SOUL in mouse eyes is a new hexameric heme-binding protein with characteristic optical absorption, resonance Raman spectral, and heme-binding properties

SOUL is specifically expressed in the retina and pineal gland and displays more than 40% sequence homology with p22HBP, a heme protein ubiquitously expressed in numerous tissues. SOUL was purified as a dimer in the absence of heme from the Escherichia coli expression system but displayed a hexameric structure upon heme binding. Heme-bound SOUL displayed optical absorption and resonance Raman spectra typical of 6-coordinate low-spin heme protein, with one heme per monomeric unit for both the Fe(III) and Fe(II) complexes. Spectral data additionally suggest that one of the axial ligands of the Fe(III) heme complex is His. Mutation of His42 (the only His of SOUL) to Ala resulted in loss of heme binding, confirming that this residue is an axial ligand of SOUL. The K(d) value of heme for SOUL was estimated as 4.8 x 10(-9) M from the association and dissociation rate constants, suggesting high binding affinity. On the other hand, p22HBP was obtained as a monomer containing one heme per subunit, with a K(d) value of 2.1 x 10(-11) M. Spectra of heme-bound p22HBP were different from those of SOUL but similar to those of heme-bound bovine serum albumin in which heme bound to a hydrophobic cavity with no specific axial ligand coordination. Therefore, the heme-binding properties and coordination structure of SOUL are distinct from those of p22HBP, despite high sequence homology. The physiological role of the new heme-binding protein, SOUL, is further discussed in this report.     

Identification and expression of soul/p22HBP genes in zebrafish

The SOUL/p22HBP family is an evolutionarily ancient group of heme binding proteins with a main function as cytosolic buffer against tetrapyrrole accumulation. Structural and biochemical evidence suggest specialized roles in blood formation, necrotic cell death and chemotaxis. To date, nothing is known about the precise activity and expression patterns of this class of heme binding proteins during development. The zebrafish genome possesses five soul genes belonging to two subgroups, and no p22HBP orthologous gene. Here, spatial and temporal expression patterns are reported for zebrafish soul1, soul2 and soul4 genes. All three soul genes are maternally transcribed, and their zygotic expression takes place in unique (heart, pharynx, yolk syncytial layer, brain, eyes, lateral line) and overlapping (pronephros, pituitary gland, olfactory and otic vesicle) regions of the zebrafish embryo. Our study constitutes the first detailed analysis of soul gene expression in metazoan development, and provides the basis to understand the genetics of tetrapyrrole metabolism in a wide range of embryonic processes.     

A novel haem-binding interface in the 22 kDa haem-binding protein p22HBP

The 22 kDa haem-binding protein, p22HBP, is highly expressed in erythropoietic tissues and binds to a range of metallo- and non-metalloporphyrin molecules with similar affinities, suggesting a role in haem regulation or synthesis. We have determined the three-dimensional solution structure of p22HBP and mapped the porphyrin-binding site, which comprises a number of loops and a alpha-helix all located on a single face of the molecule. The structure of p22HBP is related to the bacterial multi-drug resistance protein BmrR, and is the first protein with this fold to be identified in eukaryotes. Strikingly, the porphyrin-binding site in p22HBP is located in a similar position to the drug-binding site of BmrR. These similarities suggest that the broad ligand specificity observed for both BmrR and p22HBP may result from a conserved ligand interaction mechanism. Taken together, these data suggest that the both the fold and its associated function, that of binding to a broad range of small hydrophobic molecules, are ancient, and have been adapted throughout evolution for a variety of purposes.     

Protein-mediated efflux of heme from isolated rat liver mitochondria

Proteins are required for the efflux of heme from mitochondria and liposomes. The efflux from liposomes is independent of the heme-binding affinity of the protein (Biochem. 23:3715, 1984). We tested whether heme-binding proteins increase efflux of newly synthesized heme from structurally and functionally intact rat liver mitochondria. Mitochondria whose heme was labeled with 14C-delta-aminolevulinic acid, were incubated in the presence of glutathione transferases (GSTs), serum albumin (RSA) or heme-binding protein (HBP), all from the rat. HBP caused a 6-8 fold increase in efflux of newly synthesized heme as compared to that effected by RSA or GSTs. This result indicates that heme efflux from intact mitochondria, unlike that from liposomes, depends on the type of protein present and that HBP may specifically facilitate heme efflux from mitochondria.     

Characterization of a human and mouse tetrapyrrole-binding protein

The cDNA for p22HBP has been cloned from human and mouse, and the protein expressed, purified, and characterized. Both mouse and human proteins bind heme and porphyrins with micromolar K(d)s, are highly homologous, monomeric, and soluble, and have a cytoplasmic location. The proteins bind metalloporphyrins, free porphyrins, and N-methylprotoporphyrin with similar affinities, and mutations of a selected set of putative metal ligating residues did not have any significant effect on the measured K(d)s. That the presence or absence of metal in the porphyrin has no effect on the binding constants and the observation that the EPR signal for heme does not change upon binding to the protein strongly suggest that p22HBP is a generic tetrapyrrole-binding protein rather than a dedicated heme-binding protein. A role for p22HBP in cellular porphyrin metabolism is discussed.     

Dimerization and heme binding are conserved in amphibian and starfish homologues of the microRNA processing protein DGCR8

Human DiGeorge Critical Region 8 (DGCR8) is an essential microRNA (miRNA) processing factor that is activated via direct interaction with Fe(III) heme. In order for DGCR8 to bind heme, it must dimerize using a dimerization domain embedded within its heme-binding domain (HBD). We previously reported a crystal structure of the dimerization domain from human DGCR8, which demonstrated how dimerization results in the formation of a surface important for association with heme. Here, in an attempt to crystallize the HBD, we search for DGCR8 homologues and show that DGCR8 from Patiria miniata (bat star) also binds heme. The extinction coefficients (ε) of DGCR8-heme complexes are determined; these values are useful for biochemical analyses and allow us to estimate the heme occupancy of DGCR8 proteins. Additionally, we present the crystal structure of the Xenopus laevis dimerization domain. The structure is very similar to that of human DGCR8. Our results indicate that dimerization and heme binding are evolutionarily conserved properties of DGCR8 homologues not only in vertebrates, but also in at least some invertebrates.     

DiGeorge critical region 8 (DGCR8) is a double-cysteine-ligated heme protein

All known heme-thiolate proteins ligate the heme iron using one cysteine side chain. We previously found that DiGeorge Critical Region 8 (DGCR8), an essential microRNA processing factor, associates with heme of unknown redox state when overexpressed in Escherichia coli. On the basis of the similarity of the 450-nm Soret absorption peak of the DGCR8-heme complex to that of cytochrome P450 containing ferrous heme with CO bound, we identified cysteine 352 as a probable axial ligand in DGCR8. Here we further characterize the DGCR8-heme interaction using biochemical and spectroscopic methods. The DGCR8-heme complex is highly stable, with a half-life exceeding 4 days. Mutation of the conserved proline 351 to an alanine increases the rate of heme dissociation and allows the DGCR8-heme complex to be reconstituted biochemically. Surprisingly, DGCR8 binds ferric heme without CO to generate a hyperporphyrin spectrum. The electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance spectra of the DGCR8-heme complex suggest a ferric heme bearing two cysteine ligands. This model was further confirmed using selenomethionine-substituted DGCR8 and mercury titration. DGCR8 is the first example of a heme-binding protein with two endogenous cysteine side chains serving as axial ligands. We further show that native DGCR8 binds heme when expressed in eukaryotic cells. This study provides a chemical basis for understanding the function of the DGCR8-heme interaction in microRNA maturation.     

Human and rodent amyloid-β peptides differentially bind heme: Relevance to the human susceptibility to Alzheimer’s disease

Amyloid-β (Aβ) peptides are implicated in the neurodegeneration of Alzheimer’s disease (AD). We previously investigated the mechanism of neurotoxicity of Aβ and found that human Aβ (huAβ) binds and depletes heme, forming an Aβ-heme complex with peroxidase activity. Rodent Aβ (roAβ) is identical to huAβ, except for three amino acids within the proposed heme-binding motif (Site-H). We studied and compared heme-binding between roAβ and huAβ. Unlike roAβ, huAβ binds heme tightly (K_d = 140 ± 60 nM) and forms a peroxidase. The plot of bound (huAβ-heme) vs. unbound heme fits best to a two site binding hyperbola, suggesting huAβ possesses two heme-binding sites. Consistently, a second high affinity heme-binding site was identified in the lipophilic region (site-L) of huAβ (K_d = 210 ± 80nM). The plot of (roAβ-heme) vs. unbound heme, on the other hand, was different as it fits best to a sigmoidal binding curve, indicating different binding and lower affinity of roAβ for heme (K_d = 1 μM). The effect of heme-binding to site-H on heme-binding to site-L in roAβ and huAβ is discussed. While both roAβ and huAβ form aggregates equally, rodents lack AD-like neuropathology. High huAβ/heme ratio increases the peroxidase activity. These findings suggest that depletion of regulatory heme and formation of Aβ-heme peroxidase contribute to huAβ’s neurotoxicity in the early stages of AD. Phylogenic variations in the amino acid sequence of Aβ explain tight heme-binding to huAβ and likely contribute to the increased human susceptibility to AD.     

Purification and Characterization of a New Heme-Binding （HBP59） from the Mutant Strain DJ35 of Azotobacter vinelandii

A new protein, an approximately 59-kDa monomer containing iron atoms, was first isolated from the mutant strain DJ35 of Azotobacter vinelandii Llpmann. After analysis by matrix-assisted laser desorptlon ionization time-of-flight mass spectrometry, the protein was Identified as the product of a predicted gene. Thus, the protein was tentatively called HBP59. Its absorption spectra （ABS） In the reduced state exhibited three peaks at 421,517, and 556 nm and the maximal peak was shifted from 421 to 413 nm after exposure of HBP59 to air. The Soret circular dichrolsm （CD） spectrum of HBP59 In the reduced state displayed four positive peaks at 364, 382, 406, and 418 nm and two negative peaks at 398 and 433 nm; the △ε （CD extinction coefficient） values of these peaks were found to be 0.92, 0.58, 0.87, 0.72, -0.65 and -1.12 L/mol per cm, respectively. Titration with heme showed that the protein has 0.1 heme molecules/protein molecule. After HBP59 had fully Interacted with heme, Its maximal ABS value and Soret CD Intensity were increased by approximately 10-fold compared with values before Interaction. Therefore, It seems that one molecule of HBP59 can be interacted with only one heme. These results indicate that HBP59 contains heme with low spin and may be Involved In heme utilization or adhesion.     

A protein with multiple heme-binding sites from rabbit serum

A 93,000 molecular weight protein (HBP.93) which binds hemin and protoporphyrin IX with high affinity has been isolated from rabbit serum using affinity chromatography on hemin-conjugated agarose. The amino acid composition of this protein is unique in that the proline and histidine contents are remarkably high (16.6 and 9.9 mol %, respectively). A large increase in the absorbance of the Soret region arises from the heme-protein interaction. The spectrophotometric titration showed that the protein can bind 25-35 mol of hemin/mol of protein. The apparent dissociation constant was estimated to be 1-4 X 10(-7) M for hemin at pH 7.4 and approximately 10(-6) M for protoporphyrin IX at pH 9.2. The similarity of the difference spectrum of heme-HBP.93 complex to that of heme-hemopexin complex suggests that a bisimidazol-type coordination of heme iron is involved in the binding. The extremely high capacity of HBP.93 to bind heme is also demonstrated by a large increase in the sedimentation velocity of the protein upon heme binding. The native heme-protein complex migrates faster than the heme-free protein in a polyacrylamide gel at pH 8.8; the increased mobility appears to be due to the charge on the carboxyl groups of the bound heme. Although the use of a hemin-agarose column has failed to reveal a protein of similar size and heme affinity in the sera of a number of other species, including man, the heme-binding properties and high histidine level of the human alpha 2-histidine-rich glycoprotein raise the possibility that the two proteins are related.     

Heme-peptide/protein ions and phosphorous ligands: search for site-specific addition reactions

High-resolution Fourier transform ion cyclotron resonance mass spectrometry is employed to gain thorough kinetics and thermodynamics information on the reaction of free and ligated heme-type ions with selected ligands, with the aim of obtaining an insight into the coordination environment of the prosthetic group in a variety of biomolecular ions. Adopting a stepwise approach towards systems of increasing complexity, we examined the reactivity of free gaseous iron(III) protoporphyrin IX ions, Fe(III)-heme(+), of the charged species from microperoxidase-11 (MP11) (covalently peptide bound heme), and of the multiply charged ions from heme proteins, namely, cytochrome c (cyt c) and myoglobin (examples of noncovalently protein bound hemes). Among an array of test compounds allowed to react with Fe(III)-heme(+), OP(OMe)(3) and P(OMe)(3) proved to be similarly efficient ligands in the first addition step, yet displayed markedly distinct reactivity towards heme iron already engaged in axial coordination. The ease with which P(OMe)(3) acts as a second axial ligand is exploited to probe structural and conformational features of biomolecular ions. In this way, circumstantial evidence is gained of a folded conformation of +2 charge state ions from MP11 and an elongated one for the +3 charge state ions. Similarly, both the general reaction pattern and detailed kinetics and thermodynamics data point to a regiospecific addition reaction of P(OMe)(3) directed at the heme iron within multiply charged ions from cyt c. This unprecedented example of ion-molecule reaction which specifically involves a prosthetic group belonging to protein ions stands in contrast to the multiple, nonspecific interactions established by OP(OMe)(3) molecules with the protonated sites of multiply charged cyt c and apomyoglobin ions. This finding may develop and provide sensitive probes of the structure and bonding features of protein ions in the gas phase.     

Activation of heme-regulated eukaryotic initiation factor 2alpha kinase by nitric oxide is induced by the formation of a five-coordinate NO-heme complex: optical absorption, electron spin resonance, and resonance raman spectral studies

Heme-regulated eukaryotic initiation factor 2alpha kinase (HRI) regulates the synthesis of hemoglobin in reticulocytes in response to heme availability. HRI contains a tightly bound heme at the N-terminal domain. Earlier reports show that nitric oxide (NO) regulates HRI catalysis. However, the mechanism of this process remains unclear. In the present study, we utilize in vitro kinase assays, optical absorption, electron spin resonance (ESR), and resonance Raman spectra of purified full-length HRI for the first time to elucidate the regulation mechanism of NO. HRI was activated via heme upon NO binding, and the Fe(II)-HRI(NO) complex displayed 5-fold greater eukaryotic initiation factor 2alpha kinase activity than the Fe(III)-HRI complex. The Fe(III)-HRI complex exhibited a Soret peak at 418 nm and a rhombic ESR signal with g values of 2.49, 2.28, and 1.87, suggesting coordination with Cys as an axial ligand. Interestingly, optical absorption, ESR, and resonance Raman spectra of the Fe(II)-NO complex were characteristic of five-coordinate NO-heme. Spectral findings on the coordination structure of full-length HRI were distinct from those obtained for the isolated N-terminal heme-binding domain. Specifically, six-coordinate NO-Fe(II)-His was observed but not Cys-Fe(III) coordination. It is suggested that significant conformational change(s) in the protein induced by NO binding to the heme lead to HRI activation. We discuss the role of NO and heme in catalysis by HRI, focusing on heme-based sensor proteins.     

Identification of heme binding protein complexes in murine erythroleukemic cells: study by a novel two-dimensional native separation -- liquid chromatography and electrophoresis

In the current postgenomic era there is a growing interest in analysis of protein complexes in their native state. Here we present a novel two-dimensional separation technique for assessment of native protein complexes. The method combines native chromatography with native electrophoresis. The approach was used to study heme-binding protein complexes in murine erythroleukemia cells. The cells were metabolically labeled with [(59)Fe]-heme and cellular lysates were separated by anion-exchange chromatography. Fractions containing the (59)Fe isotope were collected, concentrated and further separated by native gel electrophoresis. A total of 13 radioactive protein bands were detected and analyzed by liquid chromatography-tandem mass spectrometry. Thirty-three individual proteins were identified and attributed to four novel multiprotein complexes representing four different 'snapshots' of cellular events involved in hemoglobin biosynthesis.     

Unique heme-iron coordination by the hemoglobin receptor IsdB of Staphylococcus aureus

Iron is an essential requirement for life for nearly all organisms. The human pathogen Staphylococcus aureus is able to acquire iron from the heme cofactor of hemoglobin (Hb) released from lysed erythrocytes. IsdB, the predominant Hb receptor of S. aureus, is a cell wall-anchored protein that is composed of two NEAT domains. The N-terminal NEAT domain (IsdB-N1) binds Hb, and the C-terminal NEAT domain (IsdB-N2) relays heme to IsdA for transport into the cell. Here we present the 1.45 ? resolution X-ray crystal structure of the IsdB-N2-heme complex. While the structure largely conforms to the eight-strand β-sandwich fold seen in other NEAT domains such as IsdA-N and uses a conserved Tyr residue to coordinate heme-iron, a Met residue is also involved in iron coordination, resulting in a novel Tyr-Met hexacoordinate heme-iron state. The kinetics of the transfer of heme from IsdB-N2 to IsdA-N can be modeled as a two-step process. The rate of transfer of heme between the isolated NEAT domains (82 s(-1)) was found to be similar to that measured for the full-length proteins. Replacing the iron coordinating Met with Leu did not abrogate high-affinity heme binding but did reduce the heme transfer rate constant by more than half. This unusual Met-Tyr heme coordination may also bestow properties on IsdB that help it to bind heme in different oxidation states or extract heme from hemoglobin.     

β-淀粉样蛋白-血红素复合物和蛋白质酪氨酸硝化及其与阿尔茨海默症的关系

β-淀粉样蛋白(Amyloid-β,Aβ)是阿尔茨海默症(Alzheimer’s disease,AD)病人大脑中淀粉样斑块的主要组成部分。β-淀粉样蛋白级联假说指出,Aβ在脑实质的沉积是最终导致阿尔茨海默症的一个关键步骤。目前的大量研究表明,相对于高度聚集的Aβ,可溶性的Aβ低聚物可能与认知功能障碍的关联性更强。血红素(heme)的代谢在AD患者大脑中发生了改变。近来发现heme可与Aβ结合,形成一个复合物Aβ-heme,该复合物拥有显著高于heme的过氧化物酶活性,具有比heme更强的催化蛋白质酪氨酸硝化的能力。这个结果提示,Aβ-heme可能是联系Aβ与AD中大量蛋白质发生硝化的关键分子。同时,Aβ与heme的结合改变了heme催化蛋白质硝化的位点选择性。这些研究对于阐明Aβ和heme在体内可能的生理作用具有重要意义。