Metal Ions and Electrolytes Regulate the Dissociation of Heme from Human Hemopexin at Physiological pH

Abstract

The stability of the hemopexin-heme (Hx-heme) complex to dissociation of the heme prosthetic group has been examined in bicarbonate buffers in the presence and absence of various divalent metal ions. In NH 4 HCO 3 buffer (pH 7.4, 20 mM, 25°C) containing Zn 2؉ (100 M), 14% of the heme dissociates from this complex (4.5 M) within 10 min, and 50% dissociates within 2 h. In the absence of metal ions, the rate of dissociation of this complex is far lower, is decreased further in KHCO 3 solution, and is minimal in NaHCO 3 . In NH 4 HCO 3 buffer, dissociation of the Hx-heme complex is accelerated by addition of divalent metals with decreasing efficiency in the order ZnAddition of Ca 2؉ prior to addition of Zn 2؉ stabilizes the Hx-heme complex to dissociation of the heme group, and addition of Ca 2؉ after Zn 2؉ -induced dissociation of the Hxheme complex results in re-formation of the Hx-heme complex. These effects are greatly accelerated at 37°C and diminished in other buffers. Overall, the solution conditions that promote formation of the Hx-heme complex are similar to those found in blood plasma, and conditions that promote release of heme are similar to those that the Hx-heme complex should encounter in endosomes following endocytosis of the complex formed with its hepatic receptor.The plasma protein hemopexin (Hx, 2 MW ϳ58,000) is a positive acute phase reactant glycoprotein with the primary role of scavenging heme released from methemoglobin and other heme proteins as the result of hemolysis, rhabdomyolysis, or ischemia-reperfusion injury (1-3). In doing so, Hx prevents the oxidative damage and pro-inflammatory effects of free heme (4 -6) as demonstrated by the increased sensitivity of Hx-null mice (4, 7) to heme overload and development of heme-catalyzed oxidative damage to the vasculature, liver, and kidneys. Hx may also have a role in protection of neural tissue, and it exhibits other activities that include inhibition of necrosis and adhesion of polymorphonuclear leukocytes (8 -10). The dominant form of circulating Hx is the apoprotein 3 (6 -25 M (1)), which upon binding heme forms the Hx-heme complex that is recognized by hepatic receptors and removed from circulation by endocytosis (3,11,12). Subsequent to endocytosis, heme is dissociated from the Hx-heme complex and oxidized by heme oxygenase-1 to release iron that is then added to ferritin stores (13,14). The protein component of the endocytosed Hx-heme complex that is liberated in this manner either returns to circulation (15-18) or undergoes lysosomal degradation (11,18).Heme binds to Hx with extremely high affinity (19, 20) even though the crystal structure for the rabbit Hx-heme complex indicates that the heme is more exposed to solvent than is the case for many heme proteins (21, 22). The Hx-heme complex is an unusual b-type hemeprotein in that the axial ligands that coordinate the heme iron are not located in a structurally constrained region of the protein, and one of the axial histidine ligands is located in the flexible linker region that connects...

Ion flux and the function of endosomes and lysosomes: pH is just the start

“…In fact, recently Byrne et al [24] identified an anion-binding site that regulates iron dissociation from the transferrin protein implicating increasing Cl À concentrations, in addition to a lower pH, as key regulators of transferrin function in the early endosome. In addition to the orthodoxy of the cation-dependent and cation-independent mannose-6-phosphate receptors [25], ionic modulation of receptor-ligand interactions has also been reported for hemopexin-heme [26], LDLR-LDL [27], and CD163-haptoglobin-hemoglobin [28]. Furthermore, apart from affecting receptor sorting, changes in the luminal ions could impact on the degradative pathway.…”

Silver and Copper Acute Effects on Membrane Proteins and Impact on Photosynthetic and Respiratory Complexes in Bacteria

“…Indeed, substitution of Mg 2+ affects the LHCII and the photosystem PSII, thereby causing a decrease in photosynthesis ( 20 ). Likewise, it was shown that Ni 2+ , Cu 2+ , and Zn 2+ induced a destabilization of heme binding to b -type hemoproteins and led to the release of heme from myoglobin, ferricytochrome b 5 , indoleamine-dioxygenase, hemopexin, and cytochrome P450 ( 45 , 46 ). Formation of a bioconjugate of human hemoglobin with Ag + ions was also reported ( 47 , 48 ).…”

An alternative view of the proposed alternative activities of hemopexin

“…48 Other chemical modifications have been reported for human hemopexin, for example, exposure of hemopexin to Ni 2þ can result in hydrolysis of a susceptible peptide bond in the linker sequence. 68 Carbonylation of hemopexin has been detected in patients with sporadic Alzheimer's disease 69 and with idiopathic pulmonary fibrosis. 70 Although the cause of this oxidation remains unknown, it raises the possibility that hemopexin could also be susceptible to oxidation under the conditions of some purification protocols.…”

“…1,8 An homology model for the structure of the human hemopexin-heme complex based on the structure of the rabbit protein was used to identify several additional, putative binding sites for metal ions on human hemopexin 21 and has led to speculation that hemopexin and the hemopexin-heme complex might 130 On the other hand, at neutral pH Ca 2þ promotes the binding of heme 68 and increases the thermal stability (T m ) of the hemopexin-heme complex. 21 All of these observations, as well as specific ion effects on the dissociation of heme from the hemopexin-heme complex, 57,68,131 suggest a means by which the interaction of the protein with metal ions may promote transfer and binding of heme to hemopexin in blood plasma, release of heme from the hemopexin-heme complex in the endosome, and, as a result, promotion of the known anti-oxidant capacity of hemopexin. Nevertheless, there is no experimental evidence at present that metal ion binding to hemopexin comprises a new functional property of the protein in the transport of metal ions in vivo.…”