What are the functions of heme?
- Transport of oxygen (hemoglobin, myoglobin)
- Electron transport (respiratory cytochromes)
- Oxidation-reduction reactions (cytochrome P450 enzymes)
- Where are the major sites of heme synthesis?
- Where else is heme synthesized?
- What cannot synthesize heme?
- Major Sites:
- bone marrow ⇒ hemoglobin (6-7g hemoglobin are synthesized each day)
- liver ⇒ cytochrome P450 enzymes (drug detoxificaton)
- However, heme is also required for other important cellular proteins and is synthesized in virtually all cells,
- mature erythrocytes do not synthesize heme (lack mitochondria)
- What are porphyrins?
- What is the structure of heme?
- Porphyrins: cyclic tetrapyrroles capable of chelating to various metals to form essential prosthetic groups for various biological molecules
- Heme is predominantly a planar molecule
- porphyrin derrivative + a single ferrous ion (Fe2+ = reduced form of iron)
Heme = ?
- What is heme oxidized to?
Heme = Ferroprotoporphyrin IX
- Ferroprotoporphyrin IX (heme) is rapidly autooxidized to ferriprotoporphyrin IX (“hemin”; contains ferric Fe3+ iron)
7 major steps of heme biosynthesis:
- The 1st and last 3 steps occur in the ….
- The intermediate steps occur in the ….
- The 1st and last 3 steps occur in the mitochondrion
- The intermediate steps occur in the cytosol
What is the committed step of heme synthesis?
Step 1: condensation of glycine and succinyl-CoA with decarboxylation, to yield 5-aminolevulinate (ALA)
**Step 1 **of heme synthesis:
- **Reaction: **
- **Enzyme: **
- **Location: **
- **Cofactor: **
- Reaction: condensation of glycine and succinyl-CoA with decarboxylation, to yield 5-aminolevulinate (ALA)
- Enzyme: 5-aminolevulinate synthase (ALAS)
-
Location: ALAS is localized to the inner mitochondrial membrane
- encoded by a nuclear gene family
- must be imported into the mitochondrion
-
Cofactor: pyridoxal phosphate (PLP) dependent enzyme (vitamin B6)
- Condensation with succinyl-CoA takes place while the amino group of glycine is in Schiff base linkage to the PLP aldehyde
What are the **isoforms **of ALAS?
Two isoforms of ALAS:
- ALAS1 is the liver isoform
- ALAS2 is the erythroid/reticulocyte isoform
Describe the regulation of ALAS1:
- Feedback inhibition by heme or hemin regulates heme biosynthesis in the liver
- Heme (hemin) exerts multiple regulatory effects on hepatic heme biosynthesis by inhibiting ALAS1 synthesis at both transcriptional and translational levels, as well as its mitochondrial import
- drugs or metabolites can increase ALAS1 activity
- **increase the synthesis of cytochrome P450 enzymes **⇒ increasing the demand for heme
Describe the regulation of ALAS2:
- Heme biosynthesis in erythroid cells is NOT regulated by feedback repression of ALAS2 by heme
- In reticulocytes (immature RBCs), **heme stimulates synthesis of globin **and ensures that heme & globin are synthesized in the correct ratio for assembly into hemoglobin
- Drugs that cause a marked elevation in ALAS1 activity, such as phenobarbital, do not affect ALAS2
Step 2 of heme biosynthesis:
- Reaction:
- **Enzyme: **
- **Cofactor: **
- **Location: **
- Complication:
-
Reaction: condensation of two molecules of ALA to form one molecule of porphobilinogen (PBG)
- first pathway intermediate that includes a pyrrole ring
- Enzyme: ALA dehydratase (ALAD)
-
Cofactor: Zn2+
- lead and other heavy metals can displace the Zn2+ and eliminate catalytic activity
- Location: cytosol
-
Complication: lead poisoning
- increase ALA in urine
- clinical manifestations that mimic acute porphyrias
Effects of **lead poisoning: **
- Heme synthesis:
- Neurologic symptoms:
-
Inhibition of ALA dehydratase (aka, porphobilinogen synthase) by lead (Pb2+) results in elevated blood ALA
- as impaired heme synthesis leads to de-repression of transcription of the ALAS gene
-
ALA is toxic to the brain, perhaps due to:
- Similar ALA & neurotransmitter GABA (γ-aminobutyric acid) structures
- ALA autoxidation generates reactive oxygen species (ROS)
Step 3 of heme synthesis:
- Reaction:
- Enzyme:
- Coenzyme:
- **Location: **
- **Reaction: **
-
Step 1: head-to-tail condensation of 4 porphobilinogen molecules to form hydroxymethylbilane (linear tetrapyrrole)
- Each condensation ⇒ liberation of one ammonium ion
- Step 2: hydroxymethylbilane ⇒ uroporphyrinogen III
-
Step 1: head-to-tail condensation of 4 porphobilinogen molecules to form hydroxymethylbilane (linear tetrapyrrole)
- Enzyme: porphobilinogen deaminase (PBGD) or uroporphyrinogen I synthase
- Coenzyme: uroporphyrinogen III cosynthase
- Location: cytosol
What is the role of uroporphrinogen III cosynthase?
- The tetrapyrrole can spontaneously cyclize to form uroporphyrinogen I (nonenzymatic) which IS NOT in the normal pathway for heme biosynthesis
- However, PBGD is tightly associated with a second enzyme uroporphyrinogen III cosynthase (UROS)
- no enzymatic activity alone
- serves to direct the stereochemistry of the condensation reaction to yield the uroporphyrin ogen III isomer which IS on the pathway for heme biosynthesis
**Step 4 **of heme synthesis:
- Reaction:
- **Enzyme: **
- Location:
-
Reaction: uroporphyrinogen III ⇒ coprophorphyrinogen III
- decarboxylation of acetate side chains to methyl groups
- Enzyme: Uroporphyrinogen decarboxylase (UROD)
- Location: cytosol
Step 5 of heme synthesis:
- Reaction:
-
Enzyme:
- What is being converted?
-
Location:
- What does this imply?
-
Reaction: Coproporphyrinogen III ⇒ protoporphyrinogen IX
- transported into the intermembrane space
-
Enzyme: coproporphyrinogen III oxidase (CPO)
- converts specific propionic acid side chains to vinyl groups
-
Location: intermembrane space of the mitochondrion
- implying that its product or protoporphyrin IX must cross the inner mitochondrial membrane because heme is formed within the inner membrane
Step 6 of heme synthesis:
- **Reaction: **
- **Enzyme: **
- **Location: **
- **Reaction: **protoporphyrinogen IX ⇒ protoporphyrin IX (moving double bonds)
- Enzyme: protoporphyrinogen IX oxidase (PPO)
- Location: mitochondrion
Step 7 of heme synthesis:
- **Reaction: **
- **Enzyme: **
- **Location: **
- Reaction: Insertion of Fe2+ into protoporphyrin IX to generate HEME
- Enzyme: ferrochelatase
- Location: mitochondrion
- What can inhibit Step 7 of heme synthesis?
- What will result in a brillant flourescent complex?
- Ferrochelatase is inhibited by lead (lead poisoning; increase protoporphyrin in urine) and is also inhibited during iron deficiency (anemia)
-
In the absence of Fe2+:
- ferrochetalase can insert Zn2+ into the protoporphyrin ring to yield a brilliantly fluorescent complex
What are porphyrias?
inherited genetic or acquired (rarely) disorders resulting from deficiency in specific enzymes of the porphyrin/heme biosynthetic pathway
- How are porphyrias classified?
- What is the pattern of inheritance?
Either hepatic or erythroid
- reflect the principal sites of heme biosynthesis
- depend on the site of expression of the enzyme defect
- Inheritance: autosomal dominant
- Except congenital erythropoietic porphyria (autosomal recessive)
- What causes symptoms seen in porphyrias?
- What is the difference in location of the defect (early vs. late)?
- Accumulation of intermediates upstream from the enzyme defect **results in the clinical symptoms **associated with the various porphyrias
- Defects early in the biosynthetic pathway (accumulation of ALA, prophobilinogen) result in neurologic dysfunction
-
Defects later in the pathway (accumulation of cyclic tetrapyrroles, but not prophobilinogen) result in sunlight-induced cutaneous lesions:
- in the presence of molecular oxygen, UV irradiation of cyclic tetrapyrroles generates reactive oxygen species that can produce cellular damage
What are the acute porphyrias?
- Definiton:
- Symptoms:
- Examples:
- Periodic acute attacks
- Symptoms: abdominal pain, neurologic deficits, psychiatric symptoms, and reddish-colored urine.
- Examples:
- Doss porphyria (ALA dehydratase deficiency)
- Acute intermittent porphyria
- Hereditary coproporyphyria
- Variegate porphyria
What are chronic porphyrias?
- Dermatologic diseases that may or may not include the liver and nervous system
- Examples:
- Congenital erythropoietic porphyria (Gunther’s disease)
- Erythropoietic porphyria/protoporphyria
- Porphyria cutanea tarda
- What enzymes are particularly sensitive to lead poisoning?
- What will be seen in the urine during lead poisoning?
- Ferrochelatase and ALA dehydratase are particularly sensitive to lead poisoning
- Protoporphyrin and ALA accumulate in the urine
What is the function of hemoglobin?
- Hemoglobin is a specialized protein designed to transport oxygen (O2) from the lungs, a region of high O2concentration, **to peripheral **tissues, **where oxygen is low
- Metabolism in the peripheral tissues generates CO2 and H+ that are transported back to the lungs, in part, by hemoglobin.
- O2 has very low solubility in plasma
- As a consequence, >98% of the O2 that reaches tissues is carried in red blood cells (RBCs) bound to Hemoglobin
How is CO2 transport different from O2 transport?
- RBCs contain carbonic anhydrase which catalyzes the rapid reversible hydration of CO2 to carbonic acid (H2CO3).
- H2CO3 then rapidly and spontaneously dissociates to bicarbonate (HCO3-) and a H+
- CO2 and HCO3- are soluble in plasma and RBC cytosol
- most of the CO2 made in tissues returns to the lungs as those species
- about 14% of the CO2 made is carried bound to Hb
- What is the structure of hemoglobin?
- What is hemoglobin related to?
- Which form of Fe can bind O2?
- Which form of iron cannot bind O2? What is it called?
- **Hemoglobin is a heterotetrameric protein **(αβ)2
- Both subunits are evolutionarily related to myoglobin
- a monomeric protein abundant in muscle that is designed to store O2
- myoglobin: 1 heme groups
- hemoglobin: 4 heme groups
- Fe2+ is the ferrous form of iron that is capable of binding O2
-
Fe3+ is the ferric form of iron that CANNOT bind O2 and is present in an INACTIVE form
- methemoglobin (metHb)
Describe the cooperative binding curve for oxygen:
- Myoglobin gives a normal binding curve which is hyperbolic in shape
-
Hemoglobin shows sigmoidal cooperative binding of oxygen
- direct result of its more complex subunit structure
- **P50: **partial pressure of oxygen yielding 50% saturation of binding
- analogous to Km for the binding of substrates to enzymes
What kind of cooperativity does hemoglobin exhibit for oxygen?
Sequential cooperativity for oxygen binding:
- binding of oxygen to one subunit induces a conformational change that is partially transmitted to adjacent subunits
- transmission of the partial conformational change induces an increased affinity for oxygen by these adjacent subunits
- R=relaxed=high affinity; T=taut=low affinity
What happens if CO binds to hemoglobin?
- Carbon monoxide (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
- limiting oxygen release in peripheral tissues
How Does O2 Binding Change the Conformation of a Hb Subunit?
Without O2 bound:
- 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
- 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
Allosteric Regulation of O2 Binding to Hemoglobin:
- Reduces affinity:
- Increases affinity:
- **Reduces affinity: **
- H+, CO2, and 2,3-diphosphoglycerate (DPG) ALL can bind to Hb
- leftward curve shift
-
Increases affinity:
- **high O2 **
- causes H+, DPG and CO2 to dissociate from Hb
- rightward curve shift
__ and ___ are heterotropic negative allosteric effectors that decrease the affinity of Hb for O2
H+ and CO2 are heterotropic negative allosteric effectors that decrease the affinity of Hb for O2
When would you want H+ and CO2 to be high in the blood?
During catabolism
- In the blood of tissues, CO2 and H+ are high
- HbO2 will release its O2 load ⇒catabolism can continue
______ is a positive homotropic allosteric effector of O2 binding
Oxygen is a positive homotropic allosteric effector of O2 binding
Desribe the Bohr effect:
**Reciprocal relationship between O2 and H+ binding **
- Oxygen is a negative allosteric effector of H+ and CO2 binding
- Changes in H+ binding result from a shift in the pKa of specific residues (mostly histidines) due to microenvironment effects triggered by conformational changes in the hemoglobin molecule
_______________ is a negative allosteric effector of O2 binding
2,3-diphosphoglycerate is a negative allosteric effector of O2 binding
How does 2,3-DPG alter O2 affinity for Hb?
- **2,3-DPG binds to a specific site in a central cavity between the β subunits **
- Binding is by ionic interactions
- Special regulatory mechanisms exist in RBCs to control the concentration of 2,3-DPG in order to fine tune the affinity of hemoglobin for O2 in response to changes in metabolism and environment
2 important things to notice about the effect of 2,3-DPG on the O2 binding curves:
-
Without any 2,3-DPG
- Hb would be much more like myoglobin
- 2,3-DPG stabilizes the T-state, making it easier for Hb to release O2
-
2,3-DPG levels increase at high altitudes
- Because there is less O2 at high altitudes, tissues tend to become somewhat hypoxic
- By increasing the concentration of 2,3-DPG ⇒ RBC adapt to hypoxia ⇒ easier for O2 to dissociate from Hb
- anemia and smoking also cause an increase in 2,3- DPG
- Changes in 2,3-DPG occur over hours and days
- takes most people a few days to adapt to high altitude
- exercise or strenuous activity will be difficult until then
How does temperature affect cooperative O2 binding?
↑ temp = ↓O2 affinity
↓ temp = ↑ O2 affinity
- Each chromosome __ has two α-globin genes
- Each chromosome __ has a single β-globin gene
- Each chromosome 16 has two α-globin genes
- a person has 4 total, each is active and codes ~1/4 of expressed α-globin subunit
- Each chromosome 11 has a single β-globin gene
What are the levels of Hb in normal adult red blood cells?
- ~95% HbA (α2β2)
- ~3% HbA2 (α2δ2)
- ~2% HbF (α2γ2)
Note: all forms have an α-globin
- What is the most common hemoglobinopathy?
- What is the pattern of inheritance?
- most common hemoglobinopathy ⇒ sickle cell anemia
- pattern of inheritance ⇒ autosomal recessive
What causes sickle cell anemia?
- Caused by a point mutation in the adult β-globin gene that causes **substitution of valine (Val) for **glutamic acid (Glu) at amino acid 6
- Patient’s RBCs containing mainly hemoglobin S (HbS)
- two normal adult α-globin subunits and two sickle adult β-globin subunits.
-
Valine is hydrophobic and its presence creates a sticky patch on deoxyHb
- leads to polymerization of Hb tetramers into long chains
- intracellular fibers cause the sickle cell shape and reduced deformability of the RBCs
- leads to problems in their passage through the microcirculation
Rate and extent of polymer formation in a circulating SS RBCs depends primarily on three independent variables:
-
degree of deoxygenation
- which can be affected by subtle changes in pH, ionic strength and temperature
- Deoxygenated HbS forms insoluble polymers
- intracellular hemoglobin concentration
-
relative amount of HbF present
- HbF inhibits polymerization owing to a GLU residue at position 87 of the gamma chain, which prevents a critical lateral contact in the sickle cell fiber
- HbF decreases with post-partum age but varies from 1-30% of total Hb in sickle cell individuals
Define thalassemia syndromes:
a heterogeneous group of disorders caused by inherited mutations that decrease the synthesis of adult hemoglobin HbA (α2β2)
What are the major categories of thalassemias?
-
β-Thalassemias
- Caused by mutations that diminish the synthesis of β-globin chains
-
α-Thalassemias
- Caused by mutations that result in reduced or absent synthesis of α-globin chains