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Title: Heme orientational disorder in human adult hemoglobin reconstituted with a ring fluorinated heme and its functional consequences

Abstract

A ring fluorinated heme, 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2-fluoro-7,12, 18-trimethyl-porphyrin-atoiron(III), has been incorporated into human adult hemoglobin (Hb A). The heme orientational disorder in the individual subunits of the protein has been readily characterized using {sup 19}F NMR and the O{sub 2} binding properties of the protein have been evaluated through the oxygen equilibrium analysis. The equilibrated orientations of hemes in {alpha}- and {beta}- subunits of the reconstituted protein were found to be almost completely opposite to each other, and hence were largely different from those of the native and the previously reported reconstituted proteins [T. Jue, G.N. La Mar, Heme orientational heterogeneity in deuterohemin-reconstituted horse and human hemoglobin characterized by proton nuclear magnetic resonance spectroscopy, Biochem. Biophys. Res. Commun. 119 (1984) 640-645]. Despite the large difference in the degree of the heme orientational disorder in the subunits of the proteins, the O{sub 2} affinity and the cooperativity of the protein reconstituted with 2-MF were similar to those of the proteins reconstituted with a series of hemes chemically modified at the heme 3- and 8-positions [K. Kawabe, K. Imaizumi, Z. Yoshida, K. Imai, I. Tyuma, Studies on reconstituted myoglobins and hemoglobins II. Role of the heme side chains in the oxygenation of hemoglobin, J.more » Biochem. 92 (1982) 1713-1722], whose O{sub 2} affinity and cooperativity were higher and lower, respectively, relative to those of native protein. These results indicated that the heme orientational disorder could exert little effect, if any, on the O{sub 2} affinity properties of Hb A. This finding provides new insights into structure-function relationship of Hb A.« less

Authors:
 [1];  [1];  [1];  [2];  [3];  [4]
  1. Department of Chemistry, University of Tsukuba, Tsukuba 305-8571 (Japan)
  2. Department of Frontier Sciences, Faculty of Engineering, Hosei University, Koganei, Tokyo 184-8584 (Japan)
  3. Department of Materials Engineering, Nagaoka College of Technology, Nagaoka 940-8532 (Japan)
  4. Department of Chemistry, University of Tsukuba, Tsukuba 305-8571 (Japan). E-mail: yamamoto@chem.tsukuba.ac.jp
Publication Date:
OSTI Identifier:
20979830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 354; Journal Issue: 3; Other Information: DOI: 10.1016/j.bbrc.2007.01.010; PII: S0006-291X(07)00040-X; Copyright (c) 2007 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; AFFINITY; ELECTRONIC STRUCTURE; FLUORINE 19; HEME; HEMOGLOBIN; HORSES; MYOGLOBIN; NUCLEAR MAGNETIC RESONANCE; OXYGEN; PARAMAGNETISM; STRUCTURE FUNCTIONS

Citation Formats

Nagao, Satoshi, Hirai, Yueki, Kawano, Shin, Imai, Kiyohiro, Suzuki, Akihiro, and Yamamoto, Yasuhiko. Heme orientational disorder in human adult hemoglobin reconstituted with a ring fluorinated heme and its functional consequences. United States: N. p., 2007. Web. doi:10.1016/j.bbrc.2007.01.010.
Nagao, Satoshi, Hirai, Yueki, Kawano, Shin, Imai, Kiyohiro, Suzuki, Akihiro, & Yamamoto, Yasuhiko. Heme orientational disorder in human adult hemoglobin reconstituted with a ring fluorinated heme and its functional consequences. United States. doi:10.1016/j.bbrc.2007.01.010.
Nagao, Satoshi, Hirai, Yueki, Kawano, Shin, Imai, Kiyohiro, Suzuki, Akihiro, and Yamamoto, Yasuhiko. Fri . "Heme orientational disorder in human adult hemoglobin reconstituted with a ring fluorinated heme and its functional consequences". United States. doi:10.1016/j.bbrc.2007.01.010.
@article{osti_20979830,
title = {Heme orientational disorder in human adult hemoglobin reconstituted with a ring fluorinated heme and its functional consequences},
author = {Nagao, Satoshi and Hirai, Yueki and Kawano, Shin and Imai, Kiyohiro and Suzuki, Akihiro and Yamamoto, Yasuhiko},
abstractNote = {A ring fluorinated heme, 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2-fluoro-7,12, 18-trimethyl-porphyrin-atoiron(III), has been incorporated into human adult hemoglobin (Hb A). The heme orientational disorder in the individual subunits of the protein has been readily characterized using {sup 19}F NMR and the O{sub 2} binding properties of the protein have been evaluated through the oxygen equilibrium analysis. The equilibrated orientations of hemes in {alpha}- and {beta}- subunits of the reconstituted protein were found to be almost completely opposite to each other, and hence were largely different from those of the native and the previously reported reconstituted proteins [T. Jue, G.N. La Mar, Heme orientational heterogeneity in deuterohemin-reconstituted horse and human hemoglobin characterized by proton nuclear magnetic resonance spectroscopy, Biochem. Biophys. Res. Commun. 119 (1984) 640-645]. Despite the large difference in the degree of the heme orientational disorder in the subunits of the proteins, the O{sub 2} affinity and the cooperativity of the protein reconstituted with 2-MF were similar to those of the proteins reconstituted with a series of hemes chemically modified at the heme 3- and 8-positions [K. Kawabe, K. Imaizumi, Z. Yoshida, K. Imai, I. Tyuma, Studies on reconstituted myoglobins and hemoglobins II. Role of the heme side chains in the oxygenation of hemoglobin, J. Biochem. 92 (1982) 1713-1722], whose O{sub 2} affinity and cooperativity were higher and lower, respectively, relative to those of native protein. These results indicated that the heme orientational disorder could exert little effect, if any, on the O{sub 2} affinity properties of Hb A. This finding provides new insights into structure-function relationship of Hb A.},
doi = {10.1016/j.bbrc.2007.01.010},
journal = {Biochemical and Biophysical Research Communications},
number = 3,
volume = 354,
place = {United States},
year = {Fri Mar 16 00:00:00 EDT 2007},
month = {Fri Mar 16 00:00:00 EDT 2007}
}
  • Proton nuclear magnetic resonance spectroscopy has been utilized to investigate the rates of exchange with deuterium of the proximal histidyl ring protons in a series of chemically modified and mutated forms of Hb A. Differences in rates of exchange are related to differences in the stability of the deformed or partially unfolded intermediates from which exchange with bulk solvent takes place. Each modified/mutated Hb exhibited kinetic subunit heterogeneity in the reduced ferrous state, with the alpha subunit exhibiting faster exchange than the beta subunit. Modification or mutation resulted in significant increases in the His F8 ring NH exchange rates primarilymore » for the affected subunit and only if the modification/mutation occurs at the allosterically important alpha 1 beta 2 subunit interface. Moreover, this enhancement in exchange rate is observed primarily in that quaternary state of the modified/mutated Hb in which the modified/substituted residue makes the intersubunit contact. This confirms the importance of allosteric constraints in determining the dynamic properties of the heme pocket. Using modified or mutated Hbs that can switch between the alternate quaternary states within a given ligation state or ligate within a given quaternary state, we show that the major portion of the enhanced exchange rate in R-state oxy Hb relative to T-state deoxy Hb originates from the quaternary switch rather than from ligation. However, solely ligation effects are not negligible. The exchange rates of the His F8 ring labile protons increase dramatically upon oxidizing the iron to the ferric state, and both the subunit kinetic heterogeneity and the allosteric sensitivity to the quaternary state are essentially abolished.« less
  • High-resolution proton nuclear magnetic resonance spectroscopy has been used to investigate the molecular mechanism of the Bohr effect of human normal adult hemoglobin in the presence of two allosteric effectors, i.e., chloride and inorganic phosphate ions. The individual hydrogen ion equilibria of 22-26 histidyl residues of hemoglobin have been measured in anion-free 0.1 M HEPES buffer and in the presence of 0.18 M chloride or 0.1 M inorganic phosphate ions in both deoxy and carbonmonoxy forms. The results indicate that the {beta}2-histidyl residues are strong binding sites for chloride and inorganic phosphate ions in hemoglobin. The affinity of the {beta}2-histidylmore » residues for these anions is larger in the deoxy than in the carbonmonoxy form. Nevertheless, the contribution of these histidyl residues to the anion Bohr effect is small due to their low pK value in deoxyhemoglobin in anion-free solvents. The interactions of chloride and inorganic phosphate ions with the hemoglobin molecule also result in lower pK values and/or changes in the shapes of the hydrogen ion binding curves for several other surface histidyl residues. These results suggest that long-range electrostatic interactions between individual ionizable sites in hemoglobin could play an important role in the molecular mechanism of the anion Bohr effect.« less
  • High-resolution {sup 1}H and {sup 31}P nuclear magnetic resonance spectroscopy has been used to investigate the binding of 2,3-diphosphoglycerate to human normal adult hemoglobin and the molecular interactions involved in the allosteric effect of the 2,3-diphosphoglycerate molecule on hemoglobin. Individual hydrogen ion NMR titration curves have been obtained for 22-26 histidyl residues of hemoglobin and for each phosphate group of 2,3-diphosphoglycerate with hemoglobin in both the deoxy and carbonmonoxy forms. The results indicate that 2,3-diphosphoglycerate binds to deoxyhemoglobin at the central cavity between the two {beta} chains and the binding involves the {beta}2-histidyl residues. Moreover, the results suggest that themore » binding site of 2,3-diphosphoglycerate to carbonmonoxyhemoglobin contains the same (or at least some of the same) amino acid residues responsible for binding in the deoxy form. As a result of the specific interactions with 2,3-diphosphoglycerate, the {beta}2-histidyl residues make a significant contribution to the alkaline Bohr effect under these experimental conditions. These results give the first experimental demonstration that long-range electrostatic and/or conformation effects of the binding could play an important role in the allosteric effect of 2,3-diphosphoglycerate on hemoglobin. The {sup 31}P nuclear magnetic resonance titration data for each phosphate group of 2,3-diphosphoglycerate have been used to calculate the pK values of the phosphate groups in 2,3-diphosphoglycerate bound to deoxy- and carbon-monoxyhemoglobin and the proton uptake by 2,3-diphosphoglycerate upon ligand binding to hemoglobin.« less
  • Using neutron diffraction analysis, the protonation states of 35 of 38 histidine residues were determined for the deoxy form of normal human adult hemoglobin. Distal and buried histidines may contribute to the increased affinity of the deoxy state for hydrogen ions and its decreased affinity for oxygen compared with the oxygenated form. The protonation states of the histidine residues key to the function of deoxy (T-state) human hemoglobin have been investigated using neutron protein crystallography. These residues can reversibly bind protons, thereby regulating the oxygen affinity of hemoglobin. By examining the OMIT F{sub o} − F{sub c} and 2F{sub o}more » − F{sub c} neutron scattering maps, the protonation states of 35 of the 38 His residues were directly determined. The remaining three residues were found to be disordered. Surprisingly, seven pairs of His residues from equivalent α or β chains, αHis20, αHis50, αHis58, αHis89, βHis63, βHis143 and βHis146, have different protonation states. The protonation of distal His residues in the α{sub 1}β{sub 1} heterodimer and the protonation of αHis103 in both subunits demonstrates that these residues may participate in buffering hydrogen ions and may influence the oxygen binding. The observed protonation states of His residues are compared with their ΔpK{sub a} between the deoxy and oxy states. Examination of inter-subunit interfaces provided evidence for interactions that are essential for the stability of the deoxy tertiary structure.« less