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In hemolytic disorders, erythrocyte lysis results in massive release of hemoglobin and, subsequently, toxic heme. Hemopexin is the major protective factor against heme toxicity in human blood and currently considered for therapeutic use. It has been widely accepted that hemopexin binds heme with extraordinarily high affinity of <1 pM in a 1:1 ratio. However, several lines of evidence point to a higher stoichiometry and lower affinity than determined 50 years ago. Here, we re-analyzed these data. SPR and UV/Vis spectroscopy were used to monitor the interaction of heme with the human protein. The heme-binding sites of hemopexin were characterized using hemopexin-derived peptide models and competitive displacement assays. We obtained a K value of 0.32 ± 0.04 nM and the ratio for the interaction was determined to be 1:1 at low heme concentrations and at least 2:1 (heme:hemopexin) at high concentrations. We were able to identify two yet unknown potential heme-binding sites on hemopexin. Furthermore, molecular modelling with a newly created homology model of human hemopexin suggested a possible recruiting mechanism by which heme could consecutively bind several histidine residues on its way into the binding pocket. Our findings have direct implications for the potential administration of hemopexin in hemolytic disorders. D value of 0.32 ± 0.04 nM and the ratio for the interaction was determined to be 1:1 at low heme concentrations and at least 2:1 (heme:hemopexin) at high concentrations. We were able to identify two yet unknown potential heme-binding sites on hemopexin. Furthermore, molecular modelling with a newly created homology model of human hemopexin suggested a possible recruiting mechanism by which heme could consecutively bind several histidine residues on its way into the binding pocket. Our findings have direct implications for the potential administration of hemopexin in hemolytic disorders., (© 2021 Walter de Gruyter GmbH, Berlin/Boston.)

Plasma hemopexin (HPX) is the key antioxidant protein of the endogenous clearance pathway that limits the deleterious effects of heme released from hemoglobin and myoglobin (the term "heme" is used in this article to denote both the ferrous and ferric forms). During intra-vascular hemolysis, heme partitioning to protein and lipid increases as the plasma concentration of HPX declines. Therefore, the development of HPX as a replacement therapy during high heme stress could be a relevant intervention for hemolytic disorders. A logical approach to enhance HPX yield involves recombinant production strategies from human cell lines. The present study focuses on a biophysical assessment of heme binding to recombinant human HPX (rhHPX) produced in the Expi293F TM (HEK293) cell system. In this report, we examine rhHPX in comparison with plasma HPX using a systematic analysis of protein structural and functional characteristics related to heme binding. Analysis of rhHPX by UV/Vis absorption spectroscopy, circular dichroism (CD), size-exclusion chromatography (SEC)-HPLC, and catalase-like activity demonstrated a similarity to HPX fractionated from plasma. In particular, the titration of HPX apo-protein(s) with heme was performed for the first time using a wide range of heme concentrations to model HPX-heme interactions to approximate physiological conditions (from extremely low to more than two-fold heme molar excess over the protein). The CD titration data showed an induced bisignate CD Soret band pattern typical for plasma and rhHPX versions at low heme-to-protein molar ratios and demonstrated that further titration is dependent on the amount of protein-bound heme to the extent that the arising opposite CD couplet results in a complete inversion of the observed CD pattern. The data generated in this study suggest more than one binding site in both plasma and rhHPX. Furthermore, our study provides a useful analytical platform for the detailed characterization of HPX-heme interactions and potentially novel HPX fusion constructs.

Haemophilus influenzae HxuA is a cell-surface protein with haem-haemopexin binding activity which is key to haem acquisition from haemopexin and thus is one of the potential sources of haem for this microorganism. HxuA is secreted by its specific transporter HxuB. HxuA/HxuB belongs to the so-called two-partner secretion systems (TPSs) that are characterized by a conserved N-terminal domain in the secreted protein which is essential for secretion. Here, the 1.5 Å resolution structure of the secretion domain of HxuA, HxuA301, is reported. The structure reveals that HxuA301 folds into a β-helix domain with two extra-helical motifs, a four-stranded β-sheet and an N-terminal cap. Comparisons with other structures of TpsA secretion domains are reported. They reveal that despite limited sequence identity, strong structural similarities are found between the β-helix motifs, consistent with the idea that the TPS domain plays a role not only in the interaction with the specific TpsB partners but also as the scaffold initiating progressive folding of the TpsA proteins at the bacterial surface.

Hemopexin (HPX) is a plasma protein with the strongest binding capacity to heme and widely involved in modulation of a variety of physiological and pathological processes. The main physiological function of HPX is to bind and transport free toxic heme. Recent studies indicate that HPX also plays roles of anti-oxidant, anti-apoptosis, immune regulation and organic protection. In addition, HPX participates in regulation of cell differentiation and extracellular matrix reconstruction. In recent years, a great deal of progress has been made in studies of the mechanisms of HPX protective effects and on possible clinical application. In the past few years, especially, a number of proteomic studies have demonstrated that HPX could be served as positive molecular biomarkers for cancers of lung, liver, kidney, colon, and uterine myoma as well as osteoarthritis. In this review, recent progress in the biochemical characteristics and function of HPX and its possible clinical applications are summarized.

Hemopexin (HPX) displays a pivotal role in heme scavenging and delivery to the liver. In turn, heme-Fe-hemopexin (HPX-heme-Fe) displays heme-based spectroscopic and reactivity properties. Here, kinetics and thermodynamics of cyanide binding to ferric and ferrous hexa-coordinate human plasma HPX-heme-Fe (HHPX-heme-Fe(III) and HHPX-heme-Fe(II), respectively), and for the dithionite-mediated reduction of the HHPX-heme-Fe(III)-cyanide complex, at pH 7.4 and 20.0°C, are reported. Values of thermodynamic and kinetic parameters for cyanide binding to HHPX-heme-Fe(III) and HHPX-heme-Fe(II) are K = (4.1 ± 0.4) × 10(-6) M, k(on) = (6.9 ± 0.5) × 10(1) M(-1) s(-1), and k(off) = 2.8 × 10(-4) s(-1); and H = (6 ± 1) × 10(-1) M, h(on) = 1.2 × 10(-1) M(-1) s(-1), and h(off) = (7.1 ± 0.8) × 10(-2) s(-1), respectively. The value of the rate constant for the dithionite-mediated reduction of the HHPX-heme-Fe(III)-cyanide complex is l = 8.9 ± 0.8 M(-1/2) s(-1). HHPX-heme-Fe reactivity is modulated by proton acceptor/donor amino acid residue(s) (e.g., His236) assisting the deprotonation and protonation of the incoming and outgoing ligand, respectively., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Hemopexin (HPX) binds heme tightly, thus protecting cells from heme toxicity during hemolysis, trauma and ischemia-reperfusion injury. Heme uptake via endocytosis of heme-HPX followed by heme catabolism by heme oxygenase-1 (HMOX1) raises regulatory iron pools, thus linking heme metabolism with that of iron. Normal iron homeostasis requires copper-replete cells. When heme-HPX induces HMOX1, the copper-storing metallothioneins (MTs) are also induced whereas the copper-responsive copper chaperone that delivers copper to Cu, Zn superoxide dismutase, CCS1, is decreased; both are known responses when cellular copper levels rise. Endocytosis of heme-HPX is needed to regulate CCS1 since the signaling ligand cobalt-protoporphyrin (CoPP)-HPX, which does not induce HMOX1 but does co-localize with heme-HPX in endosomes, also decreased CCS1. These observations support that heme-HPX mobilizes copper in cells. The regulation of both hmox1 and mt1 is prevented by the copper-chelator, bathocuproinedisulfonate (BCDS), but not uptake of heme-AlexaFluor-labeled HPX into endosomes. Supporting a role for copper in HMOX1 regulation by heme-HPX, nutritional copper deficiency generated by tetraethylene pentamine or 232 tetraamine prevented HMOX1 induction. Using conditions that mimic maturing endosomes, we found that copper prevents rebinding of heme to apo-HPX. A model is presented in which copper endocytosis together with that of heme-HPX provides a means to facilitate heme export from HPX in the maturing endosomes: heme is needed for hmox1 transcription, while cytosolic copper and CCS1 provide a link for the known simultaneous regulation of hmox1 and mt1 by heme-HPX.

Hemopexin (HPX) has two principal roles: it sequesters free heme in vivo for the purpose of preventing the toxic effects of this moiety, which is largely due to heme's ability to catalyze free radical formation, and it transports heme intracellularly thus limiting its availability as an iron source for pathogens. Spectroelectrochemistry was used to determine the redox potential for heme and meso-heme (mH) when bound by HPX. At pH 7.2, the heme-HPX assembly exhibits E (1/2) values in the range 45-90 mV and the mH-HPX assembly in the range 5-55 mV, depending on environmental electrolyte identity. The E (1/2) value exhibits a 100 mV positive shift with a change in pH from 7.2 to 5.5 for mH-HPX, suggesting a single proton dependent equilibrium. The E (1/2) values for heme-HPX are more positive in the presence of NaCl than KCl indicating that Na(+), as well as low pH (5.5) stabilizes ferro-heme-HPX. Furthermore, comparing KCl with K(2)HPO(4), the chloride salt containing system has a lower potential, indicating that heme-HPX is easier to oxidize. These physical properties related to ferri-/ferro-heme reduction are both structurally and biologically relevant for heme release from HPX for transport and regulation of heme oxygenase expression. Consistent with this, when the acidification of endosomes is prevented by bafilomycin then heme oxygenase-1 induction by heme-HPX no longer occurs.

Two spectroscopically distinct, non-interconverting forms of human hemopexin have been isolated by immobilized metal ion affinity chromatography and characterized spectroscopically. Form alpha (characterized by a bisignate Soret CD spectrum) and form beta (Soret CD characterized by a positive Cotton effect) exhibit different spectroscopic responses to addition of Zn2+ or Cu2+, yet both forms exhibit the same metal ion-induced decrease in Tm for the thermally induced release of the heme prosthetic group. Far UV-CD spectra indicate that the two isoforms possess essentially identical secondary structures, but their differential retention during metal ion affinity chromatography indicates slight differences in exposure of His residues on the protein surface. We propose that these observations result from the binding of heme in form beta with an orientation that differs from the crystallographically observed binding orientation for rabbit hemopexin by rotation of the heme prosthetic group by 180 degrees about the alpha-gamma meso-carbon axis and from interaction of metal ions at two separate binding sites.

Hemopexin (HPX) serves as scavenger and transporter of toxic plasma heme to the liver. HPX is formed by two four-bladed beta-propeller domains, resembling two thick disks that lock together at a 90 degrees angle. The heme is bound between the two beta-propeller domains in a pocket formed by the interdomain linker peptide. Residues His213 and His266 coordinate the heme iron atom giving a stable bis-histidyl complex. The HPX-heme geometry is reminiscent of heme-proteins endowed with ligand binding and (pseudo-)enzymatic properties. HPX-heme binds reversibly CO, (*)NO, and cyanide by detaching His213; however, O(2) induces HPX-heme(II) oxidation. Furthermore, HPX-heme(II) facilitates (*)NO/O(2) and (*)NO/peroxynitrite scavenging. Heme sequestering by HPX prevents heme-mediated activation of oxidants which induce the low-density lipoprotein oxidation. Here, ligand binding and (pseudo-)enzymatic properties of HPX-heme are reviewed. HPX, acting not only as a heme carrier but also displaying transient heme-based ligand binding and (pseudo-)enzymatic properties, could be considered a 'chronosteric' heme-protein.

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.

Hemopexin (HPX), which serves as a scavenger and transporter of toxic plasma heme, has been postulated to play a key role in the homeostasis of NO. In fact, HPX-heme(II) reversibly binds NO and facilitates NO scavenging by O(2). HPX-heme is formed by two four-bladed beta-propeller domains. The heme is bound between the two beta-propeller domains, residues His213 and His266 coordinate the heme iron atom. HPX-heme displays structural features of heme-proteins endowed with (pseudo-)enzymatic activities. In this study, the kinetics of rabbit HPX-heme(III) reductive nitrosylation and peroxynitrite-mediated oxidation of HPX-heme(II)-NO are reported. In the presence of excess NO, HPX-heme(III) is converted to HPX-heme(II)-NO by reductive nitrosylation. The second-order rate constant for HPX-heme(III) reductive nitrosylation is (1.3 +/- 0.1) x 10(1) m(-1).s(-1), at pH 7.0 and 10.0 degrees C. NO binding to HPX-heme(III) is rate limiting. In the absence and presence of CO2 (1.2 x 10(-3) m), excess peroxynitrite reacts with HPX-heme(II)-NO (2.6 x 10(-6) m) leading to HPX-heme(III) and NO, via the transient HPX-heme(III)-NO species. Values of the second-order rate constant for HPX-heme(III)-NO formation are (8.6 +/- 0.8) x 10(4) and (1.2 +/- 0.2) x 10(6) m(-1).s(-1) in the absence and presence of CO2, respectively, at pH 7.0 and 10.0 degrees C. The CO2-independent value of the first-order rate constant for HPX-heme(III)-NO denitrosylation is (4.3 +/- 0.4) x 10(-1) s(-1), at pH 7.0 and 10.0 degrees C. HPX-heme(III)-NO denitrosylation is rate limiting. HPX-heme(II)-NO appears to act as an efficient scavenger of peroxynitrite and of strong oxidants and nitrating species following the reaction of peroxynitrite with CO2 (e.g. ONOOC(O)O-, CO3-, and NO2).

Hemopexin (HPX), serving as scavenger and transporter of toxic plasma heme, has been postulated to play a key role in the homeostasis of NO. Here, kinetics of HPX-heme(II) nitrosylation and O2-mediated oxidation of HPX-heme(II)-NO are reported. NO reacts reversibly with HPX-heme(II) yielding HPX-heme(II)-NO, according to the minimum reaction scheme: HPX-heme(II)+NO kon<-->koff HPX-heme(II)-NO values of kon, koff, and K (=kon/koff) are (6.3+/-0.3)x10(3)M-1s-1, (9.1+/-0.4)x10(-4)s-1, and (6.9+/-0.6)x10(6)M-1, respectively, at pH 7.0 and 10.0 degrees C. O2 reacts with HPX-heme(II)-NO yielding HPX-heme(III) and NO3-, by means of the ferric heme-bound peroxynitrite intermediate (HPX-heme(III)-N(O)OO), according to the minimum reaction scheme: HPX-heme(II)-NO+O2 hon<--> HPX-heme(III)-N(O)OO l-->HPX-heme(III)+NO3- the backward reaction rate is negligible. Values of hon and l are (2.4+/-0.3)x10(1)M-1s-1 and (1.4+/-0.2)x10(-3)s-1, respectively, at pH 7.0 and 10.0 degrees C. The decay of HPX-heme(III)-N(O)OO (i.e., l) is rate limiting. The HPX-heme(III)-N(O)OO intermediate has been characterized by optical absorption spectroscopy in the Soret region (lambdamax=409 nm and epsilon409=1.51x10(5)M-1cm-1). These results, representing the first kinetic evidence for HPX-heme(II) nitrosylation and O2-mediated oxidation of HPX-heme(II)-NO, might be predictive of transient (pseudo-enzymatic) function(s) of heme carriers.

Heme released from heme-binding proteins on internal hemorrhage, hemolysis, myolysis, or other cell damage is highly toxic due to oxidative and proinflammatory effects. Complex formation with hemopexin, the high-affinity heme-binding protein in plasma and cerebrospinal fluid, dampens these effects and is suggested to facilitate cellular heme metabolism. Using a ligand-affinity approach, we purified the human hemopexin-heme receptor and identified it as the low-density lipoprotein receptor-related protein (LRP)/CD91, a receptor expressed in several cell types including macrophages, hepatocytes, neurons, and syncytiotrophoblasts. Binding experiments, including Biacore analysis, showed that hemopexin-heme complex formation elicits the high receptor affinity. Uptake studies of radio-labeled hemopexin-heme complex in LRP/CD91-expressing COS cells and confocal microscopy of the cellular processing of fluorescent hemopexin-heme complex established the ability of LRP/CD91 to mediate hemopexin-heme internalization resulting in cellular heme uptake and lysosomal hemopexin degradation. Uptake of hemopexin-heme complex induced LRP/CD91-dependent heme-oxygenase 1 mRNA transcription in cultured monocytes. In conclusion, hemopexin-heme complexes are removed by a receptor-mediated pathway showing striking similarities to the CD163-mediated haptoglobin-hemoglobin clearance in macrophages. Furthermore, the data indicate a hitherto unknown role of LRP/CD91 in inflammation.

The study compared the damage inflicted to endothelial cells (ECs) by intact hemoglobin (Hb) and isolated chains. To compare optional in vivo contact of acellular Hb with the endothelium, oxy-forms of Hb and its isolated alpha- and beta-chains existing in the thalassemias were added to primary confluent cultures of bovine aorta EC. Cell damage was followed by morphological changes or leakage of lactic dehydrogenase and pre-inserted 51Cr from the cells, followed for 27 h. Under these experimental conditions, Hb did not affect the cells but its chains inflicted damage, beta- more than alpha-chains. Based on the literature and our data, we hypothesized that hemin and/or globin should be responsible for the increased endothelial damage by beta-chains. While hemin hardly affected ECs, globin, unlike the plasma protein hemopexin, was harmful. Since hemin release leaves globin with a large hydrophobic surface, the globin-damage appears to result from adsorptive pinocytosis to endothelial membrane.

Thermal denaturation of the human hemopexin-heme complex was investigated under a variety of solution conditions to identify factors that influence heme release. The midpoint temperature for the transition between the folded and folded states, T(m), of the hemopexin-ferriheme complex exhibits a significant dependence on pH. When the pH is reduced from 7 to 5 (50 mM BisTris buffer and 50 mM NaCl), T(m) decreases by approximately 23 degrees C despite the relatively higher chloride concentration that tends to stabilize the protein. The thermal stability of the hemopexin-ferroheme complex was examined at pH 7.4 to yield a T(m) that is 3.2 degrees C lower than that of the hemopexin-ferriheme complex under identical conditions. The effect of transition metal ions, which hemopexin has recently been shown to bind [Mauk, M. R., Rosell, F. I., Lelj-Garolla, B., Moore, G. R., and Mauk, A. G. (2005) Biochemistry 44, XXXX-XXXX], was also considered. Cu(2+) and Zn(2+) had the greatest effect, reducing T(m) for the transition by 4.8 and 6.5 degrees C, respectively, relative to the value for the protein in the absence of metal ions [T(m) = 64.9 degrees C [10 mM sodium phosphate buffer (pH 7.4)]]. These metal ions also interfered significantly with the recovery of the native state from the unfolded protein when the protein on returning to 20 degrees C. The current results demonstrate how the conditions within the endosomes of hepatocytes (pH approximately 5.0, [Cl(-)] approximately 60 mM) and the potential presence of transition metal ions or heme iron reduction contribute to the membrane receptor-mediated process of heme release from hemopexin.

Binding of divalent metal ions to human hemopexin (Hx) purified by a new protocol has been characterized by metal ion affinity chromatography and potentiometric titration in the presence and absence of bound protoheme IX. ApoHx was retained by variously charged metal affinity chelate resins in the following order: Ni(2+) > Cu(2+) > Co(2+) > Zn(2+) > Mn(2+). The Hx-heme complex exhibited similar behavior except the order of retention of the complex on Zn(2+)- and Co(2+)-charged columns was reversed. One-dimensional (1)H NMR of apoHx in the presence of Ni(2+) implicates at least two His residues and possibly an Asp, Glu, or Met residue in Ni(2+) binding. Potentiometric titrations establish that apoHx possesses more than two metal ion binding sites and that the capacity and/or affinity for metal ion binding is diminished when heme binds. For most metal ions that have been studied, potentiometric data did not fit to binding isotherms that assume one or two independent binding sites. For Mn(2+), however, these data were consistent with a high-affinity site [K(A) = (15 +/- 3) x 10(6) M(-)(1)] and a low-affinity site (K(A)

Hemopexin (HPX) serves as a trap for toxic plasma heme, ensuring its complete clearance by transportation to the liver. Moreover, HPX-heme has been postulated to play a key role in the homeostasis of nitric oxide (NO). Here, the thermodynamics for NO binding to rabbit ferrous HPX-heme as well as the EPR and optical absorption spectroscopic properties of rabbit ferrous nitrosylated HPX-heme (HPX-heme-NO) are reported. The value of the dissociation equilibrium constant for NO binding to rabbit ferrous HPX-heme (i.e., H) is (1.4+/-0.2)x10(-7) M, at pH 7.0 and 10.0 degrees C; the value of H is unaffected by sodium chloride. At pH 7.0, rabbit ferrous HPX-heme-NO is a six-coordinate heme-iron species, characterized by an X-band EPR spectrum with an axial geometry and by epsilon=146 mM(-1) cm(-1) at 419 nm. At pH 4.0, rabbit ferrous HPX-heme-NO is a five-coordinate heme-iron species, characterized by an X-band EPR spectrum with three-line splitting centered at 334 mT and by epsilon=74 mM(-1) cm(-1) at 387 nm. The p K(a) value of the reversible pH-induced six- to five-coordinate spectroscopic transition is 4.8+/-0.1 in the absence of sodium chloride and 4.3+/-0.1 in the presence of 1.5x10(-1) M sodium chloride. This result is in agreement with the effect of sodium chloride on rabbit HPX-heme stability. The present data have been analyzed in parallel with those of a related heme model compound and heme-protein systems.

Iron is essential to life, but poses severe problems because of its toxicity and the insolubility of hydrated ferric ions at neutral pH. In animals, a family of proteins called transferrins are responsible for the sequestration, transport, and distribution of free iron. Comparison of the structure and function of transferrins with a completely unrelated protein hemopexin, which carries out the same function for heme, identifies molecular features that contribute to a successful protein system for iron acquisition, transport, and release. These include a two-domain protein structure with flexible hinges that allow these domains to enclose the bound ligand and provide suitable chemistry for stable binding and an appropriate trigger for release.

Hemopexin has two homologous domains (N- and C-terminal domains), binds 1 mole of heme per mole with high affinity (Kd < 1 pM) in a low-spin bis-histidyl complex, and acts as a transporter for the heme. Transport is accomplished via endocytosis without degradation of the protein. Factors that affect stability of the heme coordination complex and potentially heme release in vivo were examined. The effects of temperature on hemopexin, its N-terminal domain, and their respective ferri-, ferro-, and CO-ferro-heme complexes were studied using absorbance and circular dichroism (CD) spectroscopy. As monitored with second-derivative absorbance spectra, the higher order structure of apo-hemopexin unfolds with a Tm of 52 degrees C in 50 mM sodium phosphate buffer and is stabilized by 150 mM NaCl (Tm 63 degrees C). Bis-histidyl heme coordination by hemopexin, observed by Soret absorbance, is substantially weakened by reduction of ferri-heme-hemopexin (Tm 55.5 degrees C) to the ferro-heme form (Tm 48 degrees C), and NaCl stabilizes both complexes by 10-15 degrees C. CO binding to ferroheme-hemopexin restores complex stability (Tm 67 degrees C). Upon cooling, unfolded apo- and ferriheme-hemopexin extensively refold and recover substantial heme-binding activity, but the characteristic ellipticity of the native protein (UV region) and heme complex (Soret region) are not regained, indicating that altered refolded forms are produced. Lowering the pH from 7.4 to 6.5 has little effect on the stability of the apo-protein but increases the Tm of heme complexes by 5-12 degrees C. The stability of the apo-N-terminal domain (Tm 53 degrees C) is similar to that of intact hemopexin, and the ferri-, ferro-, and CO-ferro-heme complexes of the N-terminal domain have Tm values of 53 degrees C, 33 degrees C, and 75 degrees C, respectively.

Hemopexin binds 1 mol of heme per mol with high affinity (Kd < 1 pM) in a low-spin complex and acts as a transport vehicle for the heme. Circular dichroism (CD) spectroscopy was used to examine the heme environment in the ferri-, ferro-, and CO-ferro complexes of four iron tetrapyrroles [meso-, proto-, deutero-, and (2-vinyl, 4-hydroxymethyl)-deutero-heme] with three species (human, rabbit, and rat) of hemopexin. All ferri-heme-hemopexin complexes exhibit a band of positive ellipticity near the Soret maximum, except for the human ferri-protoheme hemopexin complex, which has a bisignate spectrum. The ferro-heme and CO-ferro-heme complexes display a variety of spectra, demonstrating redox- and ligand-linked shifts in conformation that alter the environment of the heme. The rabbit mesoheme-N-domain complexes have absorbance spectra almost indistinguishable from those of intact hemopexin, but present CD spectra that are distinctly different. However, adding the C-domain to mesoheme-N-domain restores most of the CD characteristics of the intact hemopexin complexes.

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.

Hemopexin, which acts as an antioxidant by binding heme (Kd < 1 pM), is synthesized by hepatic parenchymal cells, by neurons of the central and peripheral nervous systems, and by human retinal ganglia. Two key regulatory molecules, nitric oxide (.NO) and carbon monoxide (CO), both bind to heme proteins and since ferroheme-hemopexin binds CO, the possible role of heme-hemopexin in binding .NO was investigated. .NO binds rapidly to hemopexin-bound ferroheme as shown by characteristic changes in the Soret and visible-region absorbance spectra. Circular dichroism spectra of .NO-ferroheme-hemopexin in the Soret region exhibit an unusual bisignate feature with a zero crossover at the absorbance wavelength maximum, showing that exciton coupling is occurring. Notably, the .NO complex of ferroheme-hemopexin is sufficiently avid and stable to allow hemopexin to bind this molecule in vivo and, thus, hemopexin may protect against NO-mediated toxicity especially in conditions of trauma and hemolysis.

The kinetics of the interaction of heme with hemopexin and albumin was monitored by measuring the time dependence of changes in the Soret absorption spectra. Since the protein binding sites can only bind heme monomers, the binding kinetics apparently reflected the slow dissociation of heme dimers, resulting from dimer/monomer equilibria in aqueous heme solutions. The dissociation of heme dimers is characterized by the rate constant of (3-4) x 10(-3) s(-1). The measurements further revealed significant differences in the kinetic profiles (slowing down the binding interaction) that were dependent on the storage time of heme solutions at room temperature. These presumably responded to the gradual formation of higher aggregates of heme, which cannot dissociate into dimers/monomers. Alternatively, partial autooxidation of heme molecules could increase the stability of heme dimers and obstruct specific binding of heme to the proteins.

Heme binding to rabbit hemopexin and its domain I, obtained by proteolytic cleavage of intact hemopexin, was studied by EPR, MCD and 1H-NMR spectroscopies. The data obtained support the proposal that the heme Fe(III) is coordinated by two histidine ligands (Morgan et al. (1988) J. Biol. Chem. 263, 8220-8225; Muster et al. (1991) J. Protein Chem. 10, 123-128) and are inconsistent with recently reported mutagenesis studies indicating that bis-histidine ligation is unlikely (Satoh et al. (1994) Proc. Natl. Acad. Sci. USA 91, 8423-8427). Although the MCD data are consistent with both bis-histidine and histidine/lysine ligation, the EPR spectra are typical of bis-histidine ligation. Overall the magneto-optical spectra are characteristic for bis-histidine ligation. The EPR and NMR data indicate that there is a difference in the heme environments of the intact hemopexin and its domain I but overall the spectroscopic information suggests heme bound to domain I has the same ligands as intact hemopexin. The 1H-NMR studies indicate that heme binding to domain I perturbs at least 4 of the 5 histidines. This is consistent with axial ligation of the heme by two histidines, and a conformational change induced by heme binding affecting two more. Interestingly, resonances of the carbohydrate bound to intact hemopexin and domain I were also perturbed by heme binding. pH dependence studies showed that heme remained bound to intact hemopexin over the pH range 6.5-10.0 without any major change in the ligation or environment of the heme.

Hemopexin, a heme-binding serum glycoprotein, is thought to play an important role in the prevention of oxidative damage that may be catalysed by free heme. Through the use of EPR techniques, the generation of free radicals from organic hydroperoxides by heme and heme-hemopexin complexes, and the concomitant formation of high oxidation-state iron species has been studied; these species are implicated as causative agents in processes such as cardiovascular disease and carcinogenesis. From the rates of production of these species from both n-alkyl and branched hydroperoxides, it has been inferred that the dramatic reduction in the yield of oxidising species generated by heme upon its complexation with hemopexin arises from steric hindrance of the access of hydroperoxide to the bound heme.

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)

Hemopexin is a serum glycoprotein that binds heme with the highest known affinity of any characterized heme-binding protein and plays an important role in receptor-mediated cellular heme uptake. Complete understanding of the function of hemopexin will require the elucidation of its molecular structure. Previous analysis of the secondary structure of hemopexin by far-UV circular dichroism (CD) failed due to the unusual positive ellipticity of this protein at 233 nm. In this paper, we present an examination of the structure of hemopexin by both Fourier-transform infrared (FTIR) and circular dichroism spectroscopy. Our studies show that hemopexin contains about 55% beta-structure, 15% alpha-helix, and 20% turns. The two isolated structural domains of hemopexin each have secondary structures similar to hemopexin. Although there are significant tertiary conformational changes indicated by the CD spectra, the overall secondary structure of hemopexin is not affected by binding heme. However, moderate changes in secondary structure do occur when the heme-binding domain of hemopexin associates with heme. In spite of the exceptionally tight binding at neutral pH, heme is released from the bis-histidyl heme-hemopexin complex at pH 5.0. Under this acidic condition, hemopexin maintains the same overall secondary structure as the native protein and is able to resume the heme-binding function and the native structure of the heme-protein (as indicated by the CD spectra) when returned to neutral pH. We propose that the state of hemopexin identified in vitro at pH 5.0 resembles that of this protein in the acidic environment of the endosomes in vivo when hemopexin releases heme during receptor-mediated endocytosis.

Hemopexin (Hx), the major heme-binding plasma glycoprotein, scavenges circulating heme and performs an antioxidant function. In the present study, human Hx was expressed in a baculovirus system and its presumed essential His residues were mutated to Thr as a means of investigating their participation in heme binding. The recombinant Hx proteins were purified by sequential chromatography on Con A-agarose and SP-Sepharose. The purified recombinant wild-type Hx retained its heme binding. The binding constant for heme was considerably reduced, however, suggesting that glycosylation contributes critically to the heme binding property of Hx. Mutation either at His-127 or at His-56 plus His-127, but not at His-56 per se, reduced the affinity for heme by an order of magnitude relative to wild-type Hx. It is concluded that His-127 contributes to the high affinity for heme. We recorded proton NMR spectra to investigate the possibility that the degree of high-spin content is increased by deletion of an axial His-iron coordination. 1H NMR data indicate that each of the single-mutant heme-Hx complexes is predominantly low-spin, perhaps owing to coordination of the heme iron by the Thr side-chain oxygen or water oxygen coordinating to the iron.

Proton nuclear magnetic resonance spectroscopy of the complex of heme with hemopexin, a plasma protein with an exceptionally high affinity for heme, is reported. Characteristic spectra are shown for heme.hemopexin of cow, human, rabbit, and rat. Each of these spectra demonstrate that the iron of heme bound by hemopexin is paramagnetic and low-spin. Rabbit heme.hemopexin, which exhibits the best signal-to-noise ratio, is studied in detail. Deuterium isotope labeling experiments indicate that the methyls in heme positions 1-, 3-, and 8- are resolved downfield from the protein envelope of resonances; the 5-methyl may lie in the -5 to +12 ppm region. Two-dimensional nuclear Overhauser effect spectroscopy locates other protons of the heme periphery, including from the 2-vinyl. Strongly relaxed upfield resonances are identified and assigned to protons on the axial ligands. Cyanide interaction with heme.hemopexin produces an additional low-spin adduct.

Hemopexin is a plasma glycoprotein that has two structural domains (I and II) and binds and transports heme particularly to liver cells. Differential scanning calorimetry (DSC) studies show that hemopexin is largely stabilized by heme, which binds exclusively to domain I. The melting temperature (Tm) of heme-hemopexin is 66.4 +/- 0.7 degrees C as compared with 53.9 +/- 0.3 degrees C for apohemopexin, and this Tm increase is accompanied by a 100 kcal increase in molar enthalpy. Heme stabilizes hemopexin by stabilizing domain I. This is demonstrated by the 26 degrees C increase in Tm from 51.9 +/- 0.3 to 77.6 +/- 0.6 degrees C and the over 3-fold increase in molar enthalpy when domain I associates with heme. A moderate change in domain I secondary structure is indicated by an increase in negative molar ellipticity at 206 nm. However, there is no net effect on the secondary structure of holo-hemopexin caused by heme binding as indicated by both far-UV circular dichroism (CD) and Fourier-transform infrared spectra. The characteristic positive ellipticity of hemopexin at 233 nm, ascribed to tryptophan residues in domain II, is dramatically increased, suggesting a change in teritary structure for domain II of hemopexin. DSC and CD results show that isolated domain I and domain II interact both in the presence and absence of heme. Moreover, domain II destabilizes heme-domain I, which may be an important factor in facilitating heme release to the hemopexin receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Rabbit hemopexin cDNA was cloned from a rabbit liver lambda gt11 cDNA expression library using a mixture of five monoclonal antibodies raised against rabbit hemopexin, and the entire rabbit hemopexin sequence was determined. The heme-binding domain I of rabbit hemopexin (Smith, A., and Morgan, W. T. (1984) J. Biol. Chem. 259, 12049-12053) contains only 4 histidine residues which are conserved in rabbit, human, rat, and mouse hemopexin. The 2 axial heme-iron coordinating histidine residues, identified by Edman microsequencing and amino acid analyses of chemically modified domain I and isolated fragments of domain I, are the conserved histidine residues at positions 56 and 127 of the mature rabbit protein. The epitope recognized by JEN-14 (a monoclonal antibody which specifically reacts with domain I and blocks the hemopexin-receptor interaction (Morgan, W. T., Muster, P., Tatum, F. M., McConnell, J., Conway, T. P., Hensley, P., and Smith, A. (1988) J. Biol. Chem. 263, 8220-8225) was shown to lie between residues 122 and 142 by Western blotting of protease-digested domain I and transposon-insertion mutants of domain I expressed in a plasmid vector system. The location of this epitope near the heme-binding histidine residue 127 is compatible with a transport mechanism in which the release of heme from hemopexin is accompanied by a concomitant transfer of heme to the hemopexin receptor or the membrane heme-binding protein (Smith, A., and Morgan, W. T. (1985) J. Biol. Chem. 260, 8325-8329).