Nagai Masako, Nagai Yukifumi, Aki Yayoi, Sakurai Hiroshi, Mizusawa Naoki, Ogura Takashi, Kitagawa Teizo, Yamamoto Yasuhiko, Nagatomo Shigenori
Research Center for Micro-Nano Technology, Hosei University, Koganei, Tokyo, Japan.
Department of Health Sciences, Kanazawa University School of Medicine, Kanazawa, Japan.
Chirality. 2016 Aug;28(8):585-92. doi: 10.1002/chir.22620. Epub 2016 Jul 18.
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.
天然人类成人血红蛋白（Hb A）的血红素大多具有正常取向，而在大肠杆菌中表达的重组Hb A（rHb A）含有正常和反向取向的血红素。具有正常血红素的Hb A在Soret和260 nm区域均呈现正圆二色性（CD）带，而具有反向血红素的rHb A则显示负Soret带且260 nm CD带减弱。为了研究α或β亚基的近端组氨酸（His F8）在确定血红素取向中的作用，我们制备了两种腔突变体血红蛋白，rHb(αH87G)和rHb(βH92G)，在咪唑存在下用甘氨酸取代His F8。两种腔突变体血红蛋白的CD光谱均未显示负Soret带，而是在Soret和260 nm区域均呈现强度较强的正带，这表明腔突变体血红蛋白中几乎不存在反向血红素。我们通过¹H NMR和共振拉曼（RR）光谱证实，腔突变体血红蛋白在突变和天然亚基中主要具有正常的血红素取向。这些结果表明，α和β亚基中的血红素Fe-His F8连接影响血红素取向，并且一种亚基的血红素取向与互补亚基的血红素取向相同。本研究表明，除了常用的¹H NMR技术外，CD和RR光谱也是检测Hb A血红素旋转无序的有力工具。《手性》28:585 - 592，2016年。© 2016威利期刊公司。
