BIOCHEM A

Porphyrin and Respiration – Dr. Bravo

HEMOGLOBIN  Hydrogen bonding – between polar amino acid

(serine, threonine)
Characteristics:  Van der Waal forces – between hydrophobic amino
 It is a metalloprotein because it is a molecule acid
 Composed of 4 subunits  Ionic interaction/salt bridges
 Each subunit has its own heme prosthetic group which is iron  Most important because this is the one that can
be reversed by oxygen binding
RBCs and Hemoglobin:  Interaction between the charged amino acids
 Hemoglobin are carried by RBCs like arginine, aspartic acid, lysine, histidine…
 Each RBC contains about 280 million molecules of hemoglobin  8 ionic interactions are possible
 Each hemoglobin carries 3 molecules of oxygen  Ionic interactions can be intrapeptide which is
 Mature RBC does not have any nucleus and organelles to the interaction within a chain (histidine and
maximize hemoglobin carrying capacity aspartic acid) or interpeptide which is between 2
different peptide chains (arginine and aspartic
3D Structure of Hemoglobin acid on 2 different alpha subunit)
 The amino terminal group (terminus) also
contribute to the ionic interaction

 Hemoglobin is a tetramer (4 subunits) containing 2 alpha and 2

beta
 Best describes as a dimer of a dimer
o A1 and B1 will dimerize together as well as your a2 and b2
o This two dimers will dimerize again which is the reason why
we call it dimer of a dimer
Composition of Hemoglobin
o 1st dimer: a1 b1
 Can be summarized as X2Y2
o 2nd dimer: a2 b2
 It changes throughout an organisms life
 Primary Structure
 Adults
o Alpha – contains 141 amino acids
o Hgb A – composed of 2 alpha and 2 beta subunits; most
o Beta – contains 146 amino acids
common form
 Secondary Structure
o Hgb A2 – composed of 2 alpha and 2 delta subunits
o The helical structure is mostly an alpha helix
 Delta and beta are slightly different from each other
o Divided into 8 helical region from letter A to H
 Early embryo
o In between these letters there is proline, which is why there
o 2 zeta and 2 epsilon
is a break in the alpha helix
 Zeta is the counterpart of alpha, while epsilon is the
counterpart of beta
 Late embryo
o 2 alpha and 2 gamma
 Also known as the fetal hemoglobin which has higher
affinity to oxygen
 Alpha and Zeta
o Homologous
o Both are composed of 141 aa but are slightly different
 Gamma and delta
o Homologous (both contains 145 aa but are slightly different)
 Quaternary Structure
o There are certain forces that stabilize the quaternary
structure of the hemoglobin molecule
 Hydrophobic interaction – between hydrophobic
amino acid

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Heme HEMOGLOBIN SATURATION CURVE
 Prosthetic group
 Tucked in the hydrophobic crevice of the individual polypeptide
 There are 4 hemes present in one hemoglobin
 In the protoporphyrin ring, iron is located at the center
 Composed of 4 pyrrole rings linked together by methyl ridges

Iron
 Reason why hemoglobin is able to bind to oxygen
 Capable of forming 6 ligands
o First 4 attachment: are at the nitrogen in the porphyrin ring
o Where are ligands 5 and 6?
 Refer to the picture below
 Imagine this as one polypeptide
 2 amino acids are present: proximal and distal
histidine which is a ring similar to the porphyrin
because of nitrogen
 The nitrogen of the histidine forms an attachment
with the iron of the porphyrin  ligand 5
 Oxygen that is attached to iron, connects iron to the
nitrogen of distal histidine.  ligand 6
 X axis (Partial Pressure of Oxygen)
o Represents the availability of oxygen
 Y axis (Percent Saturation of Hemoglobin)
o Represents how much oxygen is present within the
hemoglobin
 INTERPRETATION:
o In the lungs, since there are a lot of oxygen, the saturation
of hemoglobin is near to 100% because it is carrying a lot of
oxygen
o In the peripheral tissues, the saturation of hemoglobin
drops because it releases the oxygen that it contains

Hemoglobin acts like a sponge. It soaks up oxygen when there is a lot

of it and it squeezes it out when there is little amount in the peripheral
tissues

HEMOGLOBIN CONFORMATION

Functions of Hemoglobin
 Major: transport oxygen from lungs to the tissues
 Minor: transport carbon dioxide from tissues to the lungs

Hemoglobin has two conformational change:

 Taut state
o There is a lot of ionic bonds
o Low affinity to O2
 Relaxed state
o Ionic bonds are destroyed
o High affinity to O2

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Conditions that would favor the Taut State:
 High temperature
 High BPG Cooperativity
 High CO2  In the taut state, the initial binding of oxygen is difficult. The
 Low pH conformational change makes the second binding easier…

Conditions that would favor the relaxed state:

 Low temperature
 Low BPG
 Low CO2
 High pH

How does hemoglobin change from one state to another? (Taut to

Relaxed)
 Hemoglobin is still in the taut state, then it encounters oxygen
 When hemoglobin enters the lungs (1), by random chance one of
the oxygen molecule will bind (2) to it thus creating a
o Change in hemoglobin molecules
conformational change in the structure of the hemoglobin
 In the taut state, the a1b1 subunits are obscuring the
molecule and transform to the relaxed state (3), the molecule will
a2b2
now have a high affinity to oxygen (4)
 The taut state will rotate 15 degrees once oxygen
binds to it thus exposing the a2b2
 The change is in the entire hemoglobin molecule
because of the disruption in the ionic bonds

 How did this occur? (2 major changes in structure)

o Change in the heme conformation
 In the taut state, the iron is slightly off the plane of the
porphyrin ring. When oxygen binds to the iron, it
connects to the distal histidine causing iron to move
towards the plane. Iron brings along the rest of the
HOW DOES HEMOGLOBIN KNOW WHEN TO TAKE UP OR RELEASE
molecule and proximal histidine with it
OXYGEN?
 The movement will cause the amino acids to move
away from each other causing the disruption of the
Upon reaching the peripheral tissues, the hemoglobin needs to restore
ionic bonds  shift of taut to relaxed state
its ionic bonds. What would be its signal to restore ionic bonds?
 High hydrogen ions, BPG and CO2

Allosteric Regulators of Oxygen Binding

 pH
o when pH is low, there is a shift to the right, meaning
hemoglobin has lower affinity to oxygen
o Explanation:
 When pH is very low, there is a lot H+ ions. That ions
will protonate the basic side chains (histidine). When
histidine is protonated, it will assume a positive
charge. When it assumes a positive charge, it can now
interact with acidic aa (aspartic). Since they interact,
there will now be restoration of ionic bonds  shift
from relaxed to taut

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  Fetal hemoglobin has serine rather than histidine
causing it to have no effect when BPG binds

 Temperature
 Oxygen
o High oxygen = high affinity

SICKLE CELL ANEMIA

 Problem in sickle cell anemia: there is a mutation in the gene that

codes for hemoglobin thus causing an alteration in the amino
acids

Which of the ff AA substitution in the β subunit of Hgb will most likely

result to abnormal RBC?
o Low pH favors the taut state A. Glu to Asp (A to A)
o pH also explains the Bohr effect B. Lys to Arg (B to B)
 Bohr effects describe the inverse effect of carbon C. Val to Leu (NP to NP)
dioxide and hydrogen ion concentration on the affinity D. Glu to Val (P to NP)
of hemoglobin to oxygen
 In the peripheral tissues, there is a lot of CO2. The CO2  Why? Since valine is a non polar, it needs to be buried within the
will diffuse from the tissue to the plasma. In the molecule. This will now cause a conformational change in the
plasma, CO2 will fuse with water  carbonic acid molecule in order to bury valine
 Carbonic acid will ionize to form bicarbonate and H+.  This now changes the solubility of the molecule
The H+ comes out in exchange for chloride (chloride o Normally, when the oxygen is released, the Hgb remains
shift) soluble
 H+ will bind to the histidine which causes the shift of o But when it’s a sickle RBC, when oxygen is release it forms
relaxed to taut polymers which will cause the distortion of RBCs
 The more CO2, the more H+ ions, thus favoring more  Why does it form polymers?
the taut state o On a normal Hbg: when O2 dissociates, a receptor site is
formed
o On a sickle Hgb: a receptor & sticky site
 Because of the mutation, there is a change in
conformation, a protrusion is formed--- the sticky site
 Because of the sticky site, they would stick w/ each
other forming polymer that will result to long
filaments which will distort the shape of RBC
becoming sickle
 Consequence of sickle shape: it loses its flexibility that causes
plugging of capillaries causing micro infarction
 Complications:
o Leg ulcer
o Pain Episodes
o Stroke
o Increase infection rate
o Bone damage
 CO2
o Jaundice
o Carbon dioxide has a two-fold effect
o Gallstones
o It can bind to the N terminals of the polypeptide. When it
o Low RBC count
binds, it will form carbamates which are negatively charged
o Lung blockage
o The negative charge can form a soft bridge of arginine
o Kidney damage
o Favors the taut state
o Priapism
o Eye damage
 BPG (Biphosphogycerate)
o ↑ concentration of BPG = shift to the RIGHT
o In the taut state, the pockets are wide enough to
accommodate BPG and it will covalently stabilize the Taut MYOGLOBIN
state of Hgb = ↓affinity to O2  Heme protein found in the muscle tissue
o Source of BPG: Glycolysis  Its tertiary structure is typical of water soluble globular protein
o Does BPG have an effect on fetal hemoglobin? NO  It is largely composed of alpha helical structure with 8 alpha
 Fetal hemoglobin should always be in the relaxed helices
state so that it can bind oxygen from the mother

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  Porphyrin is usually linked to a metal in the center. When the
metal is iron, it now becomes your heme

Heme
 Prosthetic group for hemoglobin
 Heme and all other porphyrin RARELY exist in their FREE FORM in
nature
 They are usually packaged together with proteinHEMOPROTEIN
o Hemoglobin
Hemoglobin vs. Myoglobin o Myoglobin
 Hemoglobin is a tetramer. Myoglobin is a monomer o Cytochrome P450 – Liver enzyme
 The conformation of myoglobin is the same as the beta subunit, o Chlorophyll – Instead of iron, it has magnesium
so you can say that they come from the same molecule o Catalase

Function of Myoglobin PROPERTIES OF PORPHYRIN

 For storage of oxygen  Excitable molecules
o It does not release oxygen oxygen immediately (under  Absorb light at 450 nm
normal circumstances)  If they absorb UV light they become very excitable/reactive

BIOSYNTHESIS OF HEME
 Largest producer of heme: bone marrow in erythroid precursor
cells (80%), liver in hepatocytes (15%)
 Synthesis is a cytosolic and mitochondrial event

o The pathway begins in the mitochondria, the middle part

takes place in the cytoplasm, and in the end it goes back to
the mitochondria

Seven major steps in the synthesis of heme:

PORPHYRIN  ALAS
o Key regulatory enzyme
o ALAS 1 – inducible; not always present but can be
stimulated or inhibited
o ALAS 2 – constituted; always present
 1st and 2nd step includes:
o Formation of key regulatory enzyme: ALA
o Formation of pyrrole
o Formation of tetrapyrrole

 Porphyrin is a tetra pyrrole

o House shaped – imagine nitrogen as the roof
o The nitrogen is located at the center of the pyrrole
o Each pyrrole is linked together by methyl ridges
o It has two distinctive side chain

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 Middle part 2nd step:
o Modification of side chains

 D-ALA will be pumped out into the cytoplasm

 2 ALA are needed and they will be condensed together to form
your porphobilinogen (PBG)
 Last step
o Occurs in the mitochondria
3rd step:
o Modification of the methyl bridges

 4 PBG are needed to form your tetrapyrrole

 4 PBG will be link together in a head to tail like manner with the
1st step: help of uropophyrinogen I synthase
 However PBG #4 will not be linked to the head of PBG #1. This is
the reason why it is called a linear tetrapyrrole referred to as your
hydroxymethylbilane
 There are two possibilities:
o Non-enzymatic closure to form a true porphyrin ring
o Enzymatic closure catalyzed by uroporphyrinogen III
cosynthase to form your first TRUE CLOSED TETRAPYRROLE
which is uroporphyrinogen III

4th step:
 Occurs in the mitochondria
 2 components: Succinyl CoA and Glycine

 Succinyl CoA condenses with Glycine which is catalyzed by the

enzyme delta aminolenulinic acid synthase (ALA Synthase)
 This will now form an intermediate substance called alpha amino
beta ketoadipate. This substance is not stable
 Its carboxyl group will be continuously cleaved off in the form of
carbon dioxide (decarboxylation process) leaving now your delta
 Involves addition of side chains
amino levulinic acid

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 What are the side chains of uropophyrinogen? 7th step:

 The side chain that will be first modified are the acetate
o They will be decarboxylated catalyzed by uroporphyrinogen
decarboxylase  There will be an insertion of iron at the center of the molecule
o The carboxyl group will be removed leaving now own (catalyzed by ferrochelatase) forming now your heme
carbon group which is now your methyl group 
coproporphyrinogen III
THIS IS NOW YOUR HEME
5th step:

 Modification of the first 2 proprionate side chains (not all 4 will be

modified)
 The 2 proprionate will be carboxylated liberated in the form of
carbon dioxide SUMMARY:
 At the same time, there will be oxidation there will be loss of
electrons double bond will be formed
 Enzyme will be Coproporphyrinogen II Oxidase
 Proprionate side chains will now forming vinyl Side Chain
 Protoporphyrinogen IX

6th step:

3 points of control
 There will be modification of the methyl ridges
 1st step – controlled in the same manner as lac operon
 The central part will be oxidized (catalyzed by
o Excess amount of heme will combine with aporepressor and
protoporphyrinogen IX oxidase – according to cleofe trans) so that
will go to the gene that transcribes ALAS and will stop its
there will be introduction of double bonds forming now your
transcription
protoporphyrin IX
 Formation of hydroxymethylbilane
 Last step

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DISORDER OF HEME METABOLISM o Variegate Porphyria
 Protoporphyrinogen Oxidase is deficient
Porphyria  You cannot form Protoporphyrin IX
 Extremely rare genetic disorder that is transmitted in an  Accumulation of ALA, PBG and Uroporphyrinogen III
autosomal recessive manner  Neuropsychiatric symptoms, abdominal pain,
 Results in the absence of enzymes in the heme biosynthesis photosensitivity

2 Clinical Forms: Hematopoetic

Hepatic

 The liver enzymes are the one usually affected

 Bone marrow enzymes are affected
 Usual symptoms: abdominal pain, neuropsychiatric symptoms,
 Major symptoms: abdominal pain, neuropsychiatric symptoms,
photosensitivity
photosensitivity, anemia
o These symptoms depend on when the defect is
 Types:
o There is not anemia because liver is not involves in
o X-Linked Siderolastic Anemia
hemoglobin production
 ALA Synthase is missing
 Types:
 You cannot form all especially heme
o ALA Dehydrogenase Deficiency
 ANEMI is the only symptom here
 Deficiency of ALA dehydratase leading to
 No neuropsychiatric symptoms because ALA is not yet
accumulation of ALA which are neurotoxic
formed
 Leads to neuropsychiatric symptoms and abdominal
o Congenital Erythropoietic Porphyria
pain
 Uroporphyrinogen III synthase is missing
o Acute Intermittent Porphyria
 You cannot form Uroporphyrinogen III
 Uroporphyrinogen I Synthase is missing
 Accumulation of ALA (Toxic) and PBG
 Uroporphyrinogen III cannot be formed leading to
 Anemia, neuropsychiatric symptoms, abdominal pain
accumulation of ALA (Toxic) and PBG
 Neuropsychiatric symptoms and abdominal pain is o Protoporphyria
present  Typical
 Ferrochelatase is missing
o Porphyria Cutanea Tarda
 You cannot form heme
 Most common form of hepatic porphyria
 Accumulation of ALA, PBG and Uroporphyrinogen III
 Uroporphyrinogen Decarboxylase is missing
 Anemia, neuropsychiatric symptoms, abdominal pain,
 You cannot form Coproporphyrinogen III
photosensitivity
 Accumulation of ALA, PBG and Uroporphyrinogen III
 The growth of teeth is affected also because there is
 Neuropsychiatric symptoms, abdominal pain ,
deposition of porphyrin in the enamel of teeth
photosensitivity
 Presence of photosensitivity is due to the
HEME CATABOLISM
accumulation of UBG which is a closed
 The life span of RBC is 120 days. The old RBCs will be engulfed by
tetrapyrrole that is highly reactive when
the reticuloendothelial system
exposed to UV light
 What happens to heme?
 There will be a 2nd degree burn and are
o Iron is conserved
susceptible to infection
o Amino acid is conserved
o Hereditary Coproporphyria o Porphyrin is catabolized
 Difficult to identify with Porphyria Cutanea Tarda
 Deficiency in Coproporphyrinogen Oxidase Heme catabolism is summarized in this photo:
 You cannot form Protoporphyrinogen IX
 Accumulation of ALA, PBG and
Uroporphyrinogen III
 Neuropsychiatric symptoms, abdominal pain,
photosensitivity

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 RBC is engulfed by the phagocytes and breaks it down to its
components:
o Globin – the amino acids in the globin will be recycled
o Iron – recycled
o Heme – catabolized to bilirubin which will be conjugated in
the liver and eventually secreted in the bile

Phase of Heme Catabolism:

 Heme catabolism has 3 steps. 2 of those occurs in the spleen
while the last one occurs in the liver

Phase 1: occurs in the spleen

 Step 1
o Heme will enter the heme oxygenase system where it will
be catabolized
o Iron will be released and conserved
o The methylene bridge between pyrrole 1 and 2 will undergo
oxidation thus forming your biliverdin
 Step 2
o The side chain between the 3rd and 4th pyrrole of the
biliverdin will be reduced thus removing your double bonds
o Removal of the double bond cause the formation of your
linear tetrapyrrole
o Bilverdin is now reduced to bilirubin
 Step 3
o Bilirubin is transported to the liver through binding with
albumin
o The proprionate side chain will serve as the attachment site
for gluronic acid to yield bilirubin diglucorinide
 Source of glucorinic acid: UDP-glucoronic acid
catalyzed by UDP-glucoronyl transferase
o The bilirubin diglucorinide is a polar substance thus it can
now be excreted in the bile

BILIRUBIN CATABOLISM
 READ ON CONFERENCE TOPIC REGARDING JAUNDICE. Hindi nadin
kasi diniscuss ng ayos ni Doc Bravo ito

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