Kloek A P, Yang J, Mathews F S, Frieden C, Goldberg D E
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
J Biol Chem. 1994 Jan 28;269(4):2377-9.
The parasitic nematode Ascaris suum has a gene encoding a two-domain hemoglobin with remarkable oxygen avidity. The strong interaction with oxygen is a consequence of a particularly slow oxygen off-rate. The single polypeptide chain consists of two domains, each of which can be expressed separately in Escherichia coli as a globin-like protein exhibiting oxygen binding characteristics comparable with the native molecule. Site-directed mutagenesis was performed on the gene segment encoding domain one. The E7 position, involved in forming a hydrogen bond with the liganded oxygen in vertebrate globins, is a glutamine in both Ascaris domains. Conversion of this residue to leucine or alanine produced a hemoglobin variant with an oxygen off-rate 5- or 60-fold faster than that of unaltered domain one. Replacement of the tyrosine B10 with either phenylalanine or leucine (as found in vertebrate globins) yielded hemoglobin mutants with oxygen off-rates 280- or 570-fold faster, approaching rates found with vertebrate myoglobins. The data suggest that the distal glutamine hydrogen bonds with the liganded oxygen and that the tyrosine B10 hydroxyl contributes an additional hydrogen bond that appears substantially responsible for the extreme oxygen avidity of Ascaris hemoglobin.
寄生线虫猪蛔虫有一种基因，编码一种具有显著氧亲和力的双结构域血红蛋白。与氧的强烈相互作用是氧解离速率特别慢的结果。单条多肽链由两个结构域组成，每个结构域都可以在大肠杆菌中单独表达为一种类珠蛋白，其氧结合特性与天然分子相当。对编码结构域一的基因片段进行了定点诱变。在脊椎动物珠蛋白中与结合氧形成氢键的E7位点，在蛔虫的两个结构域中都是谷氨酰胺。将该残基转换为亮氨酸或丙氨酸产生了一种血红蛋白变体，其氧解离速率比未改变的结构域一快5倍或60倍。用苯丙氨酸或亮氨酸（如在脊椎动物珠蛋白中发现的）取代酪氨酸B10，产生了氧解离速率快280倍或570倍的血红蛋白突变体，接近脊椎动物肌红蛋白的速率。数据表明，远端谷氨酰胺与结合氧形成氢键，酪氨酸B10羟基贡献了一个额外的氢键，这似乎是蛔虫血红蛋白极高氧亲和力的主要原因。
