Significance of Heme and Heme Degradation in the Pathogenesis of Acute Lung and Inflammatory Disorders

The heme molecule serves as an essential prosthetic group for oxygen transport and storage proteins, as well for cellular metabolic enzyme activities, including those involved in mitochondrial respiration, xenobiotic metabolism, and antioxidant responses. Dysfunction in both heme synthesis and degradation pathways can promote human disease. Heme is a pro-oxidant via iron catalysis that can induce cytotoxicity and injury to the vascular endothelium. Additionally, heme can modulate inflammatory and immune system functions. Thus, the synthesis, utilization and turnover of heme are by necessity tightly regulated. The microsomal heme oxygenase (HO) system degrades heme to carbon monoxide (CO), iron, and biliverdin-IXα, that latter which is converted to bilirubin-IXα by biliverdin reductase. Heme degradation by heme oxygenase-1 (HO-1) is linked to cytoprotection via heme removal, as well as by activity-dependent end-product generation (i.e., bile pigments and CO), and other potential mechanisms. Therapeutic strategies targeting the heme/HO-1 pathway, including therapeutic modulation of heme levels, elevation (or inhibition) of HO-1 protein and activity, and application of CO donor compounds or gas show potential in inflammatory conditions including sepsis and pulmonary diseases.

Heme Binding to HupZ with a C-Terminal Tag from Group A Streptococcus

HupZ is an expected heme degrading enzyme in the heme acquisition and utilization pathway in Group A Streptococcus. The isolated HupZ protein containing a C-terminal V5-His6 tag exhibits a weak heme degradation activity. Here, we revisited and characterized the HupZ-V5-His6 protein via biochemical, mutagenesis, protein quaternary structure, UV–vis, EPR, and resonance Raman spectroscopies. The results show that the ferric heme-protein complex did not display an expected ferric EPR signal and that heme binding to HupZ triggered the formation of higher oligomeric states. We found that heme binding to HupZ was an O2-dependent process. The single histidine residue in the HupZ sequence, His111, did not bind to the ferric heme, nor was it involved with the weak heme-degradation activity. Our results do not favor the heme oxygenase assignment because of the slow binding of heme and the newly discovered association of the weak heme degradation activity with the His6-tag. Altogether, the data suggest that the protein binds heme by its His6-tag, resulting in a heme-induced higher-order oligomeric structure and heme stacking. This work emphasizes the importance of considering exogenous tags when interpreting experimental observations during the study of heme utilization proteins.

Heme Degradation in Pathophysiology of and Countermeasures to Inflammation-Associated Disease

The class of tetrapyrrol “coordination complexes” called hemes are prosthetic group components of metalloproteins including hemoglobin, which provide functionality to these physiologically essential macromolecules by reversibly binding diatomic gasses, notably O2, which complexes to ferrous (reduced/Fe(II)) iron within the heme porphyrin ring of hemoglobin in a pH- and PCO2-dependent manner—thus allowing their transport and delivery to anatomic sites of their function. Here, pathologies associated with aberrant heme degradation are explored in the context of their underlying mechanisms and emerging medical countermeasures developed using heme oxygenase (HO), its major degradative enzyme and bioactive metabolites produced by HO activity. Tissue deposits of heme accumulate as a result of the removal of senescent or damaged erythrocytes from circulation by splenic macrophages, which destroy the cells and internal proteins, including hemoglobin, leaving free heme to accumulate, posing a significant toxicogenic challenge. In humans, HO uses NADPH as a reducing agent, along with molecular oxygen, to degrade heme into carbon monoxide (CO), free ferrous iron (FeII), which is sequestered by ferritin protein, and biliverdin, subsequently metabolized to bilirubin, a potent inhibitor of oxidative stress-mediated tissue damage. CO acts as a cellular messenger and augments vasodilation. Nevertheless, disease- or trauma-associated oxidative stressors sufficiently intense to overwhelm HO may trigger or exacerbate a wide range of diseases, including cardiovascular and neurologic syndromes. Here, strategies are described for counteracting the effects of aberrant heme degradation, with a particular focus on “bioflavonoids” as HO inducers, shown to cause amelioration of severe inflammatory diseases.

Glutathione and the intracellular labile heme pool

One candidate for the cytosolic labile iron pool is iron(II)glutathione. There is also a widely held opinion that an equivalent cytosolic labile heme pool exists and that this pool is important for the intracellular transfer of heme. Here we describe a study designed to characterise conjugates that form between heme and glutathione. In contrast to hydrated iron(II), heme reacts with glutathione, under aerobic conditions, to form the stable hematin–glutathione complex, which contains iron(III). Thus, glutathione is clearly not the cytosolic ligand for heme, indeed we demonstrate that the rate of heme degradation is enhanced in the presence of glutathione. We suggest that the concentration of heme in the cytosol is extremely low and that intracellular heme transfer occurs via intracellular membrane structures. Should any heme inadvertently escape into the cytosol, it would be rapidly conjugated to glutathione thereby protecting the cell from the toxic effects of heme.

Xanthine Oxidase Has a Protective Role during Heme Crisis By Binding and Degrading Heme

Xanthine oxidase (XO) is a key enzyme in the purine degradation pathway, catalyzing the catabolism of hypoxanthine to xanthine and xanthine to uric acid. A byproduct of these reactions is the generation of the reactive oxygen species (ROS), hydrogen peroxide and superoxide. XO is produced primarily in the liver; however, following hepatic stressors such as inflammation, hypoxia, or ischemia, XO is released from the liver and enters the circulation. XO can then bind distal endothelium via electrostatic interactions with glycosaminoglycans (GAGs). Current dogma believes that XO plays a harmful role in pathologies due to the increase in ROS production that can alter signaling pathways and damage endothelial cells. XO activity has been shown to be elevated in a number of hemolytic conditions including, sickle cell disease, malaria, and sepsis; however, the involvement of XO in these pathological conditions has not been fully elucidated. These conditions result in increased hemolysis, releasing free heme and hemoglobin into the circulation, inducing an inflammatory response, and damaging endothelial cells. Identifying the involvement of XO during heme crisis could improve our understanding of the pathologies associated with hemolytic conditions and lead to the identification or development of more effective treatment options. We hypothesized that XO has damaging properties under basal conditions; however, the presence of XO is crucial and protective during heme crisis by serving as a secondary mechanism of heme degradation when canonical heme degradation pathways are saturated. In order to explore the role of XO in heme crisis, we developed a novel heme crisis model in which we pre-treated mice with the clinically relevant dose of 10 mg/kg/day febuxostat, an FDA approved XO inhibitor, for five days in drinking water. Following inhibition of XO, the mice were challenged with two identical doses of hemin one hour apart and monitored for 24 hours. We observed a 20-fold increase in XO activity in the mice treated with 50 μmol/kg hemin. This increase was completely inhibited in the mice treated with febuxostat. Surprisingly, the febuxostat treated mice had worsened survival compared to the untreated mice. This suggests a protective role for XO during heme crisis. We hypothesized that XO has a protective role by preventing platelet activation and degrading excess free heme. To investigate this hypothesis, we used flow cytometry to quantify heme-induced platelet activation. Healthy human platelets were isolated and treated with 2.5 μM hemin, 10 mU/mL XO, 200 μM hypoxanthine, and 20 μM febuxostat. We observed 72% activation with heme alone, while incubation with XO and hypoxanthine resulted in almost complete prevention of platelet activation (16%). We were also able to partially restore platelet activation (45%) when febuxostat was added. Based on these results, we hypothesize that XO binds GAGs on the platelet surface and degrades heme in order to protect platelets from heme-induced activation. To assess the ability of XO to degrade heme, we tested whether XO binds heme. We performed computational modeling in which we identified a potential heme binding site in the FAD domain of XO with a kd = 128 nM. We confirmed heme-XO binding by performing a heme binding assay. We incubated heme (25 μM) alone, heme + XO (50 μM), and heme + XO + hypoxanthine (100 μM) for 20 minutes. Heme binding was assessed by dot blotting in nitrocellulose followed by chemiluminescent detection and dot density quantification. We observed a 2-fold increase in dot density when heme and XO were incubated and a 4-fold increase with heme, XO, and hypoxanthine. These results support a potential heme-XO interaction that is amplified when the enzyme is active. Lastly, we measured XO's ability to degrade heme using UV visible spectrophotometry. We incubated hemin, XO, and hypoxanthine together and measured the absorbance over 20 minutes. We observed a decrease in absorbance at 618 nm, indicative of heme degradation. In conclusion, contrary to the current dogma, we have identified a potential protective role for XO during hemolytic crisis. We found that febuxostat treatment worsened survival in heme challenged mice, XO prevented heme-induced platelet activation, identified a potential heme-XO binding site, and observed XO-induced heme degradation. XO may have a protective role in hemolytic conditions by serving as a secondary mechanism of heme degradation. Disclosures No relevant conflicts of interest to declare.