Proton nuclear magnetic resonance studies of hemoglobins M Boston (.alpha.58E7 His .fwdarw. Tyr) and M Milwaukee (.beta.67E11 Val .fwdarw. Glu): spectral assignments of hyperfine-shifted proton resonances and of proximal histidine (E7) NH resonances to the .alpha. and .beta. chains of normal human adult hemoglobin

Takahashi, Shunsuke; Lin, Kuo; Ho, Chien

doi:10.1021/bi00564a007

Cited by 87 publications

(72 citation statements)

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“…The hyperfine-shifted proton resonances of the N ␦ H exchangeable proton of proximal histidines are between 60 and 80 ppm from DSS. The resonances at 63 and 76 ppm have been assigned to ␣His-87 and ␤His-92 of the deoxy Hb A (48,49), respectively. For the three ␣-subunit E11 mutants, the resonance at 76 ppm remains unchanged.…”

Section: Resultsmentioning

confidence: 99%

Autoxidation and Oxygen Binding Properties of Recombinant Hemoglobins with Substitutions at the αVal-62 or βVal-67 Position of the Distal Heme Pocket

Tam

Rice

Maillett

et al. 2013

Journal of Biological Chemistry

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Background: Tertiary structure of the ligand binding pocket influences oxygen binding and autoxidation of hemoglobin. Results: E11 mutants have increased autoxidation rate. ␤E11Phe increases, whereas ␤E11Ile decreases the oxygen binding affinity of hemoglobin. Conclusion: Bulky residues at ␤E11 affect ligand binding and cause noticeable tertiary structural changes at the heme pockets. Significance: Hemoglobin distal heme pocket mutations alter oxygen binding properties without changing the quaternary structure.

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Section: Resultsmentioning

confidence: 99%

Autoxidation and Oxygen Binding Properties of Recombinant Hemoglobins with Substitutions at the αVal-62 or βVal-67 Position of the Distal Heme Pocket

Tam

Rice

Maillett

et al. 2013

Journal of Biological Chemistry

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“…6). The resonance at =63 ppm has been assigned to the hyperfineshifted N8H-exchangeable proton of the proximal histidine residue of the a-chain of deoxy-Hb A and the one at =77 ppm has been assigned to the corresponding residue of the P chain of deoxy-Hb A (33,35). In Fig.…”

Section: Resultsmentioning

confidence: 99%

Production of unmodified human adult hemoglobin in Escherichia coli.

Shen

Simplăceanu

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

160

235

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We have constructed a plasmid (pHE2) in which the synthetic human a-and 3-globin genes and the methionine aminopeptidase (Met-AP) gene from Escherichia coli are coexpressed under the control of separate tac promoters. The Hbs were expressed in E. coli JM109 and purified by fast protein liquid chromatography, producing two major components, a and b. Electrospray mass spectrometry shows that at least 98% and about 90% of the expressed a and (3 chains of component a, respectively, have the expected masses.The remaining 10% of the P chain in component a corresponds in mass to the .8 chain plus methionine. In component b, both a and (3 chains have the correct masses without detectable N-terminal methionine (<2%). These results have been confirmed by Edman degradation studies of the amino-terminal sequences of the a and ,B chains of these two recombinant Hb (rHb) samples. rHbs from components a and b exhibit visible optical spectra identical to that of human normal adult Hb (Hb A). Component a and Hb A have very similar oxygen-binding properties, but component b shows somewhat altered oxygen binding, especially at low pH values. 1H-NMR spectra of component a and Hb A are essentially identical, whereas those of component b exhibit altered ring current-shifted and hyperfine-shifted proton resonances, indicating altered heme conformation in the (3 chain. These altered resonance patterns can be changed to those of Hb A by converting component b to the ferric state and then to the deoxy state and finally back to either the carbonmonoxy or oxy form. Thus, our E. coli expression system produces native, unmodified Hb A in high yield and can be used to produce desired mutant Hbs.To make use of our ability to rationally design mutant human Hbs needed for research on structure-function relationships, an efficient expression system for producing unmodified human Hbs in high yields is needed. Human adult Hb (Hb A) is a tetrameric protein containing two a chains and two (3 chains having 141 and 146 amino acid residues each, respectively. Human globins and Hbs have been expressed in transgenic mice (1-4), transgenic swine (5), insect cell cultures (6), yeast (7, 8), and Escherichia coli (9-11). In many respects, the E. coli system is the best choice for our purposes because of its high expression efficiency and the ease ofperforming site-directed mutagenesis. The first E. coli system to express human a-and 3-globin as a fusion protein was developed by Nagai and Th0gersen (9,12), but the product processing procedure is very laborious and gives low yield. Thus, this expression system has limitations, especially when large amounts of recombinant Hb (rHb) are required for biochemical-biophysical studies. Hoffman et al.

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“…the broad anisotropic EPR signal of the Co-porphyrin protein (42), the small hyperfine shift of the N ␦ proton resonance (43) and the unusually low frequency of the Fe-His mode (39). Interestingly, these features are shared by the ␣ chains in the ferrous HbA tetramer (44,45). Thus, the heme coordination geometry and the properties of the exogenous ligand both determine whether or not the proximal histidine bond will be ruptured.…”

Section: Discussionmentioning

confidence: 98%

Hydroxide Rather Than Histidine Is Coordinated to the Heme in Five-coordinate Ferric Scapharca inaequivalvisHemoglobin

Das¹,

Boffi²,

Chiancone³

et al. 1999

Journal of Biological Chemistry

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The ferric form of the homodimeric Scapharca hemoglobin undergoes a pH-dependent spin transition of the heme iron. The transition can also be modulated by the presence of salt. From our earlier studies it was shown that three distinct species are populated in the pH range 6 -9. At acidic pH, a low-spin six-coordinate structure predominates. At neutral and at alkaline pHs, in addition to a small population of a hexacoordinate high-spin species, a pentacoordinate species is significantly populated. Isotope difference spectra clearly show that the heme group in the latter species has a hydroxide ligand and thereby is not coordinated by the proximal histidine. The stretching frequency of the Fe-OH moiety is 578 cm ؊1 and shifts to 553 cm ؊1 in H 2 18 O, as would be expected for a Fe-OH unit. On the other hand, the ferrous form of the protein shows substantial stability over a wide pH range. These observations suggest that Scapharca hemoglobin has a unique heme structure that undergoes substantial redox-dependent rearrangements that stabilize the Fe-proximal histidine bond in the functional deoxy form of the protein but not in the ferric form.The homodimeric hemoglobin (HbI) 1 isolated from arcid clam Scapharca inaequivalvis possesses unique structural features (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Although HbI has a low sequence homology with tetrameric mammalian hemoglobins (Hb), it has a conserved globin fold. It displays a unique structural basis for cooperative ligand binding in that the two hemes face each other across the intersubunit contact and are able to communicate directly through their propionate groups (2,4,15,16). As a consequence of this unusual structural linkage, cooperativity is accompanied by major tertiary changes in the heme environment with only minor rearrangements of the quaternary structure, in contrast to mammalian Hbs (2, 13). The subunit interface in HbI is formed mostly by the helices E and F, which are solventexposed in tetrameric mammalian Hbs.Although a wealth of structural and functional information is available on the ferrous deoxy and ligand-bound forms of HbI (2-14), little is known about the oxidized form (1). The heme structure in the ferric form undergoes a spin transition that is dependent on pH and salt concentration (17,18). From our earlier studies by optical and resonance Raman spectroscopy, it was shown that a mixture of three distinct species are populated in the pH range 6 -9 (17, 18). The formation of a sixcoordinate low-spin heme is favored at acidic pH and high ionic strength and is accompanied by the reversible dissociation of the HbI dimer into monomers. This species is likely to be formed by coordination of the distal histidine to the heme (in addition to the proximal histidine) as shown by EPR studies (18). At neutral pH values, a dimeric pentacoordinate species appears and becomes the dominant form at alkaline pH and low salt. A population of dimeric six-coordinate high-spin heme exists over the entire 6 -9 pH-range and in both low and high salt. Thi...

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Cited by 87 publications

References 28 publications

Autoxidation and Oxygen Binding Properties of Recombinant Hemoglobins with Substitutions at the αVal-62 or βVal-67 Position of the Distal Heme Pocket

Autoxidation and Oxygen Binding Properties of Recombinant Hemoglobins with Substitutions at the αVal-62 or βVal-67 Position of the Distal Heme Pocket

Production of unmodified human adult hemoglobin in Escherichia coli.

Hydroxide Rather Than Histidine Is Coordinated to the Heme in Five-coordinate Ferric Scapharca inaequivalvisHemoglobin

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