
stops.
Further degradation by glycogen phosphorylase can occur only after the debranching enzyme,
formally known as oligo (_1n6) to (_1n4) glucan-transferase, catalyzes two successive
reactions that transfer branches. Once these branches are transferred and the glucosyl residue
at C-6 is hydrolyzed, glycogen phosphorylase activity can continue.
Glucose 1-Phosphate Can Enter Glycolysis or, in Liver,Replenish Blood Glucose
Glucose 1-phosphate, the end product of the glycogen phosphorylase reaction, is converted to
glucose 6-phosphate by phosphoglucomutase, which catalyzes the reversible reaction
Initially phosphorylated at a Ser residue, the enzyme donates a phosphoryl group to C-6 of the
substrate, then accepts a phosphoryl group from C-1.
The glucose 6-phosphate formed from glycogen in skeletal muscle can enter glycolysis and
serve as an energy source to support muscle contraction.
In liver, glycogen breakdown serves a different purpose: to release glucose into the blood when
the blood glucose level drops, as it does between meals. This requires the enzyme glucose 6-
phosphatase, present in liver and kidney but not in other tissues. The enzyme is an integral
membrane protein of the endoplasmic reticulum, predicted to contain nine transmembrane
helices, with its active site on the lumenal side of the ER. Glucose 6-phosphate formed in the
cytosol is transported into the ER lumen by a specific transporter (T1) and hydrolyzed at the
lumenal surface by the glucose 6-phosphatase. The resulting Pi and glucose are thought to be
carried back into the cytosol by two different transporters (T2 and T3), and the glucose leaves
the hepatocyte via the plasma membrane transporter, GLUT2.
Notice that by having the active site of glucose 6-phosphatase inside the ER lumen, the cell
separates this reaction from the process of glycolysis, which takes place in the cytosol and
would be aborted by the action of glucose 6-phosphatase. Genetic defects in either glucose 6-
phosphatase or T1 lead to serious derangement of glycogen metabolism, resulting in type Ia
glycogen storage disease.
Glucose 1-phosphate to glucose 6-phosphate
Because muscle and adipose tissue lack glucose 6-phosphatase, they cannot convert the glucose
6-phosphate formed by glycogen breakdown to glucose, and these tissues therefore do not
contribute glucose to the blood.
The Sugar Nucleotide UDP-Glucose Donates Glucose for Glycogen Synthesis
Many of the reactions in which hexoses are transformed or polymerized involve sugar
nucleotides, compounds in which the anomeric carbon of a sugar is activated by
attachment to a nucleotide through a phosphate ester linkage. Sugar nucleotides are the
substrates for polymerization of monosaccharides into disaccharides, glycogen, the product of
this reaction is converted to UDP-glucose by the action of UDP-glucose pyrophosphorylase,
in a key step of glycogen biosynthesis:
Notice that this enzyme is named for the reverse reaction; in the cell, the reaction proceeds in
the direction of UDPGlucose1-phosphate _ UTPUDP-glucose _ PPi glucose formation,
because pyrophosphate is rapidly hydrolysed by inorganic pyrophosphatase.
UDP-glucose is the immediate donor of glucose residues in the reaction catalyzed by glycogen
synthase, which promotes the transfer of the glucose residue from UDP-glucose to a
nonreducing end of a branched glycogen molecule.
The overall equilibrium of the path from glucose 6-phosphate to glycogen lengthened by one
glucose unit greatly favors synthesis of glycogen.
Glycogen synthase cannot make the (α1,6) bonds found at the branch points of glycogen; these
are formed by the glycogen-branching enzyme, also called amylo (1,4) to (1,6)
transglycosylase, or glycosyl-(4,6)-transferase. The glycogen-branching enzyme catalyzes
transfer of a terminal fragment of 6 or 7 glucose residues from the nonreducing end of a
glycogen branch having at least 11 residues to the C-6 hydroxyl group of a glucose residue at
a more interior position of the same or another glycogen chain, thus creating a new branch.
Further glucose residues may be added to the new branch by glycogen synthase.
The biological effect of branching is to make the glycogen molecule more soluble and to
increase the number of nonreducing ends. This increases the number of sites accessible to
glycogen phosphorylase and glycogen synthase, both of which act only at nonreducing ends.
Glycogenin Primes the Initial Sugar Residues in Glycogen

Glycogen synthase cannot initiate a new glycogen chain de novo. It requires a primer, usually
a preformed (_1→4) polyglucose chain or branch having at least eight glucose residues. So,
how is a new glycogen molecule initiated? The intriguing protein glycogenin is both the primer
on which new chains are assembled and the enzyme that catalyzes their assembly.
The first step in the synthesis of a new glycogen molecule is the transfer of a glucose residue
from UDP-glucose to the hydroxyl group of Tyr194 of glycogenin, which is catalyzed by the
protein’s intrinsic glucosyltransferase activity. The nascent chain is extended by the sequential
addition of seven more glucose residues, each derived from UDP-glucose; the reactions are
catalyzed by the chain-extending activity of glycogenin. At this point, glycogen synthase takes
over, further extending the glycogen chain. Glycogenin remains buried within the β-particle,
covalently attached to the single reducing end of the glycogen molecule.
 Pentose Phosphate Pathway

Also known as:

• Pentose shunt
• Hexose monophosphate shunt
• Phosphogluconate pathway

• It occurs in the cytosol.

One fate of G6P is the
pentose pathway.
 The pentose pathway is a shunt.

• The pathway begins with the glycolytic

intermediate glucose 6-P.
• It reconnects with glycolysis because
two of the end products of the pentose
pathway are glyceraldehyde 3-P and
fructose 6-P; two intermediates further
down in the glycolytic pathway.
• It is for this reason that the pentose
pathway is often referred to as a shunt.
 Moderate glucose flux

Glycolysis
only
 Large glucose flux

Pentose
Phosphate
Glycolysis Pathway
It’s a shunt
 What does the pentose phosphate
pathway achieve?
• The pathway yields reducing potential in
the form of NADPH to be used in
anabolic reactions requiring electrons.
• The pathway yields ribose 5-phosphate.
– Nucleotide biosynthesis leading to:
•DNA
•RNA
•Various cofactors (CoA, FAD, SAM,
NAD+/NADP+).
NADPH is a
phosphorylated
form of NADH.

In general, with some

exceptions, NADH is
used to drive the
phosphorylation of
ADP to ATP. NADPH
is used where
reducing potential is
required for synthetic
reactions.
The pentose
pathway can be
divided into two
phases.

Non-oxidative
interconversion of
sugars
NADPH + H+ is formed
from two separate
reactions.

The glucose 6-
phosphate DH (G6PD)
reaction is the rate
limiting step and is
essentially irreversible.

There is a medical story

for this enzyme.

Cells have a greater

need for NADPH than
ribose 5-phosphate.
 Regulatory enzyme

5 carbon atoms
 Regulatory enzyme

The enzyme is highly specific for NADP+; the

Km for NAD+ is 1000 greater than for NADP+.
 The nonoxidative phase of the
pentose pathway
This entails extensive carbon atom
rearrangement.

Transketolase requires the

coenzyme thiamine
pyrophosphate (TPP), the
transaldolase does not.
• Transketolase (TPP) and transaldolase
are the link back to glycolysis.
• Glyceraldehyde 3-phosphate
• Fructose 6-phosphate
• Net result:
3C5  2C6 + C3
Ingested ribose
can enter the
glycolytic
pathway through
the pentose
pathway.
 Transketolases and transaldoses
• Transketolase catalyzes the transfer of a two-carbon
fragment from a ketose donor to an aldose acceptor. In
its first appearance in the pentose phosphate pathway,
transketolase transfers C-1 and C-2 of xylulose 5-
phosphate to ribose-5-phosphate, forming the seven-
carbon product, sedoheptulose 7-phosphate. The
remaining three-carbon fragment from xylulose is
glyceraldehyde-3-phosphate.
• Next, transaldolase catalyzes a reaction similar to
the aldolase reaction of glycolysis: a three-carbon fragment
is removed from sedoheptulose 7-phosphate and condensed
with glyceraldehyde 3-phosphate, forming fructose 6-
phosphate and the tetrose erythrose-4-phosphate. Now
transketolase acts again, forming fructose 6-phosphate and
glyceraldehyde-3-phosphate from erythrose 4-phosphate
and xylulose 5-phosphate
 Regulation of the Pentose Pathway

• Glucose 6-phosphate DH is the

regulatory enzyme.
• NADPH is a potent competitive inhibitor
of the enzyme.
• Usually the ratio NADPH/NADP+ is high
so the enzyme is inhibited.
• But, with increased demand for NADPH,
the ratio decreases and enzyme activity
is stimulated.
• The reactions of the non-oxidative
portion of the pentose pathway are
readily reversible.

• The concentrations of the products and

reactants can shift depending on the
metabolic needs of a particular cell or
tissue.
Rapidly dividing cells require more ribose 5-
phosphate than NADPH.
The need for NADPH and ribose 5-phosphate is
balanced.
 More NADPH is needed than ribose 5-
phosphate; Fatty acid synthesis in adipose
cells.
The cell needs both NADPH and ATP
 Glutathione and NADPH

• What is glutathione?
• Why is it important?
• How is it related to NADPH?
 Glutathione is a
tripeptide composed
of glutamate,
cystein, glycine.

Reduced glutathione
(GSH) maintains the
normal reduced
state of the cell.

Reduced
glutathione
(GSH)
 Glutathione Functions -1

• It serves as a reductant.
• Conjugates to drugs making them
water soluble.
• Involved in amino acid transport
across cell membranes.
• Cofactor in some enzymatic
reactions.
– rearrangement of protein disulfide
bonds.
 Glutathione Functions -2

• The sulfhydryl of GSH is used to reduce

peroxides (ROS) formed during oxygen
transport.
– Reactive oxygen species (ROS)
damage macromolecules (DNA, RNA,
and protein) and ultimately lead to
cell death.
• The resulting oxidized form of GSH is
two molecules linked by a disulfide
bridge (GSSG).
The enzyme
glutathione
reductase uses
NADPH as a
cofactor to reduce
GSSG back to two
moles of GSH.
Thus, the pentose
pathway is linked
to the supply of
adequate amounts
of GSH.
 So, what happens if glucose 6-
phosphate DH is defective?

Insufficient production of NADPH.

Which translates into insufficient

glutathione.

Is this a medical problem?

YES
 Glutathione and Erythrocytes -1
• GSH is extremely important particularly
in the highly oxidizing environment of
the red blood cell.
• Mature RBCs have no mitochondria and
are totally dependent on NADPH from
the pentose phosphate pathway to
regenerate GSH from GSSG via
glutathione reductase.
• In fact, as much as 10% of glucose
consumption, by erythrocytes, is
mediated by the pentose pathway.
 Glutathione and Erythrocytes -2
• The reduced form of glutathione serves
as a sulfhydryl buffer.
• It maintains cysteine residues in
hemoglobin and other proteins in a
reduced state.
• GSH is essential for normal RBC
structure and keeping hemoglobin in
Fe++ state.
 Glutathione and Erythrocytes -3

• Reduced glutathione also detoxifies

peroxides.
2GSH + ROOH  GSSG + H2O + ROH
• Cells with low levels of GSH are
susceptible hemolysis.
• Individuals with reduced GSH are
subject to hemolysis.
• This is often clinically seen as black
urine under certain conditions.
 Conditions for hemolytic anemia
related G6PD deficiency.
• The ingestion of oxidative agents that
generate peroxides or reactive oxygen
species (ROS).
– Antimalarials - pamaquine
– purine glycoside from fava beans.
• Individules with G6PD deficiency can
not produce sufficient GSH to cope with
the ROS.
• Proteins become cross linked leading to
Heinz body formation and cell lysis.
Glucose 6-phosphate DH deficiency
and nonspherocytic hemolytic
anemia.
• Over 300 genetic variants of the G6PD
protein are known.
• Thus, there is a remarkable variation in
the clinical spectrum.
• G6PD deficiency is an inheritable X-
linked recessive disorder.
• Approximately 10-14% of the male
African American population is affected.
• It is also seen in Caucasians from the
Mediterranean Basin.
• People with the disorder are not normally
anemic and display no evidence of the disease
until the red cells are exposed to an oxidant or
stress.
Drugs that can precipitate this reaction:
• antimalarial agents
• sulfonamides (antibiotic)
• aspirin
• nonsteroidal antiinflammatory drugs (NSAIDs)
• nitrofurantoin
• quinidine
• quinine
• exposure to certain chemicals - mothballs
 FAVISM

• Individuals with G6PD deficiency must

not eat Fava beans.
• Pythagoras
• Erythrocytes lyse=dark or black urine.
• Interesting
– The growth Plasmodium falciparum
(malaria parasite) fails in G6PD
deficient individuals.
Accessory Pigments Extend the Range of Light Absorption
In addition to chlorophylls, thylakoid membranes contain secondary light-absorbing pigments, or
accessory pigments, called carotenoids. Carotenoids may be yellow, red, or purple. The most
important are _-carotene, which is a red-orange isoprenoid, and the
yellow carotenoid lutein (Fig. 19–47c, d). The carotenoid pigments absorb light at wavelengths not
absorbed by the chlorophylls (Fig. 19–48) and thus are supplementary light receptors.
Experimental determination of the effectiveness of light of different colors in promoting
photosynthesis yields an action spectrum (Fig. 19–51), often useful in identifying the pigment
primarily responsible for a biological effect of light. By capturing light in a region of the spectrum not
used by other organisms, a photosynthetic organism can claim a unique ecological niche. For
example, the phycobilins in red algae and cyanobacteria
absorb light in the range 520 to 630 nm (Fig. 19–48), allowing them to occupy niches where light of
lower or higher wavelength has been filtered out by the pigments of other organisms living in the
water above them, or by the water itself.
Chlorophyll Funnels the Absorbed Energy to Reaction Centers by Exciton
Transfer
The light-absorbing pigments of thylakoid or bacterial membranes are arranged in functional arrays
called photosystems. In spinach chloroplasts, for example, each photosystem contains about 200
chlorophyll and 50 carotenoid molecules. All the pigment molecules in a photosystem can absorb
photons, but only a few chlorophyll molecules associated with the photochemical reaction center
are specialized to transduce light into chemical energy. The other pigment molecules in a
photosystem are called light-harvesting
or antenna molecules. They absorb light energy
Photophosphorylation is the synthesis of ATP from ADP and inorganic phosphate in the
presence of light (energy). When the two photosystems work in a series, first photosystem II
(PS II) and then the PS I, a process called non-cyclic photo-phosphorylation occurs. The two
photosystems are connected through an electron transport chain, as seen in the Z scheme. Both
ATP and NADPH + H+ are synthesised by this kind of electron flow.
When only PS I is functional, the electron is circulated within the photosystem and the
phosphorylation occurs due to cyclic flow of electrons is called cyclic photo phosphorylation.
A possible location where this could be happening is in the stroma lamellae. While the
membrane or lamellae of the grana have both PS I and PS II the stroma lamellae membranes
lack PS II as well as NADP reductase enzyme. The excited electron does not pass on to NADP+
but is cycled back to the PS I complex through the electron transport chain. The cyclic flow
hence, results only in the synthesis of ATP, but not of NADPH + H+. Cyclic
photophosphorylation also occurs when only light of wavelengths beyond 680 nm are available
for excitation.
In plants, both the water-splitting reaction and electron flow through the cytochrome b6 f
complex are accompanied by proton pumping across the thylakoid membrane. The proton-
motive force thus created drives ATP synthesis by a CFoCF1 complex similar to the
mitochondrial FoF1 complex.
The ATP synthase of chloroplasts (CFoCF1) is very similar in both structure and catalytic
mechanism to the ATP synthases of mitochondria and bacteria. Physical rotation driven by the
proton gradient is accompanied by ATP synthesis at sites that cycle through three
conformations, one with high affinity for ATP, one with high affinity for ADP and Pi, and
one with low affinity for both nucleotides.
Photosynthesis in vascular plants takes place in chloroplasts. In the CO2-assimilating reactions
(the Calvin cycle), ATP and NADPH are used to reduce CO2 to triose phosphates. These
reactions occur in three stages: the fixation reaction itself, catalysed by rubisco; reduction of
the resulting 3-phosphoglycerate to glyceraldehyde 3-phosphate; and regeneration of ribulose
1,5-bisphosphate from triose phosphates.
■ Rubisco condenses CO2 with ribulose 1,5-bisphosphate, forming an unstable hexose
bisphosphate that splits into two molecules of 3-phosphoglycerate.
Rubisco is activated by covalent modification (carbamoylation of Lys201) catalyzed by rubisco
activase and is inhibited by a natural transition state analog, whose concentration rises in the
dark and falls during daylight.
■ Stromal isozymes of the glycolytic enzymes catalyse reduction of 3-phosphoglycerate to
glyceraldehyde 3-phosphate; each molecule reduced requires one ATP and one NADPH.
■ Stromal enzymes, including transketolase and aldolase, rearrange the carbon skeletons of
triose phosphates, generating intermediates of three,four, five, six, and seven carbons and
eventually yielding pentose phosphates. The pentose phosphates are converted to ribulose 5-
phosphate, then phosphorylated to ribulose 1,5-bisphosphate to complete the Calvin cycle.
■ The cost of fixing three CO2 into one triose phosphate is nine ATP and six NADPH, which
are provided by the light-dependent reactions of photosynthesis.
■ An antiporter in the inner chloroplast membrane exchanges Pi in the cytosol for 3-
phosphoglycerate or dihydroxyacetone phosphate produced by CO2 assimilation in the stroma.
Oxidation of dihydroxyacetone phosphate in the cytosol generates ATP and NADH, thus
moving ATP and reducing equivalents from the chloroplast to the cytosol.
■ Four enzymes of the Calvin cycle are activated indirectly by light and are inactive in the
dark, so that hexose synthesis does not compete with glycolysis—which is required to provide
energy in the dark.