Heme & Hemoglobin - Dahms

Question 1

Name three functions of heme.

Answer 1

1) Oxygen transport
2) Electron transport (cellular respiration)
3) Redox reactions (cyt P450)

Question 2

Name two major sites of heme synthesis.

What is the major utilization of the heme generated at these sites?

Answer 2

1) Bone marrow - Hemoglobin

2) Liver - Cytochrome P450 enzymes

Question 3

About how much heme is synthesized by the liver each day to replace the amount lost via normal RBC turnover?

Answer 3

6-7g

Question 4

Due to its usefulness in electron transport, heme is synthesized by all cell types EXCEPT __________, because __________.

Answer 4

Mature erythrocytes, because they lack mitochondria.

Question 5

Heme is a porphyrin, which is a cyclic molecule made up of how many units? What are these units called?
What is the overall molecular geometry of heme?

Answer 5

“cyclic tetrapyrrole” - 4 pyrrole rings

Heme is essentially a planar molecule

Question 6

What is the “full” chemical name of heme?

What ion is chelated by the porphyrin of heme?

Answer 6

Ferroprotoporphyrin IX

Heme is a ferrous chelate (Fe2+)

Question 7

What is hemin? How does it differ from heme?

Answer 7

Hemin’s full name is Ferriprotoporphyrin IX. It differs from heme in that its chelated ion is the more oxidized ferric (Fe3+) ion instead of the ferrous (Fe2+) ion.

Question 8

The pyrrole groups of various porphyrin molecules contain variable substituent groups that extend off the cyclic ring. What are these groups important for?

Answer 8

Provide binding sites for the porphyrins to their respective apoproteins.

Question 9

How many major steps are there in heme biosynthesis?

Which steps occur in the mitochondria, and which occur in the cytoplasm?

Answer 9

7 major steps
The 1st and 5th-7th steps occur in the mitochondrion.
The 2nd-4th steps occur in the cytosol.

Question 10

1) Step 1 of heme biosynthesis involves a condensation of what two molecules?
2) What is the name of the product of this reaction?
3) What parts of the porphyrin ring end up being composed by these two molecules?

Answer 10

1) succinyl CoA & glycine
2) ALA (5-aminolevulinate)
3) All C & N atoms of porphyrin are provided by (multiple pairs of) these two molecules

Question 11

1) What enzyme carries out Step 1 of heme synthesis?
2) What cofactor does this enzyme require?
3) What special chemical intermediate does the cofactor participate in during the reaction?
4) What is important about this reaction regarding heme’s biosynthetic pathway?

Answer 11

1) ALAS (5-aminolevulinate synthase)
2) Cofactor is PLP
3) Schiff base intermediate
4) The first step is also the COMMITTED step of heme biosynthesis.

Question 12

The ALAS enzyme is localized to what compartment of the cell? How does it get there?

Answer 12

ALAS is located at the inner mitochondrial membrane. However, it is a nuclear protein and so must be imported into the mitochondrion.

Question 13

How many isoforms of ALAS are there? Where are they located?

Answer 13

Two isoforms:
ALAS1 - liver isoform
ALAS2 - erythroid/reticulocyte isoform

Question 14

How is ALAS1 regulated? Is it readily affected by drugs?

How is ALAS2 regulated? Is it readily affected by drugs?

Answer 14

ALAS1: Negative feedback from heme & hemin that inhibits ALAS1 synthesis & its transport into the mitochondria. Affected by 100s of drugs (it makes heme for the cyt P450s, and we know those get affected by just about everything!)

ALAS2: NO NEGATIVE FEEDBACK by heme. Heme stimulates an appropriate amount of globin synthesis. Many drugs that affect ALAS1 do NOT affect ALAS2.

Question 15

1) What is Step 2 of hemoglobin biosynthesis?

2) What is special about the product of this reaction?

Answer 15

1) Condensation of 2x ALA to form 1x PBG (porphobilinogen)

2) PBG is the first intermediate that contains a pyrrole ring

Question 16

1) What enzyme carries out Step 2 of heme synthesis?
2) What cofactor does this enzyme use?
3) What can shut down this enzyme’s activity?

Answer 16

1) ALA dehydratase (ALAD)
2) Zn2+ (complexed to active site Cys)
3) Lead and other heavy metals

Question 17

1) Why does lead poisoning cause an increase of ALA in the blood?
2) Where else would ALA be noted in this scenario?

Answer 17

1) Inhibition of ALAD causes a buildup of ALA that is unable to be condensed into PBG. The lack of heme synthesis will also activate the ALAS gene, resulting in further ALA buildup.
2) ALA can be seen in the urine

Question 18

Are are the effects of lead poisoning due to lead itself? What else is thought to contribute to the pathology, and in what way?

Answer 18

While Pb2+ may directly affect the nervous system, ALA is toxic to the brain itself. This is perhaps due to ALA’s similar structure to the neurotransmitter GABA, as well as the ability of ALA to generate ROS.

Question 19

1) What is Step 3 of hemoglobin biosynthesis?
2) What is the product of this reaction?
3) What enzyme catalyzes this step?

Answer 19

1) Head-to-tail condensation of 4x PBG
2) A linear tetrapyrrole (hydroxymethylbilane)
3) PBGD (Porphobilinogen deaminase)

Question 20

1) What enzyme is closely associated with PBGD (from Step 3), and why is it important?

Answer 20

UROS (uroporphyrinogen III cosynthase), which serves to direct the stereochemistry of the cyclization of the tetrapyrrole in the correct fashion (the III isomer).

Without UROS, the linear tetrapyrrole can auto-cyclize to form an incorrect isomer (the I isomer), not useful in heme biosynthesis.

Question 21

1) What is Step 4 of heme biosynthesis and its product?

2) What enzyme carries out Step 4?

Answer 21

1) The decarboxylation of side chains on uroporphyrinogen III, yielding coproporphyrinogen III.
2) UROD (uroporphyrinogen decarboxylase)

Question 22

1) What is Step 5 of heme biosynthesis and its product?

2) What enzyme carries out Step 5?

Answer 22

1) Further modification of side chains to vinyl groups, to produce protoporphyrinogen IX
2) CPO (coproporphyrinogen III oxidase)

Question 23

1) What is Step 6 of heme biosynthesis and its product?

2) What enzyme carries out Step 6?

Answer 23

1) Movement of double bonds to form protoporhyrin IX

2) PPO (Protoporphyrinogen IX oxidase)

Question 24

1) What is the 7th and final step of heme biosynthesis?

2) What enzyme is this aided by?

Answer 24

1) Insertion of Fe2+ to form heme. A slow but spontaneous processes
2) Ferrocheletase - enhances the rate of Fe2+ insertion

Question 25

1) What condition can inhibit ferrocheletase activity?

2) What laboratory finding can be seen as a consequence of this?

Answer 25

1) Iron poisoning (ALSO affects this enzyme)

2) Increased protoporphyrin in the urine

Question 26

Broadly, what are porphyrias?

What two major etiological subtypes are there?

Answer 26

1) A group of inherited or (rarely) acquired disorders resulting from defects in the porphyrin/heme biosynthetic pathway. Accumulation of intermediates upstream of the defect cause the clinical symptoms.
2) Hepatic vs. Eryhtroid porphyrias, reflecting the principal sites of heme synthesis. Different sites may be affected depending of the isoform(s) of the defective enzyme(s).

Question 27

What is the inheritance pattern of most porphyrias?

What is the notable exception to this?

Answer 27

All are autosomal dominant, except for the autosomal recessive congenital erythropoietic porphyria (CEP). CEP is extremely rare (<1:1,000,000).

Question 28

What is the most common porphyria? How common is it?

Answer 28

Porphyria cutanea tarda (PCT). Even as the most common, its incidence is only 1:10,000. Porphyrias are rare diseases in general.

Question 29

What is the most common porphyria of childhood?

Answer 29

Erythropoietic protoporphyria (EPP). Still only a 1:50,000-75,000 incidence.

Question 30

Defects early in the heme biosynthetic pathway result in what type of symptoms?
Name two intermediates that would cause these symptoms when accumulated.

Answer 30

Neurologic dysfunction

e.g. ALA or PBG accumulation

Question 31

Defects late in the heme biosynthetic pathway result in what type of symptoms?
Name the intermediates that would cause these symptoms when accumulated.

Answer 31

Sunlight-induced cutaneous lesions

Accumulation of cyclic tetrapyrroles (but not PBG)

Question 32

How does accumulation of heme biosynthetic intermediates cause the sunlight-induced skin lesions seen with some porphyrias?

Answer 32

UV irradiation of cyclic tetrapyrroles (later intermediates) generates ROS, which cause cellular damage.

Question 33

Porphyrias can also be divided into two groups based on their symptomatology: acute and chronic. Describe the symptoms seen in each type.

Answer 33

Acute: Periodic, acute attacks including abdominal pain, neurologic deficits, psychiatric symptoms, and reddish urine.

Chronic: Dermatologic diseases; may or may not include the liver and nervous system.

Question 34

What factors are believed to be factors that bring about acute porphyria attacks?

Answer 34

Nutrition, esp. hypoglycemia
Smoking
Drugs: barbituates, sulfa abx, steroid hormones (esp.p progesterone)
Some women develop attacks during 2nd half of menstrual cycle, when progesterone is high.

Question 35

Name four porphyrias that present with acute symptoms.

Answer 35

1) Doss porphyria (ALAD deficiency)
2) AIP (acute intermittent porphyria)
3) HCP (hereditary coproporphyria)
4) Variegate porphyria

Question 36

Name three porphyrias that present with chronic symptoms.

Answer 36

1) CEP / Gunther’s Disease
2) EPP
3) PCT

Question 37

Years ago, what fungicide was implicated as the cause of an “outbreak” of acquired porphyria cutanea tarda (PCT) in Turkey?

Answer 37

Hexachlorobenzene (HCT)

Question 38

Again, what two enzymes of the heme synthetic pathway are sensitive to lead poisoning? What two intermediates will accumulate if these enzymes are inhibited?

Answer 38

1) ALAD (ALA accumulates)

2) Ferrochelatase (Protoporphyrin accumulates)

Question 39

What porphyria, rare in the rest of the world, has a high incidence in South Africa due to a founder effect?

Answer 39

Variegate porphyria (deficiency in PPO / Step 6)
-Photosensitivity

Question 40

Doss porphyria is due to a deficiency of which enzyme?

Answer 40

ALAD

Question 41

Porphyria Cutanea Tarda is due to a deficiency of which enzyme?

Answer 41

UROD

Question 42

1) About how much CO2 is carried to the lungs bound to Hb?
2) How is this CO2 bound to Hb?
3) How else does it reach the lungs?

Answer 42

1) Only 14%
2) Bound to alpha-amino groups of Hb
3) The remainder is directly dissolved in plasma or RBC cytosol as CO2 or HCO3-.

Question 43

What is methemoglobin? Is it useful for carrying O2?

Answer 43

Methemoglobin denotes Hb that is chelated to the ferric (Fe3+) ion instead of the normal ferrous (Fe2+) ion. It is inactive, as Fe3+ cannot bind O2.

Question 44

Describe the structure of the hemoglobin protein.

Answer 44

Hb is a heterotetrameric protein consisting of two alpha and two beta subunits, which have similar structures. Each subunit contains a heme prosthetic group used to bind O2 (4 O2 per Hb).

Question 45

On a curve of SaO2% vs. pO2, the monomeric myoglobin yields a normal binding curve with a hyperbolic shape. What is the shape of the Hb binding curve? What is it due to and why is it useful?

Answer 45

Sigmoidal curve due to cooperative binding, which provides Hb with much of its efficiency in loading O2 in the lungs and releasing a large fraction of its O2 load at the pO2 levels found in the blood of working muscle.

Question 46

Define: R state, T state. Which state is favored when?

Answer 46

R state = relaxed state of Hb subunit. High O2 affinity. Favored as more O2 is bound.
T state = taut state of Hb subunit. Low O2 affinity. Favored as less O2 is bound.

Question 47

Briefly describe how cooperative O2 binding works in Hb.

Answer 47

Binding of O2 to one subunit induces a conformational change that affects adjacent subunits, which increases the O2 affinity in those subunits as well.

Question 48

What is the effect of CO on the cooperative binding of Hb?

Answer 48

CO, with its high affinity for Hb, binds nigh-irreversibly to the heme group of one subunit. This causes all four subunits to “lock” in the R conformation, which limits the release of O2 in peripheral tissues.

Question 49

At the molecular level, how does O2 binding change the conformation of Hb subunits (T vs. R state)?

Answer 49

Without O2 bound, the heme Fe2+ is pulled AWAY from the plane of the porphyrin ring by a His residue. When O2 binds, it pulls the Fe2+ back into the plane of the ring. This moves the His residue and its whole section of the polypeptide chain, which is partially transferred to the Hb subunits around it.

Question 50

Name three heterotropic negative allosteric regulators of O2 binding to Hb. Can you name a positive allosteric regulator?

Answer 50

1) H+
2) CO2
3) 2,3-DPG

O2 is a positive allosteric regulator (this is the basis of cooperativity! The O2 binding site of one Hb subunit is an allosteric site from the perspective of another subunit!)

Question 51

When H+, CO2, and/or DPG are high, the O2 binding curve shifts to the (left/right), which favors (binding/release) of O2. This occurs physiologically in the (lungs/peripheral tissues).

When H+, CO2, and/or DPG are low, the O2 binding curve shifts to the (left/right), which favors (binding/release) of O2. This occurs physiologically in the (lungs/peripheral tissues).

Answer 51

Right, release, peripheral tissues

Left, binding, lungs

Question 52

What is the name for the reciprocal relationship between O2 and H+ binding to Hb?

Answer 52

The Bohr effect, or isohydric shift

Question 53

How does H+ binding affect O2 binding at the molecular level?

Answer 53

In short, H+ binding shifts the pKa of various residues (mostly His!), causing microenvironment effects and conformational shifts.

Question 54

Where does 2,3-DPG bind on the Hb protein?

Answer 54

A central cavity between the beta subunits. (Stabilizes the T state)

Question 55

What evolutionary advantage is granted by having a molecule like DPG, that shifts the binding curve of Hb?

Answer 55

Obviates the need to synthesize different Hb isoforms of varying O2 affinity for different circumstances - just fine tune Hb with a small molecule instead!

Question 56

Aside from high altitude, what other conditions would cause an increase in the synthesis of 2,3-DPG?

Answer 56

Other causes of tissue hypoxia, e.g. anemia & smoking

Question 57

How does temperature affect O2 binding to Hb? Under what physiological conditions is this useful?

Answer 57

Increased temperature decreases O2 affinity for Hb (right shift.) This is useful during the elevated metabolic rates seen in fever [and exercise?]

Question 58

How many alpha globin genes does a person have? What chromosome are they found on? What about beta globin genes?

Answer 58

Alpha: 4 total. 2 on each chr 16.
Beta: 2 total. 1 on each chr 11

Question 59

Does fetal Hb (HbF) have a higher or lower affinity for O2 than adult Hb? Why?

Answer 59

Higher - allows the fetus’s circulation to draw O2 from the maternal blood at the pO2 present in the placenta.

Question 60

For the one-hundreth time, what mutation causes SCD? How does it do so?

Answer 60

Glutamate 6 becomes Valine (E6V). The hydrophobic Val creates a sticky patch on deoxyHb, leading to Hb polymerization into long chains/fibers. The fibers cause the sickled shape and reduced deformability of RBCs.

Question 61

Name three factors that affect the rate and extent of HbS polymerization in the setting of SCD/

Answer 61

1) Degree of deoxygenation (deoxy-HbS polymerizes)
2) Intracellular Hb concentration
3) Amount of HbF present (HbF inhibits polymerization)

Question 62

Which are generally more severe, alpha or beta thalassemias? Why?

Answer 62

Beta are more severe because:

1) Only 2 beta globin genes as opposed to 4 alpha.
2) Accumulated beta chains (seen in alpha thalassemias) are more soluble than accumulated alpha chains (seen in beta thalassemias)

Question 63

Which is worse: b0 or b+ thalassemia? Why?

Answer 63

b+: reduced beta-globin chain synthesis from that gene

b0: absent beta-globin chain synthesis from that gene. b0 is thus worse.

Question 64

Which is worse: beta thalassemia major or minor? Why?

Answer 64

Major: both beta alleles are mutated (homozygote). Worse.
Minor: one beta allele is mutated (heterozygote).