CHAPTER 32: HEME BIOSYNTHESIS

Heme biosynthesis occurs primarily in the bone marrow and liver, although all nucleated cells have the capacity to synthesize this compound. It is derived from porphyrins, which are cyclic compounds that have a high affinity for binding metal ions, usually ferrous (Fe++) or ferric (Fe+++) iron. They contain four pyrrole rings linked together by methylene (- CH2-) bridges. Metalloporphyrins in nature are conjugated to proteins, and form many important biologic compounds. Hemoglobin (Hb), for example, is an erythrocytic iron porphyrin attached to the protein, globin. Myoglobin, which imparts red color to aerobic muscle fibers, has a structure similar to Hb, and both compounds possess the ability to combine reversibly with O2. Cytochromes are iron porphyrin proteins that act as electron transfer agents in cellular oxidation-reduction reactions. Important examples are the mitochondrial cytochromes associated with oxidative phosphorylation, the hepatic cytochrome P-450 system that participates in drug detoxification (i.e., hydroxylation), and the mitochondrial cytochromes involved with steroid hormone biosynthesis. Tissues rich in cytochromes include muscle, liver, kidney, and the steroidogenic tissues (e.g., adrenal cortices, testes, ovaries, and placenta). Catalases are ubiquitous iron porphyrin enzymes that degrade H2O2 in mammals (similarly to peroxidases in plants), and tryptophan pyrrolase, another iron porphyrin enzyme, catalyzes oxidation of tryptophan to formyl kynurenine. Chlorophyll, a magnesium (Mg++)-containing porphyrin, is an important photosynthetic pigment in plants, and also a source of porphyrins in some animal diets. With the exception of Fe++, all atoms in heme are derived from succinyl-CoA, derived from the mitochondrial tricarboxylic acid (TCA) cycle, and glycine (Gly). The initial and last three reactions in heme biosynthesis occur in mitochondria, with the intermediate steps being extramitochondrial. The first step is catalyzed by D-aminolevulinate (D-ALA) synthase, a vitamin B6- dependent enzyme, and involves condensation of succinyl-CoA and Gly to form D-ALA. This enzyme is located in the outer mitochondrial membrane, and its turnover rate is rapid (about 1 hr in mammalian liver), a common feature of an enzyme catalyzing a rate-limiting reaction. The activity of D-ALA synthase is subject to feedback inhibition by heme, and it is induced by hypoxia and a variety of compounds including alcohol (i.e., ethanol), steroids, and drugs such as the barbiturates (phenobarbital and pentobarbital). Once D-ALA reaches the cytoplasm, two molecules condense to form porphobilinogen (PBG), the basic pyrrole ring system used to assemble other porphyrins. Formation of PBG is catalyzed by D-ALA dehydratase, a zinc containing enzyme that is strongly inhibited by lead (Pb– ). Lead toxicity results in anemia due to decreased heme biosynthesis, and increased amounts of D-ALA appear in urine. Four molecules of PBG next combine to form uroporphyrinogen III through the action of two enzymes, uroporphyrinogen I synthase and uroporphyrinogen III cosynthase. Uroporphyrins were first identified in urine, but they are also found elsewhere in the organism. Uroporphyrinogen III is next converted to coproporphyrinogen III by decarboxylation of all its acetate units to methyl groups. Coproporphyrins were first isolated from feces, but they are also found in urine. Coproporphyrinogen III then enters mitochondria, where it is oxidized to protoporphyrinogen III, and then to protoporphyrinogen IX. These combined reactions alter the side chains attached to the pyrrole rings, giving them their characteristic one carbon methyl (M), two carbon vinyl (V), and three carbon propionyl (P) constituents. The final step in heme biosynthesis involves incorporation of Fe++ into protoporphyrin IX in a reaction catalyzed by ferrochelatase (also called heme synthase). This enzyme is also inhibited by Pb– . The various porphyrin intermediates in heme biosynthesis are colorless, but they can be oxidized to red or brown porphyrins. In abnormal porphyrin metabolism, oxidized products can accumulate and stain tissues. These products also absorb light energy, which can lead to photosensitization (if animals stay out in UV light), and damage to the skin. The appearance of persons with congenital erythropoietic porphyria, who have deformed faces and venture out only at night to avoid further skin damage, may have been the beginning of the werewolf legend. There are three principal types of photosensitization recognized in domestic animals. One type is caused by ingestion of significant amounts of photodynamic substances not normally present in the diet (e.g., certain poisonous plants), another is due to aberrant pigment production from endogenous sources (e.g., pink tooth of cattle or Siamese cats due to excessive presence of porphyrin intermediates), and a third type is due to liver disease in herbivores. In the latter instance, the photosensitizing agent is phylloerythrin, a natural porphyrin derivative of chlorophyll produced by intestinal microbes. This photodynamic substance is normally removed from portal blood by the liver, and secreted into bile. Hemoglobin (Hb) Myoglobin and Hb are evolutionarily conserved globular proteins. Adult Hb (HbA) has 4 subunits, each containing a heme moiety conjugated to a polypeptide chain, and a 64,450 MW. There are normally two pairs of polypeptide chains in each Hb molecule (designated and b-chains). Conversely, myoglobin contains only one polypeptide chain, and a single heme group. In uncontrolled diabetes mellitus, the erythrocytic glucose concentration rises, and glucose molecules attach to the terminal valine (Val) in each b-chain of Hb (commonly referred to as glycosylated Hb, or HbA1C. This interaction is thought to have little effect upon the O2 carrying capacity of Hb; however, HbA1C levels in blood are a useful long-term index of how well the blood glucose concentration of the diabetic animal is being managed. When Hb binds O2 (i.e., oxyhemoglobin), it attaches to the Fe++ of heme which is contained in a pocket of hydrophobic amino acids. This is the same site where carbon monoxide (CO) or nitric oxide (NO) attach, thus forming carboxyhemoglobin or nitrosylhemoglobin, respectively. The binding affinity of Hb for CO is some 230 times greater than it is for O2. Usually, less than 5% of Hb molecules contain bound CO. However, if the percentage is increased through excessive CO exposure, O2 transport can be severely compromised. Nitric oxide is an important vasodilator, and normally little is bound to Hb. However, in cases of hemolysis, where Hb floats freely in plasma, significant quantities of NO may become Hb-bound, thus leading to hypertension (Liao JC, 2002). Each heme group of Hb is attached to the globin chain by linkage of Fe++ to a histidine residue (Fig. 32-2). The histidines are arranged so that O2 can bind with Hb reversibly. Carbon dioxide (CO2) does not compete with O2 for binding to Hb. Rather, small amounts (~ ~ 20%) of capillary CO2 bind nonenzymatically to the amine (NH2) terminal of the globin polypeptide chain, thus forming carbaminohemoglobin. Most of the CO2 that enters the RBC in capillaries of the body is converted to H+ and HCO3 - through the enzymatic action of carbonic anhydrase, and then HCO3 - diffuses down its concentration gradient into plasma. Because deoxyhemoglobin has a higher pK than oxyhemoglobin, the extra H+ generated can be immediately bound (i.e., buffered) by the Hb molecule itself, a process which results in O2 release in capillary blood. Generally, one H+ tends to be bound for each two O2 molecules released, and this buffering action contributes significantly to the overall buffering capacity of blood. This change in O2 binding with a slight decrease in pH is called the Bohr effect, a phenomenon that is accompanied by a shift in the O2 dissociation curve to the right (i.e., Hb becomes less saturated at any given partial pressure of O2). Two other factors that contribute to the Bohr effect in erythrocytes are 2,3-bisphosphoglycerate (2,3- BPG), and carbaminohemoglobin formation. With the exception of horses and pigs, the blood of mammalian fetuses contains fetal Hb (HbF). The structure of HbF is similar to that of HbA, except that the b-chains are replaced by g-chains (differing amino acid sequences). Fetal Hb binds 2,3-BPG less avidly than does HbA, which helps in utero to assure that proper O2 exchange occurs between the dam and fetus. Soon after birth, however, HbF is normally replaced by HbA. Anemias and Polycythemia Common anemias (i.e., reductions in the amount of red blood cells or of hemoglobin in the circulation) result from excessive intra- or extravascular hemolysis, from impaired biosynthesis of hemoglobin (e.g., iron deficiency), or impaired production of erythrocytes (e.g., erythropoietin (EPO), folic acid, or vitamin B12 deficiency. In humans the thalassemias and sickle cell anemia are a group of hereditary hemolytic anemias resulting from variations in the amino acids comprising the a- or b-polypeptide chains of hemoglobin. Anemia classification is generally under[1]taken by determining whether evidence of a bone marrow response to the condition is present in blood. With the exception of horses, this is routinely accomplished by determining whether circulating reticulocyte numbers are increased. Regenerative anemia will usually show an increase, whereas nonregenerative anemia will not. Examples of conditions leading to regenerative anemia include blood loss and increased erythrocyte destruction (hemolysis). Hemolytic anemia usually results in a more dramatic regenerative response than hemorrhagic anemia due to greater iron availability. Nonregenerative anemia may result from several chronic disease conditions, from drug or toxin exposure, or from inflammation. For example, chronic renal failure, hypothyroidism, or liver failure can precipitate a nonregenerative anemia. Polycythemia (i.e., the presence of excessive amounts of red blood cells in the circulation) may be either relative, or absolute. A relative polycythemia may result in an increased hematocrit (Hct; the percentage of the blood volume made up of erythrocytes), but perhaps not an increase in the total red blood cell mass. Dehydration with a loss of plasma volume would be an example. An absolute polycythemia may result from increased renal EPO secretion as a response to hypoxemia, or because of oversecretion from malignancy. Polycythemia rubra vera results from a malignancy in red blood cell production despite normal or low concentrations of EPO. Any one of these polycythemic conditions causes an increase in blood viscosity, decreased cerebral perfusion, and potentially heart failure.

Study Plan: Chapter 32 - Heme Biosynthesis

Day 1: Introduction and Overview

Read and understand the general concepts of heme biosynthesis.
Focus on the primary sites of heme biosynthesis (bone marrow and liver) and the involvement of all nucleated cells.
Learn about porphyrins and their affinity for binding metal ions.
Understand the structure and function of metalloporphyrins in nature, including hemoglobin, myoglobin, and cytochromes.

Day 2: Enzymes and Reactions in Heme Biosynthesis

Study the step-by-step process of heme biosynthesis, starting from porphobilinogen (PBG) formation.
Focus on the enzymes involved in each reaction, such as D-aminolevulinate (D-ALA) synthase, D-ALA dehydratase, and uroporphyrinogen I synthase.
Understand the role of succinyl-CoA and glycine in heme biosynthesis.
Learn about the mitochondrial and extramitochondrial locations of the reactions and the significance of feedback inhibition and induction of D-ALA synthase.

Day 3: Porphyrin Intermediates and Final Steps

Study the formation of uroporphyrinogen III and coproporphyrinogen III from porphobilinogen.
Understand the oxidation reactions in mitochondria that lead to the formation of protoporphyrinogen III and protoporphyrinogen IX.
Learn about the side chain modifications in the pyrrole rings, including the constituents methyl (M), vinyl (V), and propionyl (P).
Focus on the final step of heme biosynthesis, which involves the incorporation of Fe++ into protoporphyrin IX catalyzed by ferrochelatase.

Day 4: Abnormal Porphyrin Metabolism and Photosensitization

Study the consequences of abnormal porphyrin metabolism, including the accumulation of oxidized porphyrins and tissue staining.
Understand the role of photosensitization and its connection to abnormal porphyrin metabolism.
Learn about the different types of photosensitization in domestic animals, including ingestion of photodynamic substances, aberrant pigment production, and liver disease.

Day 5: Hemoglobin, Anemias, and Polycythemia

SUMMARY

Heme biosynthesis occurs primarily in the bone marrow and liver, but all nucleated cells can synthesize it. Heme is derived from porphyrins, cyclic compounds that bind metal ions like iron. Hemoglobin and myoglobin are iron porphyrin proteins that bind oxygen. Cytochromes are iron porphyrin proteins involved in cellular oxidation-reduction reactions. Catalases and tryptophan pyrrolase are iron porphyrin enzymes that degrade H2O2 and oxidize tryptophan, respectively. Chlorophyll is a magnesium-containing porphyrin involved in photosynthesis. Heme biosynthesis involves several steps, with the initial and last three reactions occurring in mitochondria. Lead inhibits enzymes involved in heme biosynthesis, leading to anemia. Abnormal porphyrin metabolism can cause tissue staining and photosensitization. Hemoglobin and myoglobin are globular proteins that bind oxygen. Glycosylated hemoglobin levels are used to monitor blood glucose control in diabetes. Hemoglobin can also bind carbon monoxide and nitric oxide. Carbon dioxide binds to the globin chain, forming carbaminohemoglobin. The Bohr effect describes the release of oxygen in response to a decrease in pH. Fetal hemoglobin has a lower affinity for 2,3-BPG than adult hemoglobin. Anemias result from hemolysis, impaired hemoglobin biosynthesis, or impaired erythrocyte production. Polycythemia can be relative or absolute and may result from increased EPO secretion or malignancy.

OUTLINE

Heme biosynthesis occurs primarily in the bone marrow and liver
Porphyrins are cyclic compounds that bind metal ions, usually ferrous or ferric iron
Hemoglobin (Hb) and myoglobin are iron porphyrin proteins that bind reversibly with oxygen
Cytochromes are iron porphyrin proteins involved in oxidation-reduction reactions
Catalases are iron porphyrin enzymes that degrade hydrogen peroxide
Heme biosynthesis begins with the condensation of succinyl-CoA and glycine to form D-aminolevulinate (D-ALA)
D-ALA is synthesized by D-ALA synthase, a vitamin B6-dependent enzyme
D-ALA dehydratase catalyzes the formation of porphobilinogen (PBG) from D-ALA
Uroporphyrinogen III is formed from four molecules of PBG
Coproporphyrinogen III is formed from uroporphyrinogen III by decarboxylation
Protoporphyrinogen III is formed from coproporphyrinogen III in mitochondria
Protoporphyrinogen IX is the final intermediate in heme biosynthesis
Ferrochelatase incorporates iron into protoporphyrin IX to form heme
Abnormal porphyrin metabolism can lead to accumulation of oxidized products and tissue damage
Hemoglobin (Hb) is a globular protein with four subunits, each containing a heme group
Hb can bind oxygen, carbon monoxide, and nitric oxide
Carbon dioxide binds to Hb as carbaminohemoglobin
The Bohr effect describes the release of oxygen from Hb in response to decreased pH
Fetal Hb (HbF) has a higher affinity for oxygen than adult Hb (HbA)
Anemias can result from hemolysis, impaired hemoglobin biosynthesis, or impaired erythrocyte production
Polycythemia can be relative or absolute, and can result from increased EPO secretion or malignancy

QUESTIONS

Qcard 1:

Question: Where does heme biosynthesis primarily occur?

Answer: Heme biosynthesis primarily occurs in the bone marrow and liver.

Qcard 2:

Question: What are porphyrins?

Answer: Porphyrins are cyclic compounds that have a high affinity for binding metal ions, usually ferrous (Fe++) or ferric (Fe+++) iron.

Qcard 3:

Question: What is the structure of heme?

Answer: Heme contains four pyrrole rings linked together by methylene (- CH2-) bridges.

Qcard 4:

Question: What are some important biologic compounds formed by metalloporphyrins?

Answer: Hemoglobin, myoglobin, cytochromes, catalases, and chlorophyll are important biologic compounds formed by metalloporphyrins.

Qcard 5:

Question: What is the role of cytochromes in cellular oxidation-reduction reactions?

Answer: Cytochromes act as electron transfer agents in cellular oxidation-reduction reactions.

Qcard 6:

Question: What is the final step in heme biosynthesis?

Answer: The final step in heme biosynthesis involves the incorporation of Fe++ into protoporphyrin IX in a reaction catalyzed by ferrochelatase.

Qcard 7:

Question: What is the significance of HbA1C levels in blood?

Answer: HbA1C levels in blood are a useful long-term index of how well the blood glucose concentration of a diabetic animal is being managed.

Qcard 8:

Question: What is the Bohr effect?

Answer: The Bohr effect refers to the change in O2 binding with a slight decrease in pH, resulting in O2 release in capillary blood.

Qcard 9:

Question: What is the difference between regenerative and nonregenerative anemia?

Answer: Regenerative anemia shows an increase in circulating reticulocyte numbers, while nonregenerative anemia does not.

Qcard 10:

Question: What is the difference between relative and absolute polycythemia?

Answer: Relative polycythemia results in an increased hematocrit (Hct) due to dehydration, while absolute polycythemia is caused by increased renal EPO secretion or malignancy.