Importance of ligand-induced conformational changes in hemopexin for receptor-mediated heme transport

Hemopexin alters conformation upon binding heme as shown by circular dichroism (CD), but hemopexin binds the heme analog, iron-meso-tetra-(4-sulfonatophenyl)-porphine (FeTPPS), without undergoing concomitant changes in its CD spectrum. Moreover, FeTPPS, unlike heme, does not increase the compactness of the heme-binding domain (I) of hemopexin shown by an increased sedimentation rate in sucrose gradients. On the other hand, like heme, FeTPPS forms a bishistidyl coordination complex with hemopexin and upon binding protects hemopexin from cleavage by plasmin. Competitive inhibition and saturation studies demonstrate that FeTPPS-hemopexin binds to the hemopexin receptor on mouse hepatoma cells but with a lower affinity (Kd 125 nM) more characteristic of apo-hemopexin than heme-hemopexin (Kd 65 nM). This provides evidence that conformational changes produced in hemopexin upon binding heme, but not upon binding FeTPPS, are important for increasing the affinity of hemopexin for its receptor. The amount of cell-associated radiolabel from 55FeTPPS-hemopexin increases linearly for up to 90 min but at a rate only about a third of that of the mesoheme-complex. As expected from the recycling of hemopexin, more iron-tetrapyrrole than protein is associated with the Hepa cells, but the ratio of 55Fe-ligand to 125I-hemopexin is only 2:1 for FeTPPS-hemopexin compared to 4:1 for mesoheme complexes. [55Fe]Mesoheme was associated at 5 min with lower density fractions containing plasma membranes and at 30 min with fractions containing higher density intracellular compartments. In contrast, 55FeTPPS was found associated with plasma membrane fractions at both times and was not transported into the cell. Although FeTPPS-hemopexin binds to the receptor, subsequent events of heme transport are impaired. The results indicate that upon binding heme at least three types of conformational changes occur in hemopexin which have important roles in receptor recognition and that the nature of the ligand influences subsequent heme transport.     

Thermodynamics of heme-induced conformational changes in hemopexin: role of domain-domain interactions.

Hemopexin is a serum glycoprotein that binds heme with high affinity and delivers heme to the liver cells via receptor-mediated endocytosis. A hinge region connects the two non-disulfide-linked domains of hemopexin, a 35-kDa N-terminal domain (domain I) that binds heme, and a 25-kDa C-terminal domain (domain II). Although domain II does not bind heme, it assumes one structural state in apo-hemopexin and another in heme-hemopexin, and this change is important in facilitating the association of heme-hemopexin with its receptor. In order to elucidate the structure and function of hemopexin, it is important to understand how structural information is transmitted to domain II when domain I binds heme. Here we report a study of the protein-protein interactions between domain I and domain II using analytical ultracentrifugation and isothermal titration calorimetry. Sedimentation equilibrium analysis showed that domain I associates with domain II both in the presence and absence of heme with Kd values of 0.8 microM and 55 microM, respectively. The interaction between heme-domain I and domain II has a calorimetric enthalpy of +11 kcal/mol, a heat capacity (delta Cp) of -720 cal/mol.K, and a calculated entropy of +65 cal/mol.K. By varying the temperature of the centrifugation equilibrium runs, a van't Hoff plot with an apparent change in enthalpy (delta H) of -3.6 kcal/mol and change in entropy (delta S) of +8.1 cal/mol.K for the association of apo-domain I with domain II was obtained.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell surface receptor for hemopexin in human leukemia HL60 cells. Specific binding, affinity labeling, and fate of heme

The binding of 125I-labeled human hemopexin to human leukemia HL60 cell at 4 degrees C was saturable with time and with increasing concentrations of 125I-hemopexin. Scatchard analysis of the binding data revealed the presence of approximately 42,000 binding sites/cell with an apparent dissociation constant (Kd) of 1.0 X 10(-9) M. When cells were incubated with radioactive hemopexin at 37 degrees C, 125I-hemopexin was rapidly bound and then was dissociated after the release of heme. Treatment of surface-bound 125I-hemopexin with divalent lysine-directed cross-linking disuccinimidyl suberate revealed a membrane polypeptide of about 80,000 Da, to which hemopexin is cross-linked. To examine the fate of the internalized heme, lysates from the cells previously incubated with [59Fe]heme-hemopexin complex were analyzed by CM-cellulose and Sephacryl S-200 column chromatography. A considerable amount of the radioactivity was present in the fraction which co-eluted with the myeloperoxidase activity. When myeloperoxidase was isolated from the cells incubated with [59Fe]heme-hemopexin complex by immunoprecipitation with anti-myeloperoxidase antibody, radiolabeled iron associated with myeloperoxidase increased with time, and more than 30% of the radioactivity in the cells was present in the myeloperoxidase. These results indicate that the binding of hemopexin to the surface receptors triggers a release of heme and that this heme is incorporated into the intracellular myeloperoxidase.     

The aromatic and heme chromophores of rabbit hemopexin. Difference absorption and fluorescence spectra.

Spectrophotometric and fluorimetric techniques were employed to charcterize the environment of the heme chromophore of rabbit hemopexin and to monitor changes in the environment of aromatic amino acid residues induced by the interaction of hemopexin with porphyrins and metalloporphyrins. Difference spectra showed maxima at 292 and 285 nm when hemopexin binds heme or deuteroheme but not deuteroporphyrin. These maxima are attributed to alterations in the local environment of tryptophan and tyrosine residues. Spectro-photometric titrations of the tyrosine residues of hemopexin, heme-hemopexin and hemopexin in 8 M urea showed apparent pK values at 11.4, 11.7, and 10.9 respectively. Perturbation difference spectra produced by 20% v/v ethylene glycol are consistent with the exposure of 6-8 of the 14 tyrosine residues and 6-8 of the 15 tryptophan residues of rabbit hemopexin to this perturbant. Only small differences were found between the perturbation spectra of apo- and heme-hemopexin near 290 nm, suggesting that slight or compensating changes in the exposure to solvent of tryptophan chromophores occur. In the Soret spectral region, the exposure of heme in the heme-hemopexin complex to ethylene glycol was 0.7, relative to the fully exposed heme peptide of cytochrome c. The fluorescence quantum yields of rabbit apo- and heme-hemopexin were estimated to be 0.06 and 0.03, respectively, compared to a yield of 0.13 for L-tryptophan. Iodide quenched 50% of the fluorescence of the deuteroheme-hemopexin complex. Cesium was not an effective quencher. Modification of approximately, 4 tryptophan residues with N-bromosuccinimide also decreased the relative fluorescence of apo-hemopexin by 50% and concomitantly reduced the heme-binding ability of the protein by 70%. The existence of sterically unhindered tryptophan residues in either apo- heme-hemopexin is unlikely since no charge transfer compelxes between these proteins and N-methylnicotinamide were detected.     

Hemopexin-mediated heme transport to the liver. Evidence for a heme-binding protein in liver plasma membranes

Isolated liver plasma membranes interact with heme-hemopexin and effect the removal of heme from the complex. This heme is rapidly accumulated by a previously undescribed heme-binding membrane component (HBC). This intrinsic membrane component can be solubilized from the membrane with Triton X-100 in a form that retains the ability to bind heme. Solubilized HBC was shown to be distinct from hemopexin itself, free heme, ligandin, globin, heme oxygenase, cytochrome P-450, and albumin. Since formation of the heme-HBC complex is effected by the interaction of heme-hemopexin with its receptor, HBC may either be a subunit of the heme-hemopexin receptor or a separate protein that interacts with the receptor. HBC can also bind heme (Kd apparent 200 nM) that is presented to it in a nonprotein bound form, showing true heme-binding activity. HBC is proteinaceous since treatment with proteases, heat, and disulfide bond reducing agents diminishes its ability to bind heme. HBC and any associated detergent elutes from Sephacryl S-200 with an apparent molecular weight of 115,000 and Stokes radius of 7.5 nm. This component, which may comprise 0.5% of liver plasma membrane protein, appears to have an acidic pI since it adsorbs to DEAE-cellulose at pH 7.4 but not to CM-cellulose at pH 6.4. In sucrose gradients, HBC migrates with S values of 1.69 and 4.02, suggesting that it has subunits or that it forms multimers under these conditions.     

Mechanisms involved in the cellular uptake of hematoporphyrin by rat hepatoma cells

To clarify the mechanisms involved in the specific uptake of hematoporphyrin by cancer cells, we investigated the interaction of the heme- and/or hematoporphyrin-hemopexin complexes with rat hepatoma dRLh-84 cells. Hemopexin bound to the cells in a saturable, time- and temperature-dependent manner. The cells exhibited 0.55 nmol of binding sites/mg of protein for the heme-hemopexin complex and 0.38 nmol for the hematoporphyrin-hemopexin complex. The dissociation constants (Kd) for the heme-hemopexin and hematoporphyrin-hemopexin complexes were 0.57 and 0.54 microM, respectively. Specific binding of the labeled hemopexin was inhibited by the unlabeled heme- and hematoporphyrin-hemopexin complexes but was unaffected by albumin or neoglycoprotein. Hematoporphyrin bound to hemopexin was incorporated into the cells at 37 degrees C, but not at 4 degrees C. These results indicate that hematoporphyrin bound hemopexin was taken up by dRLh-84 cells, via the hemopexin receptors. When the hematoporphyrin-albumin complex was incubated with the cells, the hematoporphyrin-[125I]albumin complex bound to the cells in a time and temperature-dependent manner. Here the binding was not saturated up to 100 micrograms/ml of albumin. The binding of hematoporphyrin-[125I]albumin was partially inhibited by unlabeled albumin and hemopexin. Hematoporphyrin bound to albumin was taken up by the cells at 37 degrees C. Thus, the albumin-dependent uptake of hematoporphyrin by rat hepatoma dRL-84 cells could be differentiated from the hemopexin-mediated uptake of hematoporphyrin.     

Receptor-mediated heme uptake from hemopexin by human erythroleukemia K562 cells

Using human erythroleukemia K562 cells, existence of receptors for hemopexin has been investigated. Hemopexin was bound to the cells in saturable, time- and temperature-dependent manner. The cells exhibited approximately 8,400 binding sites/cell for hemopexin and apohemopexin. The dissociation constants (Kd) for hemopexin and apohemopexin were 4.79 nM and 10.8 nM, respectively. Specific binding of labeled hemopexin was inhibited with increasing concentrations of unlabeled hemopexin and apohemopexin, but unaffected by transferrin and serum albumin. Heme bound to hemopexin was incorporated into the cells at 37 degrees C, but not at 4 degrees C. These results indicate that heme in hemopexin was taken up by K562 cells via the receptors for hemopexin.     

Crystal structure of hemopexin reveals a novel high-affinity heme site formed between two beta-propeller domains.

The ubiquitous use of heme in animals poses severe biological and chemical challenges. Free heme is toxic to cells and is a potential source of iron for pathogens. For protection, especially in conditions of trauma, inflammation and hemolysis, and to maintain iron homeostasis, a high-affinity binding protein, hemopexin, is required. Hemopexin binds heme with the highest affinity of any known protein, but releases it into cells via specific receptors. The crystal structure of the heme-hemopexin complex reveals a novel heme binding site, formed between two similar four-bladed beta-propeller domains and bounded by the interdomain linker. The ligand is bound to two histidine residues in a pocket dominated by aromatic and basic groups. Further stabilization is achieved by the association of the two beta-propeller domains, which form an extensive polar interface that includes a cushion of ordered water molecules. We propose mechanisms by which these structural features provide the dual function of heme binding and release.     

The source of heme for vascular heme oxygenase I: heme uptake in rat aorta

During the last decade, heme oxygenase (HO) and carbon monoxide (CO) have garnered substantial research interest in terms of cell and organ regulation, especially as they bear on the central nervous system, organ transplantation, and the cardiovascular system. While the enzymatic mechanism, substrates, and products of HO are well known, it is not clear whether the cardiovascular system derives its supply of the heme substrate through de novo synthesis or uptake from the extracellular milieu. The objective of the present study was to test the latter possibility in rat aorta and to determine the influence of plasma proteins that bind heme in vivo, viz. hemopexin and albumin. Aortic tissue was exposed to [14C]heme in vitro, and the concentration and time dependence of heme uptake was assessed. The presence of hemopexin or albumin in the incubation medium dramatically decreased heme uptake by the aorta. Heme uptake by aortic tissue was not altered after induction of HO-1, which would be expected to increase tissue heme demand. In summary, the rat, isolated aorta was capable of obtaining heme from its external milieu, but this was obtunded in the presence of the plasma proteins hemopexin or albumin. For normal physiological situations, heme uptake may not be a usual source of substrate for vascular HO and hemoenzymes such as nitric oxide synthase, soluble guanylyl cyclase, and cyclooxygenase.     

Transport of heme by hemopexin to the liver: Evidence for receptor-mediated uptake

We used carefully defined heme-hemopexin complexes to investigate the role of hemopexin in the catabolism of heme in vivo. Uptake of rabbit [⁵⁹Fe]heme-[¹²⁵I]hemopexin by rat liver was rapid. The liver-associated ¹²⁵I reached a maximum 5 minutes after injection, nearly 7-fold higher than apo-hemopexin, whereas liver-associated ⁵⁹Fe increased with time. This together with an inverse relationship of [¹²⁵I]hemopexin in the liver and serum during the course of heme transport suggests that hemopexin was released from the liver back to the circulation. Saturation of uptake with heme-hemopexin, reaching about 170 pmol [¹²⁵I]hemopexin (gm liver)^?1 5 minutes after injection of 11 nmol, indicates a receptor-mediated process.We conclude that hemopexin delivers heme to the liver via interaction with a finite number of receptors and returns to the circulation.     

Biochemical properties of the heme oxygenase inhibitor, Sn-protoporphyrin. Interactions with apomyoglobin and human serum albumin

Sn-protoporphyrin is a strong competitive inhibitor of heme oxygenase and a potential pharmacological agent for the treatment of neonatal hyperbilirubinemia. Little is otherwise known about the biochemistry of tin porphyrins. We have investigated aspects of the chemistry of tin-protoporphyrin in aqueous solution and of its interactions with heme-binding proteins other than heme oxygenase, specifically apomyoglobin and human serum albumin. In the pH region 7-10, Soret region absorption studies of unbound Sn-protoporphyrin demonstrate a pH-dependent monomer-dimer equilibrium (KD congruent to 10(6) M-1 at pH 7) with little higher aggregation. Dissociation of the dimer is relatively slow at neutral pH, permitting interaction of protein ligands with monomeric and dimeric species to be distinguished and providing insights into kinetic mechanisms of porphyrin binding by heme-binding proteins. In the present study, the kinetics of interaction of Sn-protoporphyrin with apomyoglobin are presented as novel evidence that this binding proceeds by an induced fit mechanism. Binding of Sn-protoporphyrin to both apomyoglobin and serum albumin is unexpectedly weak. Between pH 7 and 9, the apparent affinity of Sn-protoporphyrin for apomyoglobin is less than 1/200 that of heme and, at pH 9, is also significantly less than that of protoporphyrin. The apparent affinity of Sn-protoporphyrin for human serum albumin is less than 1/1000 that of heme and 1/30 to 1/100 that of protoporphyrin. Competition studies between heme and Sn-protoporphyrin and between bilirubin and Sn-protoporphyrin indicate that Sn-protoporphyrin distributes differently among porphyrin-binding sites on serum albumin than does heme and that it is also not an effective competitor with bilirubin for bilirubin-binding sites. These results argue that Sn-protoporphyrin should not significantly alter normal mechanisms for the binding and transport of heme or of preformed bilirubin by serum albumin. From a more general perspective, the results indicate potentially unusual binding site selectivity by tin chelates; possible origins of this selectivity are discussed.     

Interaction of rabbit hemopexin with bilirubin

The interaction of hemopexin with bilirubin was characterized by spectrophotometric, fluorimetric and circular dichroic techniques. Hemopexin rapidly forms an equimolar complex with libirubin that has an apparent dissociation constant Kd, of 7.5.10(-7) M. The association alters the absorption band of bilirubin near 150 nm, quenches the fluorescence of tryptophan residues of hemopexin, enhances the fluorescence of bilirubin, and induces strong ellipticity extrema in bilirubin of --60 . 10(3) deg . cm2 . dmol-1 at 465 nm and +70 . 10(3) deg . cm2 . dmol-1 at 415 nm. However, the conformation-sensitive ellipticity aband at 231 nm of hemopexin is not altered. In displacement experiments using circular dichroism, heme readily replaced bound bilirubin, indicating that bilirubin and heme are bound at the same site on hemopexin. Even at molar ratios of hemopexin to albumin of 3 to 1, human serum albumin removes bilirubin from hemopexin. Hemopexin is thus unlikely to have a role in the transport of bilirubin in serum.     

Hemopexin-mediated heme uptake by liver. Characterization of the interaction of heme-hemopexin with isolated rabbit liver plasma membranes

Plasma membranes isolated from rabbit liver retain the ability to interact specifically with heme-hemopexin. In this system, apohemopexin does not compete effectively with heme-hemopexin for binding. The membranes bind heme-hemopexin complexes with high affinity (KD = 6.8 X 10(-7) M) and with an apparent capacity of 2.3 pmol/mg of membrane protein. These membranes also retain the ability to remove heme from heme-hemopexin. The release of heme reaches a plateau after 15-30 min at 30 degrees C and does not involve metabolic energy, proteolysis of hemopexin or pH gradients. The apohemopexin formed is rapidly released from the membranes. The accumulation of heme is saturable and is affected by pH and temperature with maximum uptake occurring between pH 5.5 and 6.5 and at 30 degrees C. Interestingly, much more heme (approximately 25 pmol/mg of membrane protein) is accumulated than hemopexin at saturation, implying that the receptor can turn over several times and that a heme-binding component exists in the rabbit liver plasma membrane.     

Serum proteins as mediators of hemin efflux from red cell membranes: specificity of hemopexin

The involvement of the serum heme-binding proteins hemopexin and albumin in the clearance of erythrocyte membranes from toxic hemin was compared. In the presence of hemopexin initial rates of hemin efflux from resealed ghosts were faster and the amount of extracted hemin larger. When hemin-containing ghosts were treated with a protein mixture of 1:45 hemopexin to albumin, as present in serum, most of the hemin was extracted in the form of heme-hemopexin. It was concluded that hemopexin is the serum protein responsible for heme extraction from cell membranes.     

The hemopexin receptor on the cell surface of human polymorphonuclear leukocytes

Promyelocytic leukemia HL-60 cells can be induced to differentiate to granulocytes, under the conditions of cultures in the presence of dimethyl sulfoxide (DMSO). Examination of the binding of 125I-labeled hemopexin to DMSO-induced HL-60 cells showed that the density of hemopexin receptors on the induced-cells was 1.35 times that on the uninduced cells. We proposed that a specific receptor for hemopexin was present on the plasma membranes of polymorphonuclear leukocytes (PMNs). The binding of human [125I]hemopexin to human PMNs at 4 degrees C was saturable with time and with increasing concentrations of [125I]hemopexin. Scatchard analysis of the binding revealed the presence of approximately 5.7 x 10(4) binding sites per cell with an apparent dissociation constant (Kd) of 2.3 x 10(-9) M. [125I]Hemopexin was rapidly bound then dissociated from the cells after the release of heme, when the cells were incubated with radioactive hemopexin at 37 degrees C. Incubation of the cells with the [59Fe]heme-hemopexin complex resulted in an accumulation of [59Fe]heme in the cells, with a temperature of 37 degrees C but not that of 4 degrees C. Ouabain or NaF inhibited not only the binding of [125I]hemopexin to PMNs but also the uptake of [59Fe]heme from [59Fe]heme hemopexin by the cells. Neither NH4 Cl nor chloroquine inhibited the uptake. Detergent extracts of 125I-labeled PMNs were incubated with a hemopexin-coupled Sepharose CL-6B. A polypeptide reacting with hemopexin-Sepharose was estimated to have a molecular weight of 80,000, as determined by polyacrylamide gel electrophoresis, in the presence of sodium dodecylsulfate. We propose that PMNs take up heme from hemopexin, as mediated by the 80,000 dalton receptor for hemopexin.     

Interaction of heme and heme-hemopexin with an extracellular oxidant system used to measure cell growth-associated plasma membrane electron transport

Since redox active metals are often transported across membranes into cells in the reduced state, we have investigated whether exogenous ferri-heme or heme bound to hemopexin (HPX), which delivers heme to cells via receptor-mediated endocytosis, interact with a cell growth-associated plasma membrane electron transport (PMET) pathway. PMET reduces the cell-impermeable tetrazolium salt, WST-1, in the presence of the mandatory low potential intermediate electron acceptor, mPMS. In human promyelocytic (HL60) cells, protoheme (iron protoporphyrin IX; 2,4-vinyl), mesoheme (2,4-ethyl) and deuteroheme (2,4-H) inhibited reduction of WST-1/mPMS in a saturable manner supporting interaction with a finite number of high affinity acceptor sites (Kd 221 nM for naturally occurring protoheme). A requirement for the redox-active iron was shown using gallium-protoporphyrin IX (PPIX) and tin-PPIX. Heme-hemopexin, but not apo-hemopexin, also inhibited WST-1 reduction, and copper was required. Importantly, since neither heme nor heme-hemopexin replace mPMS as an intermediate electron acceptor and since inhibition of WST-1/mPMS reduction requires living cells, the experimental evidence supports the view that heme and heme-hemopexin interact with electrons from PMET. We therefore propose that heme and heme-hemopexin are natural substrates for this growth-associated electron transfer across the plasma membrane.     

Hepatic uptake of heme and hemopexin but not albumin

[³H] Heme and ¹²⁵I-labeled hemopexin are taken up by the rabbit liver maximally 1 h after injection; ¹³¹I-labeled albumin however is not taken up, even when heme circulates in excess of the heme-binding capacity of hemopexin. Thus, hepatic engulfment of heme in vivo appears to be facilitated by hemopexin but not by albumin.     

Mechanisms of neuroprotection by hemopexin: modeling the control of heme and iron homeostasis in brain neurons in inflammatory states

Hemopexin provides neuroprotection in mouse models of stroke and intracerebral hemorrhage and protects neurons in vitro against heme or reactive oxygen species (ROS) toxicity via heme oxygenase‐1 (HO1) activity. To model human brain neurons experiencing hemorrhages and inflammation, we used human neuroblastoma cells, heme–hemopexin complexes, and physiologically relevant ROS, for example, H₂O₂ and HOCl, to provide novel insights into the underlying mechanism whereby hemopexin safely maintains heme and iron homeostasis. Human amyloid precursor protein (hAPP), needed for iron export from neurons, is induced ~twofold after heme–hemopexin endocytosis by iron from heme catabolism via the iron‐regulatory element of hAPP mRNA. Heme–hemopexin is relatively resistant to damage by ROS and retains its ability to induce the cytoprotective HO1 after exposure to tert‐butylhydroperoxide, although induction is impaired, but not eliminated, by exposure to high concentrations of H₂O₂ in vitro. Apo‐hemopexin, which predominates in non‐hemolytic states, resists damage by H₂O₂ and HOCl, except for the highest concentrations likely in vivo. Heme–albumin and albumin are preferential targets for ROS; thus, albumin protects hemopexin in biological fluids like CSF and plasma where it is abundant. These observations provide strong evidence that hemopexin will be neuroprotective after traumatic brain injury, with heme release in the CNS, and during the ensuing inflammation. Hemopexin sequesters heme, thus preventing unregulated heme uptake that leads to toxicity; it safely delivers heme to neuronal cells; and it activates the induction of proteins including HO1 and hAPP that keep heme and iron at safe levels in neurons.