Purification and spectroscopic characterization of a recombinant chloroplastic hemoglobin from the green unicellular alga Chlamydomonas eugametos
Purification and spectroscopic characterization of a recombinant chloroplastic hemoglobin from the green unicellular alga Chlamydomonas eugametos
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DOI：
10.1111/j.1432-1033.1996.0779r.x
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发表时间：
1996-12-15
期刊：
影响因子：
--
通讯作者：
Guertin, M
中科院分区：
文献类型：
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作者：
Couture, M;Guertin, M
血红蛋白（Hb），具有通过促进其与血红素的可逆结合来递送分子氧的重要任务，现在被认为已经在包括原核生物、真菌、植物和动物的所有生物群中进化。我们最近在绿色单细胞的衣原体真配子体中发现了光诱导的叶绿体Hb，这进一步扩展了这一想法，同时提出了Hb在高氧环境中（如叶绿体中）可能发挥的功能的问题。为了了解这种新的血红蛋白所发挥的作用，我们进行了其生化特性。为了便于表征衣原体Hb，其代表小于0.01%的可溶性蛋白质的绿色细菌，蛋白质已在大肠杆菌中表达并纯化至表观均一性。纯化的重组蛋白具有非共价结合的铁-原卟啉IX血红素。直接从细菌细胞中纯化的重组Hb的氧形式非常稳定，在pH 8下测得的半衰期为7天，并且具有与纤毛原生动物草履虫和四膜虫的相关细胞质Hb以及蓝细菌普通念珠藻的那些类似的紫外/可见光谱。与已报道的氧合肌红蛋白和氧合血红蛋白相比，在NADPH存在下，与LI 637 Hb结合的双氧分子可以被电子转移介体吩嗪硫酸甲酯还原，这表明衣原体Hb的血红素口袋可能更容易被小分子接近。关于这一点，我们发现，当小还原剂连二亚硫酸钠用于还原met形式时，必须在蛋白质氧化之前将其从Hb中厌氧除去以稳定地产生氧合形式。否则，当铁氧还蛋白和铁氧还蛋白NADP（+）还原酶用于酶促还原Hb时，在含氧气氛下容易从met形式获得氧合形式。最后，脱氧和甲硫氨酸形式的光谱是不寻常的，血红素是部分低自旋在生理pH值。这些结果证实了一个可逆的氧结合蛋白的存在，在叶绿体中的C。真配子蛋白质的不寻常的光谱和生化特性可能反映了这种Hb的特殊功能。
Hemoglobins (Hb), which have the important task of delivering molecular oxygen by facilitating its reversible binding to the heme, are now thought to have evolved in all groups of organisms including prokaryotes, fungi, plants and animals. Our recent finding of a light-inducible chloroplastic Hb in the green unicellular alga Chlamydomonas eugametos has further extend this idea, while raising questions about the function that an Hb could play in a high oxygen environment such as in the chloroplast. In order to understand the role played by this new Hb, we have undertaken its biochemical characterization. To facilitate the characterization of Chlamydomonas Hb, which represents less than 0.01% of the soluble protein in the green alga, the protein has been expressed in Escherichia coli and purified to apparent homogeneity. The purified recombinant protein possesses a non-covalently bound iron-protoporphyrin IX heme. The oxy form of the recombinant Hb, purified directly from bacterial cells, is very stable, with a measured half-life of 7 days at pH 8 and has an ultraviolet/visible spectrum similar to those of the related cytoplasmic Hbs of the ciliated protozoa Paramecium and Tetrahymena and of the cyanobacterium Nostoc commune. In contrast to what has been reported for oxymyoglobins and oxyhemoglobins, the dioxygen molecule bound to the LI637 Hb can be reduced by the electron-transfer mediator phenazine methosulfate in the presence of NADPH, indicating that the heme pocket of Chlamydomonas Hb may be more accessible to small molecules. With regard to this, we found that when the small reducing agent sodium dithionite is used to reduce the met form, it must be removed anaerobically from the Hb prior to oxygenation of the protein to stably produce the oxy form. Otherwise, the oxy form is obtained readily from the met form under an oxygenic atmosphere when ferredoxin and ferredoxin NADP(+) reductase are used to enzymically reduce the Hb. Finally, the spectra of the deoxy and met forms were unusual, the heme being partly low-spin at physiological pH. These results confirm the existence of a reversible oxygen-binding protein in the chloroplast of C. eugametos. The unusual spectral and biochemical properties of the protein may reflect a specialized function for this Hb.