Heme oxygenase-2 (HO-2) binds and buffers labile ferric heme in human embryonic kidney cells
2021; Elsevier BV; Volume: 298; Issue: 2 Linguagem: Inglês
10.1016/j.jbc.2021.101549
ISSN1083-351X
AutoresDavid A. Hanna, Courtney M. Moore, Liu Liu, Xiaojing Yuan, Iramofu M. Dominic, Angela S. Fleischhacker, Iqbal Hamza, Stephen W. Ragsdale, Amit R. Reddi,
Tópico(s)Cannabis and Cannabinoid Research
ResumoHeme oxygenases (HOs) detoxify heme by oxidatively degrading it into carbon monoxide, iron, and biliverdin, which is reduced to bilirubin and excreted. Humans express two isoforms of HO: the inducible HO-1, which is upregulated in response to excess heme and other stressors, and the constitutive HO-2. Much is known about the regulation and physiological function of HO-1, whereas comparatively little is known about the role of HO-2 in regulating heme homeostasis. The biochemical necessity for expressing constitutive HO-2 is dependent on whether heme is sufficiently abundant and accessible as a substrate under conditions in which HO-1 is not induced. By measuring labile heme, total heme, and bilirubin in human embryonic kidney HEK293 cells with silenced or overexpressed HO-2, as well as various HO-2 mutant alleles, we found that endogenous heme is too limiting a substrate to observe HO-2-dependent heme degradation. Rather, we discovered a novel role for HO-2 in the binding and buffering of heme. Taken together, in the absence of excess heme, we propose that HO-2 regulates heme homeostasis by acting as a heme buffering factor that controls heme bioavailability. When heme is in excess, HO-1 is induced, and both HO-2 and HO-1 can provide protection from heme toxicity via enzymatic degradation. Our results explain why catalytically inactive mutants of HO-2 are cytoprotective against oxidative stress. Moreover, the change in bioavailable heme due to HO-2 overexpression, which selectively binds ferric over ferrous heme, is consistent with labile heme being oxidized, thereby providing new insights into heme trafficking and signaling. Heme oxygenases (HOs) detoxify heme by oxidatively degrading it into carbon monoxide, iron, and biliverdin, which is reduced to bilirubin and excreted. Humans express two isoforms of HO: the inducible HO-1, which is upregulated in response to excess heme and other stressors, and the constitutive HO-2. Much is known about the regulation and physiological function of HO-1, whereas comparatively little is known about the role of HO-2 in regulating heme homeostasis. The biochemical necessity for expressing constitutive HO-2 is dependent on whether heme is sufficiently abundant and accessible as a substrate under conditions in which HO-1 is not induced. By measuring labile heme, total heme, and bilirubin in human embryonic kidney HEK293 cells with silenced or overexpressed HO-2, as well as various HO-2 mutant alleles, we found that endogenous heme is too limiting a substrate to observe HO-2-dependent heme degradation. Rather, we discovered a novel role for HO-2 in the binding and buffering of heme. Taken together, in the absence of excess heme, we propose that HO-2 regulates heme homeostasis by acting as a heme buffering factor that controls heme bioavailability. When heme is in excess, HO-1 is induced, and both HO-2 and HO-1 can provide protection from heme toxicity via enzymatic degradation. Our results explain why catalytically inactive mutants of HO-2 are cytoprotective against oxidative stress. Moreover, the change in bioavailable heme due to HO-2 overexpression, which selectively binds ferric over ferrous heme, is consistent with labile heme being oxidized, thereby providing new insights into heme trafficking and signaling. Heme is an essential but potentially cytotoxic metallocofactor and signaling molecule (1Chiabrando D. Mercurio S. Tolosano E. Heme and erythropoieis: More than a structural role.Haematologica. 2014; 99: 973-983Google Scholar, 2Chiabrando D. Vinchi F. Fiorito V. Mercurio S. Tolosano E. Heme in pathophysiology: A matter of scavenging, metabolism and trafficking across cell membranes.Front. Pharmacol. 2014; 5: 61Google Scholar, 3Kumar S. Bandyopadhyay U. Free heme toxicity and its detoxification systems in human.Toxicol. Lett. 2005; 157: 175-188Google Scholar, 4Poulos T.L. Heme enzyme structure and function.Chem. Rev. 2014; 114: 3919-3962Google Scholar, 5Zhang L. Hach A. Wang C. Molecular mechanism governing heme signaling in yeast: A higher-order complex mediates heme regulation of the transcriptional activator HAP1.Mol. Cell. Biol. 1998; 18: 3819-3828Google Scholar, 6Zhang L. Hach A. Molecular mechanism of heme signaling in yeast: The transcriptional activator Hap1 serves as the key mediator.Cell. Mol. Life Sci. 1999; 56: 415-426Google Scholar, 7Hou S. Reynolds M.F. Horrigan F.T. Heinemann S.H. Hoshi T. Reversible binding of heme to proteins in cellular signal transduction.Acc. Chem. Res. 2006; 39: 918-924Google Scholar, 8Hanna D.A. Martinez-Guzman O. Reddi A.R. Heme gazing: Illuminating eukaryotic heme trafficking, dynamics, and signaling with fluorescent heme sensors.Biochemistry. 2017; 56: 1815-1823Google Scholar, 9Mense S.M. Zhang L. Heme: A versatile signaling molecule controlling the activities of diverse regulators ranging from transcription factors to MAP kinases.Cell Res. 2006; 16: 681-692Google Scholar, 10Kuhl T. Imhof D. Regulatory Fe(II/III) heme: The reconstruction of a molecule's biography.Chembiochem. 2014; 15: 2024-2035Google Scholar). Consequently, cells must tightly regulate the concentration and bioavailability of heme (8Hanna D.A. Martinez-Guzman O. Reddi A.R. Heme gazing: Illuminating eukaryotic heme trafficking, dynamics, and signaling with fluorescent heme sensors.Biochemistry. 2017; 56: 1815-1823Google Scholar, 11Swenson S.A. Moore C.M. Marcero J.R. Medlock A.E. Reddi A.R. Khalimonchuk O. From synthesis to utilization: The ins and outs of mitochondrial heme.Cells. 2020; 9: 579Google Scholar, 12Reddi A.R. Hamza I. Heme mobilization in animals: A metallolipid's journey.Acc. Chem. Res. 2016; 49: 1104-1110Google Scholar, 13Donegan R.K. Moore C.M. Hanna D.A. Reddi A.R. Handling heme: The mechanisms underlying the movement of heme within and between cells.Free Radic. Biol. Med. 2019; 133: 88-100Google Scholar, 14Chambers I.G. Willoughby M.M. Hamza I. Reddi A.R. One ring to bring them all and in the darkness bind them: The trafficking of heme without deliverers.Biochim. Biophys. Acta Mol. Cell Res. 2021; 1868: 118881Google Scholar). In mammals, the total intracellular concentration of heme is governed by the relative rates of de novo synthesis, degradation, import, and export. The atomic resolution structures and chemical mechanisms of all the heme biosynthetic and catabolic enzymes are known and well understood (11Swenson S.A. Moore C.M. Marcero J.R. Medlock A.E. Reddi A.R. Khalimonchuk O. From synthesis to utilization: The ins and outs of mitochondrial heme.Cells. 2020; 9: 579Google Scholar, 15Severance S. Hamza I. Trafficking of heme and porphyrins in metazoa.Chem. Rev. 2009; 109: 4596-4616Google Scholar). Although cell surface heme importers (16Duffy S.P. Shing J. Saraon P. Berger L.C. Eiden M.V. Wilde A. Tailor C.S. The Fowler syndrome-associated protein FLVCR2 is an importer of heme.Mol. Cell. Biol. 2010; 30: 5318-5324Google Scholar) and exporters (17Quigley J.G. Yang Z. Worthington M.T. Phillips J.D. Sabo K.M. Sabath D.E. Berg C.L. Sassa S. Wood B.L. Abkowitz J.L. Identification of a human heme exporter that is essential for erythropoiesis.Cell. 2004; 118: 757-766Google Scholar, 18Quigley J.G. Gazda H. Yang Z. Ball S. Sieff C.A. Abkowitz J.L. Investigation of a putative role for FLVCR, a cytoplasmic heme exporter, in Diamond-Blackfan anemia.Blood Cells Mol. Dis. 2005; 35: 189-192Google Scholar) have been identified, their molecular mechanisms remain poorly characterized and outside of developing red blood cells in the case of heme exporters, the physiological context in which they function is unclear and controversial (19Ponka P. Sheftel A.D. English A.M. Scott Bohle D. Garcia-Santos D. Do mammalian cells really need to export and import heme?.Trends Biochem. Sci. 2017; 42: 395-406Google Scholar). The bioavailability of heme, which is comparatively less well understood, is governed by a poorly characterized network of heme buffering factors, intracellular transporters, and chaperones that ensure heme is made available for heme-dependent processes located throughout the cell. When the cells are confronted with excess heme, heme synthesis is downregulated (11Swenson S.A. Moore C.M. Marcero J.R. Medlock A.E. Reddi A.R. Khalimonchuk O. From synthesis to utilization: The ins and outs of mitochondrial heme.Cells. 2020; 9: 579Google Scholar, 20Yamamoto M. Hayashi N. Kikuchi G. Evidence for the transcriptional inhibition by heme of the synthesis of delta-aminolevulinate synthase in rat liver.Biochem. Biophys. Res. Commun. 1982; 105: 985-990Google Scholar, 21Yamamoto M. Hayashi N. Kikuchi G. Translational inhibition by heme of the synthesis of hepatic delta-aminolevulinate synthase in a cell-free system.Biochem. Biophys. Res. Commun. 1983; 115: 225-231Google Scholar), and heme can be detoxified by storage into lysosome-related organelles (22Chen A.J. Yuan X. Li J. Dong P. Hamza I. Cheng J.-X. Label-free imaging of heme dynamics in living organisms by transient absorption microscopy.Anal. Chem. 2018; 90: 3395-3401Google Scholar, 23Pek R.H. Yuan X. Rietzschel N. Zhang J. Jackson L. Nishibori E. Ribeiro A. Simmons W. Jagadeesh J. Sugimoto H. Alam M.Z. Garrett L. Haldar M. Ralle M. Phillips J.D. et al.Hemozoin produced by mammals confers heme tolerance.Elife. 2019; 8e49503Google Scholar), export (24Keel S.B. Doty R.T. Yang Z. Quigley J.G. Chen J. Knoblaugh S. Kingsley P.D. De Domenico I. Vaughn M.B. Kaplan J. Palis J. Abkowitz J.L. A heme export protein is required for red blood cell differentiation and iron homeostasis.Science. 2008; 319: 825-828Google Scholar, 25Doty R.T. Phelps S.R. Shadle C. Sanchez-Bonilla M. Keel S.B. Abkowitz J.L. Coordinate expression of heme and globin is essential for effective erythropoiesis.J. Clin. Invest. 2015; 125: 4681-4691Google Scholar), or degradation (26Sassa S. Why heme needs to be degraded to iron, biliverdin IXalpha, and carbon monoxide?.Antioxid. Redox Signal. 2004; 6: 819-824Google Scholar, 27Ayer A. Zarjou A. Agarwal A. Stocker R. Heme oxygenases in cardiovascular health and disease.Physiol. Rev. 2016; 96: 1449-1508Google Scholar, 28Desmard M. Boczkowski J. Poderoso J. Motterlini R. Mitochondrial and cellular heme-dependent proteins as targets for the bioactive function of the heme oxygenase/carbon monoxide system.Antioxid. Redox Signal. 2007; 9: 2139-2155Google Scholar, 29Gozzelino R. Jeney V. Soares M.P. Mechanisms of cell protection by heme oxygenase-1.Annu. Rev. Pharmacol. Toxicol. 2010; 50: 323-354Google Scholar, 30Hill-Kapturczak N. Jarmi T. Agarwal A. Growth factors and heme oxygenase-1: Perspectives in physiology and pathophysiology.Antioxid. Redox Signal. 2007; 9: 2197-2207Google Scholar, 31Munoz-Sanchez J. Chanez-Cardenas M.E. A review on hemeoxygenase-2: Focus on cellular protection and oxygen response.Oxid. Med. Cell. Longev. 2014; 2014: 604981Google Scholar). Arguably, the best understood mechanism for heme detoxification is through the heme catabolism pathway. The first and rate-limiting step of heme degradation is catalyzed by the heme oxygenases (HO) (32Stec D.E. Ishikawa K. Sacerdoti D. Abraham N.G. The emerging role of heme oxygenase and its metabolites in the regulation of cardiovascular function.Int. J. Hypertens. 2012; 2012: 593530Google Scholar, 33Constantin M. Choi A.J. Cloonan S.M. Ryter S.W. Therapeutic potential of heme oxygenase-1/carbon monoxide in lung disease.Int. J. Hypertens. 2012; 2012: 859235Google Scholar). Mammals encode two HO isoforms, inducible HO-1 and constitutive HO-2 (34Maines M.D. The heme oxygenase system: A regulator of second messenger gases.Annu. Rev. Pharmacol. Toxicol. 1997; 37: 517-554Google Scholar, 35Maines M.D. The heme oxygenase system: Update 2005.Antioxid. Redox Signal. 2005; 7: 1761-1766Google Scholar, 36Maines M.D. Trakshel G.M. Kutty R.K. Characterization of two constitutive forms of rat liver microsomal heme oxygenase. Only one molecular species of the enzyme is inducible.J. Biol. Chem. 1986; 261: 411-419Google Scholar, 37Trakshel G.M. Kutty R.K. Maines M.D. Purification and characterization of the major constitutive form of testicular heme oxygenase. The noninducible isoform.J. Biol. Chem. 1986; 261: 11131-11137Google Scholar). HO-1 and HO-2 are structurally similar, both in primary sequence and tertiary structure, operate using the same chemical mechanism, and exhibit similar catalytic properties, including Michaelis constants (KM) and maximal velocities (Vmax) (37Trakshel G.M. Kutty R.K. Maines M.D. Purification and characterization of the major constitutive form of testicular heme oxygenase. The noninducible isoform.J. Biol. Chem. 1986; 261: 11131-11137Google Scholar, 38Bianchetti C.M. Yi L. Ragsdale S.W. Phillips Jr., G.N. Comparison of apo- and heme-bound crystal structures of a truncated human heme oxygenase-2.J. Biol. Chem. 2007; 282: 37624-37631Google Scholar, 39Kochert B.A. Fleischhacker A.S. Wales T.E. Becker D.F. Engen J.R. Ragsdale S.W. Dynamic and structural differences between heme oxygenase-1 and -2 are due to differences in their C-terminal regions.J. Biol. Chem. 2019; 294: 8259-8272Google Scholar). HOs, which are primarily anchored into the endoplasmic reticulum (ER) membrane and whose active sites face the cytoplasm, bind oxidized ferric heme in its resting state using a histidine axial ligand. Upon reduction, using electrons from the NADPH-cytochrome P450 reductase (CPR) system and dioxygen binding (O2), HOs catalyze the oxidative degradation of heme to form biliverdin, ferrous iron (Fe2+), and carbon monoxide (CO) (40Trakshel G.M. Kutty R.K. Maines M.D. Cadmium-mediated inhibition of testicular heme oxygenase activity: The role of NADPH-cytochrome c (P-450) reductase.Arch. Biochem. Biophys. 1986; 251: 175-187Google Scholar, 41Matsui T. Iwasaki M. Sugiyama R. Unno M. Ikeda-Saito M. Dioxygen activation for the self-degradation of heme: Reaction mechanism and regulation of heme oxygenase.Inorg. Chem. 2010; 49: 3602-3609Google Scholar, 42Yoshida T. Migita C.T. Mechanism of heme degradation by heme oxygenase.J. Inorg. Biochem. 2000; 82: 33-41Google Scholar, 43Kumar D. de Visser S.P. Shaik S. Theory favors a stepwise mechanism of porphyrin degradation by a ferric hydroperoxide model of the active species of heme oxygenase.J. Am. Chem. Soc. 2005; 127: 8204-8213Google Scholar, 44Lightning L.K. Huang H. Moenne-Loccoz P. Loehr T.M. Schuller D.J. Poulos T.L. de Montellano P.R. Disruption of an active site hydrogen bond converts human heme oxygenase-1 into a peroxidase.J. Biol. Chem. 2001; 276: 10612-10619Google Scholar). Biliverdin is subsequently rapidly metabolized to bilirubin via a NADPH-biliverdin reductase and expelled from the cells (45Liu Y. Liu J. Tetzlaff W. Paty D.W. Cynader M.S. Biliverdin reductase, a major physiologic cytoprotectant, suppresses experimental autoimmune encephalomyelitis.Free Radic. Biol. Med. 2006; 40: 960-967Google Scholar, 46Baranano D.E. Rao M. Ferris C.D. Snyder S.H. Biliverdin reductase: A major physiologic cytoprotectant.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16093-16098Google Scholar). Given that heme catabolites ferrous iron, CO, biliverdin, and bilirubin have their own distinct beneficial or detrimental effects on cell physiology in various contexts, the activity of HO enzymes and availability of its heme substrate can impact metabolism in numerous ways (32Stec D.E. Ishikawa K. Sacerdoti D. Abraham N.G. The emerging role of heme oxygenase and its metabolites in the regulation of cardiovascular function.Int. J. Hypertens. 2012; 2012: 593530Google Scholar, 47Pamplona A. Ferreira A. Balla J. Jeney V. Balla G. Epiphanio S. Chora A. Rodrigues C.D. Gregoire I.P. Cunha-Rodrigues M. Portugal S. Soares M.P. Mota M.M. Heme oxygenase-1 and carbon monoxide suppress the pathogenesis of experimental cerebral malaria.Nat. Med. 2007; 13: 703-710Google Scholar, 48Ong W.Y. Farooqui A.A. Iron, neuroinflammation, and Alzheimer's disease.J. Alzheimers Dis. 2005; 8 (discussion 209-115): 183-200Google Scholar, 49Jazwa A. Cuadrado A. Targeting heme oxygenase-1 for neuroprotection and neuroinflammation in neurodegenerative diseases.Curr. Drug Targets. 2010; 11: 1517-1531Google Scholar, 50Lundvig D.M. Immenschuh S. Wagener F.A. Heme oxygenase, inflammation, and fibrosis: The good, the bad, and the ugly?.Front. Pharmacol. 2012; 3: 81Google Scholar, 51Motterlini R. Otterbein L.E. The therapeutic potential of carbon monoxide.Nat. Rev. Drug Discov. 2010; 9: 728-743Google Scholar, 52Wegiel B. Otterbein L.E. Go green: The anti-inflammatory effects of biliverdin reductase.Front. Pharmacol. 2012; 3: 47Google Scholar, 53Otterbein L.E. Bach F.H. Alam J. Soares M. Tao Lu H. Wysk M. Davis R.J. Flavell R.A. Choi A.M. Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway.Nat. Med. 2000; 6: 422-428Google Scholar, 54Ryter S.W. Tyrrell R.M. The heme synthesis and degradation pathways: Role in oxidant sensitivity. Heme oxygenase has both pro- and antioxidant properties.Free Radic. Biol. Med. 2000; 28: 289-309Google Scholar, 55Stocker R. Yamamoto Y. McDonagh A.F. Glazer A.N. Ames B.N. Bilirubin is an antioxidant of possible physiological importance.Science. 1987; 235: 1043-1046Google Scholar). Although the structures and mechanisms of HO-1 and HO-2 are largely the same (37Trakshel G.M. Kutty R.K. Maines M.D. Purification and characterization of the major constitutive form of testicular heme oxygenase. The noninducible isoform.J. Biol. Chem. 1986; 261: 11131-11137Google Scholar, 38Bianchetti C.M. Yi L. Ragsdale S.W. Phillips Jr., G.N. Comparison of apo- and heme-bound crystal structures of a truncated human heme oxygenase-2.J. Biol. Chem. 2007; 282: 37624-37631Google Scholar, 39Kochert B.A. Fleischhacker A.S. Wales T.E. Becker D.F. Engen J.R. Ragsdale S.W. Dynamic and structural differences between heme oxygenase-1 and -2 are due to differences in their C-terminal regions.J. Biol. Chem. 2019; 294: 8259-8272Google Scholar, 41Matsui T. Iwasaki M. Sugiyama R. Unno M. Ikeda-Saito M. Dioxygen activation for the self-degradation of heme: Reaction mechanism and regulation of heme oxygenase.Inorg. Chem. 2010; 49: 3602-3609Google Scholar, 43Kumar D. de Visser S.P. Shaik S. Theory favors a stepwise mechanism of porphyrin degradation by a ferric hydroperoxide model of the active species of heme oxygenase.J. Am. Chem. Soc. 2005; 127: 8204-8213Google Scholar, 56Davydov R. Fleischhacker A.S. Bagai I. Hoffman B.M. Ragsdale S.W. Comparison of the mechanisms of heme hydroxylation by heme oxygenases-1 and -2: Kinetic and cryoreduction studies.Biochemistry. 2016; 55: 62-68Google Scholar), the regulation and expression of these two enzymes is very different (27Ayer A. Zarjou A. Agarwal A. Stocker R. Heme oxygenases in cardiovascular health and disease.Physiol. Rev. 2016; 96: 1449-1508Google Scholar, 31Munoz-Sanchez J. Chanez-Cardenas M.E. A review on hemeoxygenase-2: Focus on cellular protection and oxygen response.Oxid. Med. Cell. Longev. 2014; 2014: 604981Google Scholar, 35Maines M.D. The heme oxygenase system: Update 2005.Antioxid. Redox Signal. 2005; 7: 1761-1766Google Scholar). Heme oxygenase-1, which is comparatively far better understood, is induced by excess heme, as well as several nonheme stressors like oxidative stress, infection, and exposure to various xenobiotics (57Prawan A. Kundu J.K. Surh Y.J. Molecular basis of heme oxygenase-1 induction: Implications for chemoprevention and chemoprotection.Antioxid. Redox Signal. 2005; 7: 1688-1703Google Scholar, 58Funes S.C. Rios M. Fernandez-Fierro A. Covian C. Bueno S.M. Riedel C.A. Mackern-Oberti J.P. Kalergis A.M. Naturally derived heme-oxygenase 1 inducers and their therapeutic application to immune-mediated diseases.Front. Immunol. 2020; 11: 1467Google Scholar, 59Campbell N.K. Fitzgerald H.K. Malara A. Hambly R. Sweeney C.M. Kirby B. Fletcher J.M. Dunne A. Naturally derived heme-oxygenase 1 inducers attenuate inflammatory responses in human dendritic cells and T cells: Relevance for psoriasis treatment.Sci. Rep. 2018; 8: 10287Google Scholar, 60Alam J. Shibahara S. Smith A. Transcriptional activation of the heme oxygenase gene by heme and cadmium in mouse hepatoma cells.J. Biol. Chem. 1989; 264: 6371-6375Google Scholar, 61Keyse S.M. Applegate L.A. Tromvoukis Y. Tyrrell R.M. Oxidant stress leads to transcriptional activation of the human heme oxygenase gene in cultured skin fibroblasts.Mol. Cell. Biol. 1990; 10: 4967-4969Google Scholar). HO-2, on the other hand, is constitutively expressed across all tissues and cell types, being most abundant in the brain and testis (34Maines M.D. The heme oxygenase system: A regulator of second messenger gases.Annu. Rev. Pharmacol. Toxicol. 1997; 37: 517-554Google Scholar, 35Maines M.D. The heme oxygenase system: Update 2005.Antioxid. Redox Signal. 2005; 7: 1761-1766Google Scholar). The current rationale for dual mammalian HO isoforms is that HO-2 provides a baseline level of protection from heme in the absence of cellular stressors that would otherwise induce HO-1. However, the biochemical necessity for expressing constitutive HO-2 is largely dependent on whether sufficient heme is available as a substrate under conditions in which HO-1 is not induced. Total cellular heme in yeast and various nonerythroid human cell lines is on the order of 1 to 20 μM (62Hanna D.A. Hu R. Kim H. Martinez-Guzman O. Torres M.P. Reddi A.R. Heme bioavailability and signaling in response to stress in yeast cells.J. Biol. Chem. 2018; 293: 12378-12393Google Scholar, 63Hanna D.A. Harvey R.M. Martinez-Guzman O. Yuan X. Chandrasekharan B. Raju G. Outten F.W. Hamza I. Reddi A.R. Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 7539-7544Google Scholar, 64Liu L. Dumbrepatil A.B. Fleischhacker A.S. Marsh E.N.G. Ragsdale S.W. Heme oxygenase-2 is post-translationally regulated by heme occupancy in the catalytic site.J. Biol. Chem. 2020; 295: 17227-17240Google Scholar, 65Hopp M.T. Schmalohr B.F. Kuhl T. Detzel M.S. Wissbrock A. Imhof D. Heme determination and quantification methods and their suitability for practical applications and everyday use.Anal. Chem. 2020; 92: 9429-9440Google Scholar, 66Martinez-Guzman O. Willoughby M.M. Saini A. Dietz J.V. Bohovych I. Medlock A.E. Khalimonchuk O. Reddi A.R. Mitochondrial-nuclear heme trafficking in budding yeast is regulated by GTPases that control mitochondrial dynamics and ER contact sites.J. Cell Sci. 2020; 133jcs237917Google Scholar, 67Mestre-Fos S. Ito C. Moore C.M. Reddi A.R. Williams L.D. Human ribosomal G-quadruplexes regulate heme bioavailability.J. Biol. Chem. 2020; 295: 14855-14865Google Scholar). All heme in the cell partitions between exchange inert high affinity hemoproteins, such as cytochromes and other heme enzymes, and certain exchange labile heme (LH) complexes that buffer free heme down to nanomolar concentrations (8Hanna D.A. Martinez-Guzman O. Reddi A.R. Heme gazing: Illuminating eukaryotic heme trafficking, dynamics, and signaling with fluorescent heme sensors.Biochemistry. 2017; 56: 1815-1823Google Scholar, 12Reddi A.R. Hamza I. Heme mobilization in animals: A metallolipid's journey.Acc. Chem. Res. 2016; 49: 1104-1110Google Scholar, 13Donegan R.K. Moore C.M. Hanna D.A. Reddi A.R. Handling heme: The mechanisms underlying the movement of heme within and between cells.Free Radic. Biol. Med. 2019; 133: 88-100Google Scholar, 14Chambers I.G. Willoughby M.M. Hamza I. Reddi A.R. One ring to bring them all and in the darkness bind them: The trafficking of heme without deliverers.Biochim. Biophys. Acta Mol. Cell Res. 2021; 1868: 118881Google Scholar, 63Hanna D.A. Harvey R.M. Martinez-Guzman O. Yuan X. Chandrasekharan B. Raju G. Outten F.W. Hamza I. Reddi A.R. Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 7539-7544Google Scholar, 68Song Y. Yang M. Wegner S.V. Zhao J. Zhu R. Wu Y. He C. Chen P.R. A genetically encoded FRET sensor for intracellular heme.ACS Chem. Biol. 2015; 10: 1610-1615Google Scholar, 69Leung G.C.-H. Fung S.S.-P. Gallio A.E. Blore R. Alibhai D. Raven E.L. Hudson A.J. Unravelling the mechanisms controlling heme supply and demand.Proc. Natl. Acad. Sci. U. S. A. 2021; 118e2104008118Google Scholar). The factors that buffer heme are poorly understood, but likely consist of a network of heme-binding proteins, nucleic acids, and lipid membranes (8Hanna D.A. Martinez-Guzman O. Reddi A.R. Heme gazing: Illuminating eukaryotic heme trafficking, dynamics, and signaling with fluorescent heme sensors.Biochemistry. 2017; 56: 1815-1823Google Scholar, 12Reddi A.R. Hamza I. Heme mobilization in animals: A metallolipid's journey.Acc. Chem. Res. 2016; 49: 1104-1110Google Scholar, 13Donegan R.K. Moore C.M. Hanna D.A. Reddi A.R. Handling heme: The mechanisms underlying the movement of heme within and between cells.Free Radic. Biol. Med. 2019; 133: 88-100Google Scholar, 14Chambers I.G. Willoughby M.M. Hamza I. Reddi A.R. One ring to bring them all and in the darkness bind them: The trafficking of heme without deliverers.Biochim. Biophys. Acta Mol. Cell Res. 2021; 1868: 118881Google Scholar, 63Hanna D.A. Harvey R.M. Martinez-Guzman O. Yuan X. Chandrasekharan B. Raju G. Outten F.W. Hamza I. Reddi A.R. Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 7539-7544Google Scholar, 66Martinez-Guzman O. Willoughby M.M. Saini A. Dietz J.V. Bohovych I. Medlock A.E. Khalimonchuk O. Reddi A.R. Mitochondrial-nuclear heme trafficking in budding yeast is regulated by GTPases that control mitochondrial dynamics and ER contact sites.J. Cell Sci. 2020; 133jcs237917Google Scholar, 67Mestre-Fos S. Ito C. Moore C.M. Reddi A.R. Williams L.D. Human ribosomal G-quadruplexes regulate heme bioavailability.J. Biol. Chem. 2020; 295: 14855-14865Google Scholar, 70Sweeny E.A. Singh A.B. Chakravarti R. Martinez-Guzman O. Saini A. Haque M.M. Garee G. Dans P.D. Hannibal L. Reddi A.R. Stuehr D.J. Glyceraldehyde-3-phosphate dehydrogenase is a chaperone that allocates labile heme in cells.J. Biol. Chem. 2018; 293: 14557-14568Google Scholar, 71Gray L.T. Puig Lombardi E. Verga D. Nicolas A. Teulade-Fichou M.P. Londono-Vallejo A. Maizels N. G-quadruplexes sequester free heme in living cells.Cell Chem. Biol. 2019; 26: 1681-1691.e5Google Scholar). Labile heme may act as a reservoir for bioavailable heme that can readily exchange with and populate heme-binding sites in heme dependent or regulated enzymes and proteins. The nature of LH, including its speciation, oxidation state, concentration, and distribution are not well understood but may be relevant for the mobilization and trafficking of heme. It is currently not known what the source of heme is for HOs, that is, whether it is buffered-free heme or a dedicated chaperone system that traffics and channels heme to HO in a manner that bypasses the LH pool. The recent development of fluorescence and activity-based heme sensors has offered unprecedented insights into LH and their diverse roles in physiology (63Hanna D.A. Harvey R.M. Martinez-Guzman O. Yuan X. Chandrasekharan B. Raju G. Outten F.W. Hamza I. Reddi A.R. Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 7539-7544Google Scholar, 68Song Y. Yang M. Wegner S.V. Zhao J. Zhu R. Wu Y. He C. Chen P.R. A genetically encoded FRET sensor for intracellular heme.ACS Chem. Biol. 2015; 10: 1610-1615Google Scholar, 69Leung G.C.-H. Fung S.S.-P. Gallio A.E. Blore R. Alibhai D. Raven E.L. Hudson A.J. Unravelling the mechanisms controlling heme supply and demand.Proc. Natl. Acad. Sci. U. S. A. 2021; 118e2104008118Google Scholar, 72Abshire J.R. Rowlands C.J. Ganesan S.M. So P.T. Niles J.C. Quantification of labile heme in live malaria parasites using a genetically encoded biosensor.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: E2068-E2076Google Scholar, 73Yuan X. Rietzschel N. Kwon H. Walter Nuno A.B. Hanna D.A. Phillips J.D. Raven E.L. Reddi A.R. Hamza I. Regulation of intracellular heme trafficking revealed by subcellular reporters.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: E5144-E5152Google Scholar). Strictly speaking, these probes report on the availability of heme to the sensor, not necessarily free heme coordinated by water (8Hanna D.A. Martinez-Guzman O. Reddi A.R. Heme gazing: Illuminating eukaryotic heme trafficking, dynamics, and signaling with fluorescent heme sensors.Biochemistry. 2017; 56: 1815-1823Google Scholar, 13Donegan R.K. Moore C.M. Hanna D.A. Reddi A.R. Handling heme: The mechanisms underlying the movement of heme within and between cells.Free Radic. Biol. Med. 2019; 133: 88-100Google Scholar, 74Bal W. Kurowska E. Maret W. The final frontier of pH and the undiscovered country beyond.PLoS One. 2012; 7e45832Google Scholar). In other words, the heme occupancy of the sensor is dictated by the extent to which LH can exchange with the probe. However, many investigators convert the fractional heme loading of a probe to a buffered-free heme concentration, which can be done if the heme-sensor dissociation constant is known. Although problematic in that the sensor may not be probing "free heme", it nonetheless provides a measure of labile or accessible heme because the calculated concentration of free heme is related to sensor heme occupancy. In intact living yeast and various nonerythroid human cell lines, the estimates of buffered-free heme based on genetically encoded heme sensors are on the order of ∼5 to 20 nM (63Hanna D.A. Harvey R.M. Martinez-Guzman O. Yuan X. Chandrasekharan B. Raju G. Outten F.W. Hamza I. Reddi A.R. Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 7539-7544Google Scholar, 68Song Y. Yang M. Wegner S.V. Zhao
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