Semihemoglobins, high oxygen affinity dimeric forms of human hemoglobin respond efficiently to allosteric effectors without forming tetramers.
Significant reduction in oxygen affinity resulting from interactions between heterotropic allosteric effectors and hemoglobin in not only the unligated derivative but also the fully ligated form has been reported (Tsuneshige, A., Park, S. I., and Yonetani, T. (2002) Biophys. Chem. 98, 49-63; Yonetani, T., Park, S. I., Tsuneshige, A., Imai, K., and Kanaori, K. (2002) J. Biol. Chem. 277, 34508-34520). To further investigate this effect in more detail, alpha- and beta-semihemoglobins, namely, alpha(heme)beta(apo) and alpha(apo)beta(heme), respectively, were prepared and characterized with respect to the impact of allosteric effectors on both conformation and ligand binding properties. Semihemoglobins are dimers characterized by a high affinity for oxygen and lack of cooperativity. We found that, compared with stripped conditions, semihemoglobins responded to effectors (inositol hexaphosphate and L35) by decreasing the affinity for oxygen by 60- and 130-fold for alpha- and beta-semihemoglobins, respectively. 1H NMR and sedimentation velocity experiments carried out with their ligated and unligated forms in the absence and presence of effectors revealed that semihemoglobins always remain as single-heme-carrying dimers. Recombination kinetics of their photolyzed CO derivatives showed that effectors did indeed interact with their ligated forms. Measurements of the Fe-His stretching mode show that the semihemoglobins undergo a large ligand binding-induced conformational shift and that both ligand-free and ligand derivatives respond to the presence of effectors. Contradictions to the Monod-Wyman-Changeaux/Perutz allosteric model arise since 1) the modulation of ligand affinity is not achieved in semihemoglobins by the formation of a low affinity T conformation (quaternary effect) but by direct interaction with effectors, 2) effectors do interact significantly with ligated forms of high affinity semihemoglobins, and 3) modulation of the ligand affinity and the cooperativity are not necessarily linked but instead can be separated into two distinct phenomena that can be isolated.
Tsuneshige
Antonio
A
Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine and the Johnson Research Foundation, Philadelphia, Pennsylvania 19104-6059, USA. ants@mail.med.upenn.edu
Kanaori
Kenji
K
Samuni
Uri
U
Danstker
David
D
Friedman
Joel M
JM
Neya
Saburo
S
Giangiacomo
Laura
L
Yonetani
Takashi
T
D000495:Allosteric Site; D002248:Carbon Monoxide / Q000737:chemistry; D019281:Dimerization; D004912:Erythrocytes; D006418:Heme / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D010108:Oxyhemoglobins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D013059:Spectrum Analysis, Raman; D013997:Time Factors; D014461:Ultracentrifugation
D006454:Hemoglobins; D008024:Ligands; D010108:Oxyhemoglobins; D006418:Heme; D002248:Carbon Monoxide; D010100:Oxygen