Comprehensive Study Guide on Heme Degradation and Physiology and Biochemistry

Heme Structure, Function and Chemistry

Heme molecules are prosthetic coordination complexes produced in the liver and bone marrow, which serve as an oxygen-binding component for several classes of hemoproteins. These hemoproteins belong to a family of metalloproteins essential for tissue oxygenation through the circulation. The presence of a heme complex in hemoglobin is responsible for the characteristic red pigmentation of blood. Under normal physiological conditions, this protein is intracellular, found within erythrocyte (red blood cell; RBC) cytoplasms. Its release free into the plasma is typically a feature of pathological processes, such as the hemolysis occurring in sickle cell disease. The structure of heme prominently includes a porphyrin ring bound as a tetradentate ligand to a central iron atom, with either one or two axial ligands.

The iron atom within the heme component of hemoproteins within the RBC cytoplasm binds reversibly to diatomic gasses diffusing into the cells from plasma, including but not limited to $O_2$. This binding causes conformational changes in the intracellular carrier protein which favor the particular biological function for which the protein is essential. Iron within heme groups also functions as a source of electrons for heme-mediated redox and electron transfer reactions, as well as the transport or detection of diatomic gases. The binding selectivity of $O_2$ and other ligands to iron is dependent on its oxidation state, with the ferrous form $Fe(II)$ complexing with cognate ligands differently than the ferric form $Fe(III)$. Heme-bound $Fe(II)$ also provides a source of electrons for these processes, with the associated porphyrin group contributing to a reduction in the tissue toxicity of singlet/radical electrons through delocalization effects by the conjugated porphyrin. These properties allow hemoproteins to participate in a diverse range of essential physiologic processes, with hemoglobin-mediated tissue oxygenation being the most extensively characterized.

Tissue Deposition and Toxicogenic Challenges of Heme

The hemoprotein family is defined by the presence of heme within its structures and includes endothelial nitric oxide synthase, catalases, cytochromes, heme peroxidase, and myoglobin. Each of these macromolecules mediates physiological processes vital to the health of an organism. Within vertebrate systems, a significant amount of heme is localized in circulating erythrocytes, complexed with hemoglobin. Hemoglobin releases hemin when iron in its ferrous ($Fe(II)$) form is oxidized to a ferric ($Fe(III)$) state. As erythrocytes become aged or damaged, they are removed from circulation by splenic macrophages. This process results in the further release of heme into the plasma and interstitium, posing a toxicogenic challenge through tissue accumulation where it may act as an oxidative stressor.

The lipophilicity of heme allows its intercalation into lipid bilayers, which engenders both physical and chemical disruption of organelles and cells. This compromises cellular integrity and adversely alters cytoskeletal structure. Consequently, this results in endothelial cell injury and an increased risk of vascular inflammatory disorders, accompanied by increased expression of intracellular adhesion molecules. These effects may contribute to a wide range of debilitating chronic illnesses, including pathologies of the nervous system, heart, lungs, kidneys, and liver.

The Mechanism of Heme Degradation by Heme Oxygenase

The major adaptive countermeasure evolved to meet the hazard of heme accumulation is the clearance of the molecule through its degradation by heme oxygenase (HO). Heme oxygenase is a $32\,kDa$ heat shock protein (hsp32) composed of $288$ amino acid residues. It is found in the plasma membrane, nucleus, and mitochondria of cells, with particularly high levels in the endoplasmic reticulum. Heme is the physiologic substrate for HO, which utilizes $NADPH$, molecular oxygen ($O_2$), and cytochrome p450 reductase to degrade heme into biliverdin. Biliverdin is subsequently converted by the enzyme biliverdin reductase to bilirubin, carbon monoxide ($CO$), and ferrous iron $Fe(II)$. This reaction occurs at both intra- and extracellular physiologic sites, providing comprehensive cytoprotection compared to dietary antioxidants, which act mostly in interstitial spaces. Failure of endogenous hemoglobin and heme degradation mechanisms to effectively clear these molecules from tissue spaces may result in severely pathological consequences.

HO-1 specifically degrades heme into biliverdin, which is then metabolized by biliverdin reductase into bilirubin, a potent physiological antioxidant. The processing of heme by HO-1 additionally releases $Fe(II)$ ferrous iron, which is sequestered by ferritin. Ferritin is a protein that reduces $Fe(II)$ toxicity by storing it. The reaction also releases $CO$, which mediates various activities including vasodilatory, anti-inflammatory, anti-apoptotic, anti-thrombotic, and pro-angiogenic activities. Collectively, these constitute a major cytoprotective adaptive response to both external and endogenous stressors.

The Enzymatic Pathway of Bilirubin Production and Excretion

The iron-porphyrin (heme) group of hemoglobin, released from dying erythrocytes in the spleen, is degraded to yield free $Fe^{3+}$ and, ultimately, bilirubin. The first step in this two-step pathway is catalyzed by heme oxygenase (HO), which converts heme to biliverdin. The other products of this reaction are free $Fe^{2+}$ and $CO$. The $Fe^{2+}$ is quickly bound by ferritin. Carbon monoxide is a poison that binds to hemoglobin, and the production of $CO$ by heme oxygenase ensures that, even in the absence of environmental exposure, about $1\%$ of an individual’s heme is complexed with $CO$. In the second step, biliverdin is converted to bilirubin by the enzyme biliverdin reductase. This step involves the reduction of the second bridging methylene and requires $NADPH + H^+ \rightarrow NADP^+$.

Bilirubin is largely insoluble and must travel in the bloodstream as a complex with serum albumin. In the liver, bilirubin is transformed into the bile pigment bilirubin diglucuronide through the action of glucuronyl-bilirubin transferase. This product is sufficiently water-soluble to be secreted with other components of bile into the small intestine. In the intestine, microbial enzymes convert it to several products, predominantly urobilinogen. Some urobilinogen is reabsorbed into the blood and transported to the kidney, where it is converted to urobilin, the compound responsible for the yellow color of urine. The urobilinogen remaining in the intestine is converted in another microbe-dependent reaction to stercobilin, which imparts the red-brown color to feces.

Impact of Heme Degradation Products on Physiology and Pathogenesis

Heme oxygenase catabolizes the porphyrin ring and its iron atom into carbon monoxide and ferrous $Fe(II)$ iron. The iron is captured by ferritin, an iron-sequestering protein that stores iron in its inner cavity as a ferric-oxo species ($Fe^{III}$), a form that poses a diminished toxicogenic hazard to tissues. Processing of heme also produces biliverdin, a green-colored tetrapyrrolic bile pigment, which is then metabolized by biliverdin reductase to bilirubin. Bilirubin is a potent and the most abundant antioxidant in mammalian tissues, functioning as a major physiologic cytoprotective agent and providing most of the antioxidant activity in serum. Its protective effects are especially critical in the developing brain of newborn infants.

Cell toxicity associated with jaundice may occur when bilirubin levels exceed the amount of serum albumin needed to solubilize it. Impaired liver function or blocked bile secretion causes bilirubin to leak from the liver into the blood, resulting in the yellowing of the skin and eyeballs known as jaundice. Newborn infants may develop jaundice because they have not yet produced sufficient amounts of glucuronyl bilirubin transferase. A traditional treatment involves exposure to a fluorescent lamp, which causes a photochemical conversion of bilirubin to more soluble and easily excreted compounds. While $CO$ is toxic at high concentrations, the very low concentrations generated during heme degradation appear to have regulatory or signaling functions, such as acting as a vasodilator (similar to, but less potent than, nitric oxide) and regulating neurotransmission. Ferrous iron ($Fe(II)$) is also handled by transferrins, which are metal-binding glycoproteins that transport iron in its $Fe(II)$ state to sites of utilization like erythroid red blood cell progenitors.

Regulation and Isozymes of Heme Oxygenase

Heme degradation is a vital process that prevents heme-induced tissue damage by breaking down old red blood cells. Given the diverse roles of its products, the pathway is regulated mainly at the first step. Humans possess at least three isozymes of heme oxygenase. HO-1 is highly regulated and inducible; its gene expression is triggered by various stress conditions, including shear stress, angiogenesis (the uncontrolled development of blood vessels), hypoxia, hyperoxia, heat shock, ultraviolet light exposure, hydrogen peroxide, and other metabolic insults. HO-2 is expressed continuously and is found mainly in the brain and testes. The third isozyme, HO-3, has not yet been well characterized.

Comprehensive Process of Erythrocyte and Heme Recycling

The degradation process begins within the macrophages of the spleen, where senescent erythrocytes are removed from circulation and engulfed by the reticuloendothelial system. The globin portion of hemoglobin is recycled into amino acids, which are then catabolized into intermediates for the citric acid cycle and fatty acid oxidation. The heme portion is oxidized by heme oxygenase on a specific carbon, opening the heme ring. This produces the linear tetrapyrrole biliverdin, ferric iron ($Fe^{3+}$), and $CO$. Biliverdin reductase then reduces the green pigment biliverdin into the red-orange pigment bilirubin. Subsequent transport by albumin leads to the liver, where bilirubin is conjugated with glucuronate by bilirubin glucuronyl transferase for excretion in the bile. The final conversion into urobilinogen and stercobilin occurs in the intestine, completing the cycle of excretion through urine and feces.