title: Heme Orientation of Cavity Mutant Hemoglobins (His F8 → Gly) in Either α or β Subunits: Circular Dichroism, (1) H NMR, and Resonance Raman Studies.
abstract: 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.
authors.affiliation: Research Center for Micro-Nano Technology, Hosei University, Koganei, Tokyo, Japan.|Department of Health Sciences, Kanazawa University School of Medicine, Kanazawa, Japan.
authors.forename: Masako
authors.initials: M
authors.lastname: Nagai
authors.affiliation: Department of Health Sciences, Kanazawa University School of Medicine, Kanazawa, Japan.
authors.forename: Yukifumi
authors.initials: Y
authors.lastname: Nagai
authors.affiliation: Department of Health Sciences, Kanazawa University School of Medicine, Kanazawa, Japan.
authors.forename: Yayoi
authors.initials: Y
authors.lastname: Aki
authors.affiliation: Department of Health Sciences, Kanazawa University School of Medicine, Kanazawa, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sakurai
authors.affiliation: Research Center for Micro-Nano Technology, Hosei University, Koganei, Tokyo, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Mizusawa
authors.affiliation: Picobiology Institute, Graduate School of Life Science, University of Hyogo, RSC-UH Leading Program Center, Hyogo, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Ogura
authors.affiliation: Picobiology Institute, Graduate School of Life Science, University of Hyogo, RSC-UH Leading Program Center, Hyogo, Japan.
authors.forename: Teizo
authors.initials: T
authors.lastname: Kitagawa
authors.affiliation: Department of Chemistry, University of Tsukuba, Tsukuba, Ibaraki, Japan.
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Yamamoto
authors.affiliation: Department of Chemistry, University of Tsukuba, Tsukuba, Ibaraki, Japan.
authors.forename: Shigenori
authors.initials: S
authors.lastname: Nagatomo
keywords: His F8 replaced by Gly; Soret and 260-nm CD bands; heme optical activity; heme rotational disorder; hemoglobin; subunit-subunit interaction