Egeberg K D, Springer B A, Martinis S A, Sligar S G, Morikis D, Champion P M
Department of Biochemistry, University of Illinois, Urbana 61801.
Biochemistry. 1990 Oct 23;29(42):9783-91. doi: 10.1021/bi00494a004.
Three mutant proteins of sperm whale myoglobin (Mb) that exhibit altered axial ligations were constructed by site-directed mutagenesis of a synthetic gene for sperm whale myoglobin. Substitution of distal pocket residues, histidine E7 and valine E11, with tyrosine and glutamic acid generated His(E7)Tyr Mb and Val(E11)Glu Mb. The normal axial ligand residue, histidine F8, was also replaced with tyrosine, resulting in His(F8)Tyr Mb. These proteins are analogous in their substitutions to the naturally occurring hemoglobin M mutants (HbM). Tyrosine coordination to the ferric heme iron of His(E7)Tyr Mb and His(F8)Tyr Mb is suggested by optical absorption and EPR spectra and is verified by similarities to resonance Raman spectral bands assigned for iron-tyrosine proteins. His(E7)Tyr Mb is high-spin, six-coordinate with the ferric heme iron coordinated to the distal tyrosine and the proximal histidine, resembling Hb M Saskatoon [His(beta E7)Tyr], while the ferrous iron of this Mb mutant is high-spin, five-coordinate with ligation provided by the proximal histidine. His(F8)Tyr Mb is high-spin, five-coordinate in both the oxidized and reduced states, with the ferric heme iron liganded to the proximal tyrosine, resembling Hb M Iwate [His(alpha F8)Tyr] and Hb M Hyde Park [His(beta F8)Tyr]. Val(E11)Glu Mb is high-spin, six-coordinate with the ferric heme iron liganded to the F8 histidine. Glutamate coordination to the ferric iron of this mutant is strongly suggested by the optical and EPR spectral features, which are consistent with those observed for Hb M Milwaukee [Val(beta E11)Glu]. The ferrous iron of Val(E11)Glu Mb exhibits a five-coordinate structure with the F8 histidine-iron bond intact.(ABSTRACT TRUNCATED AT 250 WORDS)
通过对抹香鲸肌红蛋白（Mb）的合成基因进行定点诱变，构建了三种轴向配体发生改变的突变蛋白。用酪氨酸和谷氨酸取代远端口袋残基组氨酸E7和缬氨酸E11，产生了His（E7）Tyr Mb和Val（E11）Glu Mb。正常的轴向配体残基组氨酸F8也被酪氨酸取代，得到His（F8）Tyr Mb。这些蛋白质在取代方面类似于天然存在的血红蛋白M突变体（HbM）。光吸收和电子顺磁共振光谱表明酪氨酸与His（E7）Tyr Mb和His（F8）Tyr Mb的三价铁血红素铁配位，并通过与为铁 - 酪氨酸蛋白指定的共振拉曼光谱带的相似性得到验证。His（E7）Tyr Mb是高自旋的，六配位，三价铁血红素铁与远端酪氨酸和近端组氨酸配位，类似于Hb M萨斯卡通[His（βE7）Tyr]，而该Mb突变体的二价铁是高自旋的，五配位，由近端组氨酸提供配体。His（F8）Tyr Mb在氧化态和还原态下都是高自旋的，五配位，三价铁血红素铁与近端酪氨酸配位，类似于Hb M岩手[His（αF8）Tyr]和Hb M海德公园[His（βF8）Tyr]。Val（E11）Glu Mb是高自旋的，六配位，三价铁血红素铁与F8组氨酸配位。光学和电子顺磁共振光谱特征强烈表明谷氨酸与该突变体的三价铁配位，这与Hb M密尔沃基[Val（βE11）Glu]观察到的特征一致。Val（E11）Glu Mb二价铁呈现五配位结构，F8组氨酸 - 铁键完整。（摘要截断于250字）
