Heme Orientation of Cavity Mutant Hemoglobins (His F8 → Gly) in Either α or β Subunits: Circular Dichroism,1H NMR, and Resonance Raman Studies
Autor: | Shigenori Nagatomo, Yayoi Aki, Masako Nagai, Yukifumi Nagai, Takashi Ogura, Naoki Mizusawa, Hiroshi Sakurai, Yasuhiko Yamamoto, Teizo Kitagawa |
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Rok vydání: | 2016 |
Předmět: |
0301 basic medicine
Pharmacology Circular dichroism 030102 biochemistry & molecular biology Stereochemistry Protein subunit Organic Chemistry Mutant Catalysis Analytical Chemistry 03 medical and health sciences chemistry.chemical_compound 030104 developmental biology chemistry Drug Discovery Proton NMR Hemoglobin Chirality (chemistry) Heme Spectroscopy Histidine |
Zdroj: | Chirality. 28:585-592 |
ISSN: | 0899-0042 |
DOI: | 10.1002/chir.22620 |
Popis: | Native human adult hemoglobin (Hb A) has mostly normal orientation of heme, whereas recombinant Hb A (rHb A) expressed in E. coli contains both normal and reversed orientations of heme. Hb A with the normal heme exhibits positive circular dichroism (CD) bands at both the Soret and 260-nm regions, while rHb A with the reversed heme shows a negative Soret and decreased 260-nm CD bands. In order to examine involvement of the proximal histidine (His F8) of either α or β subunits in determining the heme orientation, we prepared two cavity mutant Hbs, rHb(αH87G) and rHb(βH92G), with substitution of glycine for His F8 in the presence of imidazole. CD spectra of both cavity mutant Hbs did not show a negative Soret band, but instead exhibited positive bands with strong intensity at the both Soret and 260-nm regions, suggesting that the reversed heme scarcely exists in the cavity mutant Hbs. We confirmed by (1) H NMR and resonance Raman (RR) spectroscopies that the cavity mutant Hbs have mainly the normal heme orientation in both the mutated and native subunits. These results indicate that the heme Fe-His F8 linkage in both α and β subunits influences the heme orientation, and that the heme orientation of one type of subunit is related to the heme orientation of the complementary subunits to be the same. The present study showed that CD and RR spectroscopies also provided powerful tools for the examination of the heme rotational disorder of Hb A, in addition to the usual (1) H NMR technique. Chirality 28:585-592, 2016. © 2016 Wiley Periodicals, Inc. |
Databáze: | OpenAIRE |
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