Whitaker T L, Berry M B, Ho E L, Hargrove M S, Phillips G N, Komiyama N H, Nagai K, Olson J S
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005-1892, USA.
Biochemistry. 1995 Jul 4;34(26):8221-6. doi: 10.1021/bi00026a002.
All globins consist of eight helices and interconnecting loops except alpha hemoglobin subunits which lack the D-helix due to deletion of five consecutive residues. Previous site-directed mutagenesis work suggested that this deletion is a neutral modification in hemoglobin with respect to equilibrium O2 binding [Komiyama, N. H., Shih, T.-B., Looker, D., Tame, J., & Nagai, K. (1991) Nature 352, 349-351]. To examine the role of the D-helix in myoglobin, we have measured the O2 and CO binding and hemin dissociation properties of recombinant sperm whale myoglobin mutants in which residues 52-56 have been deleted, Mb(-D), replaced by five alanines, Mb(Ala52-56), and substituted with four alanines and a methionine, Mb(Ala52-55Met56). Crystal structures of aquometMb(-D) and aquometMb(Ala52-55Met56) were determined to 2.0 A resolution and show that the conformation of the distal pocket is little affected by removal of the D-helix or mutations in this region. As a result, these mutations have little effect on O2 and CO binding. Diffuse electron density is observed in the region between the C- and E-helices of Mb(-D), indicating a highly mobile or heterogeneous conformation in this portion of the tertiary structure. This flexibility provides an explanation for the 50-fold higher rate of hemin loss from Mb(-D) as compared to that from wild-type myoglobin. Hemin loss from Mb(Ala52-56) is also rapid. In contrast, Mb(Ala52-55Met56) shows a well-defined D-helix and has a rate of hemin loss identical to that of wild-type holoprotein [corrected].(ABSTRACT TRUNCATED AT 250 WORDS)
除α-血红蛋白亚基外，所有珠蛋白均由八个螺旋和相互连接的环组成，α-血红蛋白亚基由于连续五个残基的缺失而缺少D-螺旋。先前的定点诱变研究表明，就平衡O2结合而言，这种缺失对血红蛋白是一种中性修饰[小宫山，N.H.，施，T.-B.，洛克，D.，塔姆，J.，& 永井，K.（1991年）《自然》352，349 - 351]。为了研究D-螺旋在肌红蛋白中的作用，我们测量了重组抹香鲸肌红蛋白突变体的O2和CO结合以及血红素解离特性，这些突变体中残基52 - 56已被缺失，即肌红蛋白（-D），被五个丙氨酸取代，即肌红蛋白（Ala52 - 56），并用四个丙氨酸和一个甲硫氨酸取代，即肌红蛋白（Ala52 - 55Met56）。高铁肌红蛋白（-D）和高铁肌红蛋白（Ala52 - 55Met56）的晶体结构分辨率达到2.0 Å，结果表明，去除D-螺旋或该区域的突变对远端口袋的构象影响很小。因此，这些突变对O2和CO结合影响很小。在肌红蛋白（-D）的C-螺旋和E-螺旋之间的区域观察到弥散电子密度，表明三级结构的这一部分具有高度可移动或异质的构象。这种灵活性解释了与野生型肌红蛋白相比，肌红蛋白（-D）的血红素损失速率高50倍的原因。肌红蛋白（Ala52 - 56）的血红素损失也很快。相比之下，肌红蛋白（Ala52 - 55Met56）显示出明确的D-螺旋，其血红素损失速率与野生型全蛋白相同[校正后]。（摘要截断于250字）
