Proton nuclear magnetic resonance and spectrophotometric studies of nickel(II)-iron(II) hybrid hemoglobins.
Proton nuclear magnetic resonance and spectrophotometric studies of nickel(II)-iron(II) hybrid hemoglobins.
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镍（II）-铁（II）杂化血红蛋白的质子核磁共振和分光光度研究。
DOI：
10.1021/bi00382a019
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发表时间：
1987
期刊：
影响因子：
2.9
通讯作者：
Yonetani,T
中科院分区：
文献类型：
--
作者：
Shibayama,N;Inubushi,T;Morimoto,H;Yonetani,T
宾夕法尼亚大学生物化学与生物物理系，宾夕法尼亚州费城，19104-6089；大阪大学工程科学学院生物物理工程系，大阪丰中，560，日本摘要：用质子核磁共振技术对Ni (II)-Fe （II）杂交血红蛋白a (Fe) 2/3 (Ni) 2和a (Ni) 2/3 (Fe) 2进行了表征，并将Ni （II）原卟啉IX （Ni- pp）掺入载脂蛋白中作为永久脱氧血红素。a (Fe) 2/3 (Ni) 2、a (Ni) 2/3 (Fe) 2和NiHb在11ppm和14ppm时通常表现出可交换的质子共振，这是由于脱氧结构中的氢键质子。一氧化碳（CO）与（Fe） 2/？（Ni） 2，这些共振在pH 6.5 ~ pH 8.5时消失。另一方面，互补杂化的a (Ni) 2/3 (Fe-CO) 2在低pH下表现出11和14 ppm的共振。当pH升高时，两种共振的强度都降低，但这些变化并不同步。电子吸收光谱和超精细移质子共振表明，CO在0 （Fe）亚基中的连接引起了Ni-PP在a亚基中的配位和旋态的变化。在类脱氧结构中，Ni-PP在a亚基中的配位主要处于低自旋（5= 0）四配位态，而在类氧结构中，高自旋（S'= 1）五配位态的贡献显著增加。无论溶液条件和伙伴a （Fe）亚基的连接状态如何，β亚基中的Ni-PP总是采用高自旋五配位策略。在β（）亚基中，当改变杂化物的四元结构时，检测到近端组氨酸共振的显著下移和Ni-PP的吸收光谱的变化。此外，当pH值从6.5升高到8.5时，（Fe-CO） 218 (Ni) 2的近端组氨酸共振发生了轻微的上移，而作为脱氧四元结构标志信号的14 ppm共振在该pH区域几乎没有变化。此外，还研究了两种混合Hbs的环电流移位共振。对杂化Hbs中El - 1-Val甲基与卟啉环的化学位移进行了分析。尽管Hb1是一种被广泛研究的变构蛋白，但其协同氧化的机制尚不完全清楚。这是由于在氧化过程中直接描述中间物种的困难，因为在合作系统中结扎的中间物种以低浓度存在。因此，许多被视为中间物种模型的杂交Hbs被人工制备和检验（Banerjee & Cassoly, 1969； Ikeda-Saito等人，1977;Blough & Hoffman, 1984； Simolo等人，1985）。我们研究了金属取代的杂化Hbs， a (Fe) 20-(M) 2和a (M) 2/3 (Fe) 2，其中a或β亚基中的血红素被含有铁系过渡金属离子(M)的卟啉取代。这些杂化Hbs的氧平衡性质表明它们是Ni （II）原卟啉IX，本研究得到了NIH （HL 14508 to TY）的资助。所有的核磁共振光谱都是在宾夕法尼亚大学医学院的核磁共振设备中拍摄的，部分由美国国立卫生研究院的SO7-RR-05415和S07-RR-07083资助。宾夕法尼亚大学。大阪大学。
Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6089, and Department of Biophysical Engineering, Faculty of Engineering Science, OsakaUniversity, Toyonaka, Osaka 560, Japan Received September 19, 1986; Revised Manuscript Received December 12, 1986 abstract: Ni (II)-Fe (II) hybrid hemoglobins, a (Fe) 2/3 (Ni) 2 and a (Ni) 2/3 (Fe) 2, havebeen characterized by proton nuclear magnetic resonance with Ni (II) protoporphyrin IX (Ni-PP) incorporated in apoprotein, which serves as a permanent deoxyheme. a (Fe) 2/3 (Ni) 2, a (Ni) 2/3 (Fe) 2, and NiHb commonly show ex-changeable proton resonances at 11 and 14 ppm, due to hydrogen-bonded protons in a deoxy-like structure. Upon binding of carbon monoxide (CO) to a (Fe) 2/?(Ni) 2, these resonances disappear at pH 6.5 to pH 8.5. On the other hand, the complementaryhybrid a (Ni) 2/3 (Fe-CO) 2 showed the 11 and 14 ppm resonances at low pH. Upon raising pH, the intensities of both resonances are reduced, although these changes are not synchronized. Electronic absorption spectra and hyperfine-shifted proton resonances indicate that the ligation of CO in the 0 (Fe) subunits inducedchanges in the coordination and spinstates of Ni-PP in the a subunits. In a deoxy-like structure, the coordination of Ni-PP in the a subunits is predominantly in a low-spin (5= 0) four-coordination state, whereas in an oxy-like structure the contribution of a high-spin (S'= 1) five-coordination state markedly increased. Ni-PP in the ß subunits always takes a high-spin five-coordination stateregardless of solution conditions and the state of ligation in the partner a (Fe) subunits. In the ß () subunits, a significant downfield shift of the proximal histidyl resonance and a change in the absorption spectrum of Ni-PP were detected, upon changing the quaternary structure of the hybrid. Furthermore, the proximal histidyl resonance of a (Fe-CO) 2l8 (Ni) 2 undergoes a slight upfield shift upon raising the pH from 6.5 to 8.5, while the 14 ppm resonance, themarker signal of a deoxy quaternary structure, hardly changes in this pH region. In addition, ring current shifted resonances in both hybrid Hbs were examined. The chemical shifts were analyzed in terms of the El 1-Val methyls vs. the porphyrin rings in hybrid Hbs. tjven though Hb1 is a well-investigated allosteric protein, the mechanism of cooperative oxygenation is not fully un-derstood. This is causedby the difficulty in characterizing the intermediate species directly inthe course of oxygenation, since theintermediate species of ligation are present in low concentrations in a cooperative system. Thus, many kinds of hybrid Hbs, which were regarded as models for the intermediate species, have been artificially prepared and examined (Banerjee & Cassoly, 1969; Ikeda-Saito et al., 1977; Blough & Hoffman, 1984; Simolo et al., 1985). We have surveyed metal-substituted hybrid Hbs, a (Fe) 20-(M) 2 and a (M) 2/3 (Fe) 2, in which the hemes in either a or ß subunits are substituted with porphyrins containing iron-series transition metal ions (M). The oxygen equilibrium properties of these hybrid Hbs indicated that Ni (II) protoporphyrin IX, fThis work was supported by a grant from the NIH (HL 14508 to TY). All NMR spectra were taken in the medical school NMR facility at the University of Pennsylvania, which is partially supported by Grants SO7-RR-05415 and S07-RR-07083 from the NIH.* University of Pennsylvania. S Osaka University.