Heme 05: Heme Synthesis And Hemoglobin

Front 1

What are the functions of heme?

Back 1

- Transport of O2 (on hemoglobin and myoglobin)
- Electron transport (respiratory cytochromes)
- Oxidation-reduction reactions (cytochrome P450 enzymes)

Front 2

What are the sites of heme synthesis? What forms of heme are made in these locations?

Back 2

- Bone Marrow - hemoglobin
- Liver - cytochrome P450 enzymes
- Virtually all cells (except mature RBCs) - other important cellular proteins

Front 3

Where is hemoglobin synthesized? How much?

Back 3

- Bone marrow
- 6-7g synthesized per day to replace heme lost through normal turnover of RBCs

Front 4

Where are cytochrome P450 enzymes synthesized? Function?

Back 4

- Liver
- Drug detoxification

Front 5

Why can't hemoglobin be synthesized in mature erythrocytes?

Back 5

They lack mitochondria and other organelles

Front 6

What kind of molecule is Porphyrin? Use?

Back 6

- Cyclic, predominantly planar tetrapyrroles
- Capable of chelating to various metals
- Form prosthetic groups for various biological molecules

Front 7

What are the components of heme?

Back 7

- Porphyrin derivative: Protoporphyrin IX
- Single ferrous iron (Fe2+ = reduced form)

Front 8

What porphyrin is found in heme? What is the name for this form when combined with ferrous (Fe2+) iron?

Back 8

Protoporphyrin IX:
- Specific isomer of porphyrin that contains specific substituent groups on the four pyrrole rings
- Substituent groups provide important sites for binding to its apoproteins

Forms Ferroprotoporphyrin IX

Front 9

What happens if there is auto-oxidation of Ferroprotoporphyrin IX (heme)?

Back 9

Forms Ferriprotoporphyrin IX (with an "i")
- Called "Hemin"
- Contains ferric Fe3+ iron

Front 10

How many stages of heme biosynthesis are there? Where do these steps take place?

Back 10

7 major steps:
- 1st and last 3 (5, 6, and 7) take place in mitochondria
- 2, 3, and 4 take place in cytosol

Front 11

What are the molecules in the 7 steps to synthesize Heme?

Back 11

1. Succinyl CoA + Glycine
2. ALA: 5-Aminolevulinate
3a. PBG: Porphobilinogen
3b. Hydroxymethylbilane
4. Uroporphyrinogen III Isomer
5. Protoporphyrinogen IX
6. Protoporphyrin IX
7. Heme / Ferroprotoporphyrin IX

Front 12

What are the enzymes in the 7 steps to synthesize Heme?

Back 12

1. ALAS: 5-Aminolevulinate Synthase
2. ALAD: ALA Dehydratase
3a. PBGD: Porphobilinogen Deaminase
3b. UROS: Uroporphyrinogen III Cosynthase
4. UROD: Uroporphyrinogen Decarboxylase
5. CPO: Coproporphyrinogen III Oxidase
6. PPO: Protoporphyrinogen IX Oxidase
7. Ferrocheletase

Front 13

What happens in the first step of heme synthesis?

Back 13

- Substrate: Succinyl-CoA + Glycine
- Enzyme: ALAS - 5-aminolevulinate synthase
- Cofactors: PLP
- Product: ALA - 5-aminolevulinate (+ CO2 + CoA)

Front 14

What happens in the second step of heme synthesis, after formation of ALA?

Back 14

- Substrate: 2 x ALA - 5-aminolevulinate
- Enzyme: ALAD - 5-aminolevulinate dehydratase
- Cofactors: Zn2+
- Product: PBG - Porphobilinogen

Front 15

What happens in part one of the third step of heme synthesis, after formation of PBG?

Back 15

- Substrate: 4 x PBG - Porphobilinogen
- Enzyme: PBGD - Porphobilinogen Deaminase
- Cofactors:
- Product: Hydroxymethylbilane (+ NH3 x4)

Front 16

What happens in part two of the third step of heme synthesis, after formation of Hydroxymethylbilane?

Back 16

- Substrate: Hydroxymethylbilane
- Enzyme: UROS - Uroporphyrinogen III Cosynthase
- Cofactors: -
- Product: Uroporphyrinogen III Isomer

Front 17

What happens in the fourth step of heme synthesis, after formation of Uroporphyrinogen III Isomer?

Back 17

- Substrate: Uroporphyrinogen III Isomer
- Enzyme: UROD - Uroporphyrinogen Decarboxylase
- Cofactors: -
- Product: Coprophorphyrinogen III (+ CO2 x4)

Front 18

What happens in the fifth step of heme synthesis, after formation of Coprophorphyrinogen III?

Back 18

- Substrate: Coprophorphyrinogen III
- Enzyme: CPO - Coprophorphyrinogen III Oxidase
- Cofactors: -
- Product: Protoporphyrinogen IX

Front 19

What happens in the sixth step of heme synthesis, after formation of Protoporphyrinogen IX?

Back 19

- Substrate: Protoporphyrinogen IX
- Enzyme: PPO - Protoporphyrinogen IX Oxidase
- Cofactors: -
- Product: Protoporphyrin IX

Front 20

What happens in the seventh step of heme synthesis, after formation of Protoporphyrin IX?

Back 20

- Substrate: Protoporphyrin IX + Fe2+
- Enzyme: Ferrocheletase
- Cofactors: -
- Product: Heme

Front 21

Where do the carbon and nitrogen atoms of the porphyrin ring in heme originate?

Back 21

All C and N are from Succinyl-CoA and Glycine

Front 22

What is the enzyme in the first step of heme synthesis? Location?

Back 22

ALAS: 5-Aminolevulinate synthase
- Found in the inner mitochondrial membrane, but encoded by a nuclear gene family
- Therefore, nascent protein must be imported into mitochondrion

Front 23

What does the enzyme 5-Aminolevulinate synthase (ALAS) require? What step? Mechanism?

Back 23

- Step 1
- Pyridoxal Phosphate (PLP) dependent enzyme
- Condensation of glycine w/ succinyl-CoA takes place while amino group of glycine is in Schiff base linkage to PLP aldehyde; CoA and glycine carboxyl are lost during condensation

Front 24

What are the two forms of 5-Aminolevulinate synthase (ALAS)?

Back 24

- ALAS1 - liver isoform
- ALAS2 - erythroid / reticulocyte isoform

Front 25

How is feedback of the two ALAS isoforms different?

Back 25

- ALAS1 (liver) has feedback inhibitino by heme or hemin

- ALAS2 (RBCs) is not regulated by feedback repression

Front 26

How is ALAS1 regulated in the liver?

Back 26

- Feedback inhibition by heme or hemin regulates heme biosynthesis in liver (ALAS1)
- Heme inhibits ALAS1 synthesis at both transcriptional and translational level and as its mitochondrial import

- ~100 drugs or metabolites can stimulate ALAS1
- Many drugs are metabolized by cytochrome P450s in liver and thus increase synthesis of cytochrome P450 enzymes (increasing demand for heme)

Front 27

How is ALAS2 regulated in the RBCs?

Back 27

- Heme biosynthesis is not regulated by feedback repression of ALAS2
- Heme stimulates synthesis of globin and ensures that heme and globin are synthesized in correct ratio for assembly of hemoglobin

- Drugs that cause a marked elevation of ALAS1 (eg, phenobarbital) do not affect ALAS2

Front 28

What is the first pathway intermediate to include a pyrrole ring? When is it synthesized?

Back 28

- Porphobilinogen (PBG)
- Synthesized in step 2 in cytosol by ALA dehydratase (ALAD)

Front 29

What is required for ALA dehydratase (ALAD) activity in step 2 of heme synthesis?

Back 29

Zn2+ complexed to an active site cysteine

Front 30

What step of heme synthesis is affected by lead poisoning? How does it affect this step?

Back 30

- Step 2: ALA Dehydratase (ALAD)
- ALAD requires a Zn2+ in the active site, lead and other heavy metals can displace Zn2+ and eliminate the catalytic activity

Front 31

What are the implications of lead poisoning?

Back 31

- Increases ALA in urine (the substrate for step 2 of heme synthesis)
- Increases ALA in blood (causes neurological side effects)
- Lead may also directly affect the nervous system
- Clinical manifestations mimic acute porphyrias

Front 32

What can cause a buildup of ALA levels in the blood? What are the implications?

Back 32

- Caused by lead poisoning (which eliminates catalytic activity of ALA dehydratase, ALAD)
- Toxic to brain perhaps because ALA has a similar structure as GABA; also ALA autoxidation generates reactive oxygen species (ROS)

Front 33

What happens structurally in the third step of heme synthesis?

Back 33

Porphobilinogen Deaminase (PBGD)
- Head-to-tail condensation of four porphobilinogen molecules to form a linear tetrapyrrole (liberates 4 ammonium ions)

Uroporphyrinogen III Cosynthase (UROS)
- Directs the stereochemistry of the condensation reaction to yield Uroporphyrinogen III isomer

Front 34

What happens structurally in the fourth step of heme synthesis?

Back 34

Decarboxylation of acetate side chains to methyl groups (by UROD)

Front 35

What happens structurally in the fifth step of heme synthesis?

Back 35

- Substrate (Coproporphyrinogen III) transported into intermembrane space
- Oxidase (CPO) converts specific propionic acid side chains to vinyl groups
- Forms Protoporphyrinogen IX

Front 36

What happens structurally in the sixth step of heme synthesis?

Back 36

Another mitochondrial oxidase (PPO) moves double bonds in the structure to form protoporphyrin IX

Front 37

What happens structurally in the seventh step of heme synthesis?

Back 37

Insertion of Fe2+ into Protoporphyrin IX to generate heme by Ferrochelatase

Front 38

What inhibits Ferrocheletase (the last step of heme synthesis)?

Back 38

- Lead poisoning
- Iron deficiency (anemia) - not enough Fe2+ to insert into Protoporphyrin IX

Front 39

What happens if there is an absence of Fe2+?

Back 39

Ferrocheletase can insert Zn2+ into the protoporphyrin ring to yield a brilliantly fluorescent complex

Front 40

What are porphyrias?

Back 40

Defects in heme biosynthesis

Front 41

What causes Porphyrias?

Back 41

- Inherited / genetic disorders
- Acquired (rarely) disorders

- Result from deficiency in specific enzymes of the porphyrin/heme biosynthetic pathway

Front 42

How do you classify porphyrias?

Back 42

Based on the principal sites of heme biosynthesis and depending on the site of expression of the enzyme defect:
- Hepatic
- Erythroid

Front 43

How are Porphyrias inherited?

Back 43

* Autosomal dominant
- Exception: congenital erythropoietic porphyria which is autosomal recessive

Front 44

What is the most common porphyria?

Back 44

Acute Intermittent Porphyria

Front 45

What is the most common erythropoietic porphyria?

Back 45

Erythropoietic Protoporphyria (EPP)
(also most common childhood porphyria)

Front 46

What is the most common porphyria in childhood?

Back 46

Erythropoietic Protoporphyria (EPP)
(also most common erythropoietic porphyria)

Front 47

What type of pophyria is extremely rare?

Back 47

Congenital Erythropoietic Porphyria (CEP)

Front 48

What causes the clinical symptoms in porphyrias?

Back 48

- Accumulation of intermediates upstream from the enzyme defect (measure in urine, blood, feces)

- Defects early in pathway (accumulation of ALA, prophobilinogen) result in neurologic dysfunction

- Defects later in pathway (accumulation of cyclic tetrapyrroles, but not prophobilinogen) result in sunlight-induced cutaneous lesions; in presence of molecular O2, UV irradiation of cyclic tetrapyrroles generates ROS that can cause cellular damage

Front 49

What happens if there are defects early in the biosynthetic pathway for Heme?

Back 49

Porphyria:
- Accumulation of ALA and prophobilinogen
- Leads to neurological dysfunction

Front 50

What happens if there are defects later in the biosynthetic pathway for Heme?

Back 50

Porphyria:
- Accumulation of cyclic tetrapyrroles
- Sunlight-induced cutaneous lesions
- In presence of molecular O2, UV irradiation of cyclic tetrapyrroles generates ROS that can produce cellular damage

Front 51

What are the two ways that Porphyrias can present?

Back 51

Acute:
- Periodic acute attacks
- Sx include abdominal pain, neurologic deficits, psychiatric symptoms, and reddish-colored urine

Chronic:
- Dermatologic diseases
- May or may not include liver and nervous system

Front 52

What are some triggers that can bring about acute attacks of Porphyria?

Back 52

- Nutritional changes (eg, hypoglycemia)
- Smoking
- Certain drugs (barbiturates and sulfonamide antibiotics)
- Steroid hormones, especially progesterone (some women develop attacks during second half of menstrual cycle when progesterone is high)

Front 53

What is true about Ferrochelatase?
a) found in cytosol
b) rate-limiting enzyme in heme synthesis
c) requires glycine for activity
d) activity stimulated in presence of lead
e) catalyzes last step of heme synthesis

Back 53

Catalyzes last step in heme synthesis
- Found in mitochondria
- First step is rate-limiting
- Does not require glycine for activity
- Activity inhibited by lead and iron deficiency

Front 54

You are treating a patient with Porphyria Cutanea Tarda, most common porphyria. She came to your office w/ significant blistering on her hands following a day of gardening. Accumulation of what would cause photosensitivity?

Back 54

Accumulation of cyclic tetrapyrroles:
4. Uroporphyrinogen III Isomer
5. Protoporphyrinogen IX
6. Protoporphyrin IX

Front 55

What is the function of Hemoglobin?

Back 55

- Transport O2 from lungs (high O2 concentration) to peripheral tissues where O2 tension is low

- Transports some CO2 and H+ that are generated in peripheral tissues back to lungs (14% of CO2 made is carried on Hb)

Front 56

Why is it necessary for O2 to be carried on Hemoglobin? Consequences?

Back 56

- O2 has a very low solubility in plasma (non-cellular part of blood)
- As a consequence, >98% of O2 that reaches the tissues is carried bound to hemoglobin in RBCs

Front 57

Why does CO2 not have to be carried on Hemoglobin?

Back 57

- RBCs carry Carbonic Anhydrase which catalyzes the rapid reversible hydration of CO2 to H2CO3 which then dissociates to HCO3- and H+
- CO2 and HCO3- are soluble in plasma and RBC cytosol
- Most of the CO2 made in tissues returns to lung as those species, but 14% bound to Hb

Front 58

What are the components of Hemoglobin?

Back 58

Heterotetrameric protein: α2β2
- Contains 4 heme prosthetic groups responsible for binding O2

Front 59

How does hemoglobin relate to myoglobin?

Back 59

- Subunits of hemoglobin are evolutionarily related to myoglobin (monomeric protein abundant in muscle that is designed to store O2)
- Both proteins contain a heme prosthetic group (1/myoglobin and 4/hemoglobin)
- Fe2+ is ferrous form of iron responsible for binding O2
- Hemoglobin has a sigmoidal (cooperative) binding curve, whereas Myoglobin has a hyperoblic binding curve to O2

Front 60

How is hemoglobin affected by different forms of iron?

Back 60

- Fe2+ (Ferrous form) - capable of binding O2
- Fe3+ (Ferric form) - cannot bind O2, inactive form called Methemoglobin (metHb)

Front 61

What kind of binding curve do Myoglobin and Hemoglobin have for O2? How do you explain the different binding curve for Hb?

Back 61

- Myoglobin: normal, hyperbolic binding curve

- Hemoglobin: sigmoidal, cooperative binding curve
- D/t its more complex subunit structure, critical for its efficiency in loading O2 in lungs and unloading O2 in peripheral tissues

Front 62

What are the benefits of the cooperativity of O2 binding to hemoglobin?

Back 62

Allows Hb to release a much larger fraction of its own O2 load at the pO2 levels found in the blood of working and even resting muscle

Front 63

How do the four subunits of hemoglobin lead to a cooperative/sigmoidal O2 binding relationship/

Back 63

- Binding of O2 to one subunit induces a conformational change that is partially transmitted to adjacent subunits
- Transmission of partial conformational change induces an increased affinity for O2 by these adjacent subunits

Front 64

How does Carbon Monoxide compare to O2 binding to Hemoglobin?

Back 64

- CO has ~250-fold higher affinity for Hb than does O2
- When bound to the heme group of one subunit, it causes all four subunits to lock in the R conformation thereby limiting O2 release in peripheral tissues (needs to be in T state to release)

Front 65

What is the T state? What does it prefer? Same for R state?

Back 65

- T state = tense, favors dissociation (lower affinity for O2)
- R state = relaxed, favors association (higher affinity for O2)

Front 66

How does O2 binding change the conformation of a Hb subunit?

Back 66

- Without O2 bound, the heme Fe2+ is pulled away from the plane of the porphyrin ring by a His residue of the Hb polypeptide chain (a His ring N is bound to the Fe2+)
- When O2 binds, it pulls the Fe2+ back into the plane of the ring and that moves the His residue and its whole section of the polypeptide chain
- That in turn causes the Hb subunits to shift relative to one another to an arrangement that favors the R-conformation

Front 67

What is an allosteric regulator?

Back 67

Molecule that can bind to a protein and induce a conformational change that alters the affinity for substrate (or ligand such as O2) at some other site (allo means other)

Front 68

What are the allosteric regulators of O2 binding to Hb? Effects?

Back 68

All bind to Hb and reduce its affinity for O2:
- Protons (H+)
- CO2
- 2,3-Diphosphoglycerate (DPG)

Front 69

What are the effects of high amounts of H+, CO2, or 2,3-DPG?

Back 69

When these are present, the curve shifts to the right and O2 comes off of Hb

Front 70

What are the normal values for:
- pH?
- pO2?
- pCO2?
- HCO3-?

Back 70

- pH: 7.35-7.45
- pO2: 80-100 mmHg
- pCO2: 35-45 mmHg
- HCO3-: 22-26 mM

Front 71

What kind of effectors are H+ and CO2 on Hb binding of O2?

Back 71

Heterotropic Negative Allosteric Effectors that decrease the affinity of Hb for O2
- Heterotropic: they are not O2
- Negative: decrease affinity for O2
- Allosteric: bind to a site other than O2 site

Front 72

What kind of effector is O2 on Hb binding of O2?

Back 72

Homotropic Positive Allosteric Effector
- Homotropic: it is O2
- Positive: increases affinity for more O2
- Allosteric: binds to a site other than the site the next O2 could bind

Front 73

What kind of effector is O2 on Hb binding of H+ and CO2?

Back 73

Heterotropic Negative Allosteric Effector
- Heterotropic: not CO2 or H+
- Negative: decreases affinity for CO2 and H+
- Allosteric: binds to a site different than they would bind

Front 74

What is the term for the reciprocal relationship between O2 and H+ binding to hemoglobin?

Back 74

Bohr effect or isohydric shift
- Changes in H+ binding result from a shift in the pKa of specific residues (mostly histidines) d/t microenvironment effects triggered by conformational changes in Hb structure

Front 75

What is the mechanism of how increased H+ (↓pH) affects O2 binding?

Back 75

- In deoxyHb negative charge on Asp94 is near His146
- It is energetically favorable for N to be protonated (its pKa is higher)
- Having His protonated in turn makes it favorable for Asp to stay near it, increasing the stability of the deoxyHb (T-state)
- Conformation changes in going to oxyHb (R state) move His146 and Asp94 apart, pKa drops and H+ comes off

Front 76

What kind of effector is DPG on Hb binding of O2? Mechanism?

Back 76

Heterotropic Negative Allosteric Effector
- Binds to a specific site in central cavity between β subunits by ionic interactions
- This binding stabilizes the T state of deoxyHb

Front 77

What effects does DPG have on the O2 binding curves?

Back 77

1. Without any DBG, Hb would be more like myoglobin (hyperbolic) and nearly useless for delivering O2 from lungs to tissues

2. DPG levels increase at high altitudes
- There is less O2 at high altitudes, so tissues tend to become somewhat hypoxic
- Increasing DPG lets RBC adapt to hypoxia by making it easier for O2 to dissociate from Hb

Front 78

What situations/conditions stimulate DPG release?

Back 78

Causes of tissue hypoxia:
- High altitude
- Smoking
- Anemia

Front 79

How long does it take to make changes in DPG? Implications?

Back 79

- A few days
- Therefore takes a few days to adapt to high altitude
- Until then, strenuous aerobic exercise is difficult (more dyspnea)

Front 80

How does temperature affect O2 association/dissociation?

Back 80

↑T → ↓O2 affinity
- During fever there are elevated metabolic rates so need increasing unloading of O2

Front 81

What are the developmental forms of hemoglobin?

Back 81

- ~2% HbF - Fetal: α2γ2 (until ~5-6 months)
- ~95% HbA1 - Adult 1: α2β2 (majority)
- ~3% HbA2 - Adult 2: α2δ2 (very low)

Front 82

Which chromosome contains the α-globin gene(s)?

Back 82

Chromosome 16 has two α-globin genes (total of 4 / person)

Front 83

Which chromosome contains the β-globin gene(s)?

Back 83

Chromosome 11 has a single β-globin gene (total of 2 / person)

Front 84

Where is hemoglobin synthesized during the lifetime?

Back 84

- Yolk Sac
- Liver
- Spleen
- Bone marrow

Front 85

How do the different forms of hemoglobin (α2β2, α2δ2, and α2γ2) differ?

Back 85

Binding affinity for O2
- Higher affinity of fetal Hb means fetus' circulation can draw O2 from maternal blood at pO2 present in placenta

Front 86

How is Sickle Cell Anemia inherited? Cause?

Back 86

Homozygous recessive disease
- Point mutation in adult β-globin gene that causes substitution of valine for glutamic acid at amino acid 6
- Patients mainly contain HbS

Front 87

What is the most common hemoglobinopathy?

Back 87

Sickle Cell Anemia

Front 88

What are the effects of the mutation in Sickle Cell Anemia?

Back 88

- Valine substituted for glutamic acid at position 6 is on the surface of the β-chain and it should be hydrophilic
- Valine is hydrophobic and its presence creates a sticky patch on deoxyHb that leads to polymerization of Hb tetramers into long chains
- Val6 makes critical contact with hydrophobic acceptor pocket of β-subunit of another molecule formed by Leu88 and Phe85
- Intracellular fibers cause sickle cell shape and reduced deformability of RBCs that leads to problems w/ their passage through microcirculation

Front 89

What determines the amount/rate of polymerization of HbS?

Back 89

- Degree of deoxygenation (can be affected by pH, ionic strength, and temperature); deoxyHbS forms insoluble polymers
- Intracellular Hb concentration
- Relative amount of HbF present (HbF inhibits polymerization d/t Glu87 on γ-chain

Front 90

What occurs in a Thalassemia?

Back 90

Inherited mutations cause a decreased synthesis of adult hemoglobin (α2β2)

Front 91

What kind of mutations cause β-thalassemias?

Back 91

- β° mutations have absent β-globin chain synthesis
- β⁺ mutations have reduced (but detectable) β-globin chain synthesis

Front 92

What are the implications of β-thalassemias?

Back 92

- Deficit in HbA
- Unpaired α-chains precipitate in RBC precursors, resulting in apoptosis

Front 93

What are the types of β-thalassemias? Differences?

Back 93

- β-thalassemia major: two β-thalassemia alleles w/ severe, transfusion-dependent anemia

- β-thalassemia minor: heterozygotes have only one β-thalassemia allele and mild or asymptomatic microcytic anemias

Front 94

What causes α-thalassemias?

Back 94

Mutations that result in reduced or absent synthesis of α-globin chains

Front 95

What are the implications of α-thalassemias?

Back 95

Unpaired β-chains are more soluble than unpaired α-chains, thus effects less severe than in β-thalassemias

Front 96

What is true of Oxygen:
a) binding converts Hb to T state
b) dissociation from Hb is inhibited in patients w/ fever
c) dissociation from Hb is enhanced at pH values below pH 7.4
d) binding to Hb causes conversion of a β strand to a random coil
e) dissociation from Hb is inhibited in presence of CO2

Back 96

Dissociation from Hb is enhanced at pH values below 7.4

Front 97

What is true about Methemoglobin?
a) similar O2 association/dissociation curve as myoglobin
b) similar O2 association/dissociation curve as hemoglobin
c) monomer
d) cannot bind hemoglobin
e) contains ferric iron

Back 97

Cannot bind hemoglobin (it contains ferric / Fe3+ form)