ALTERATION OF AXIAL COORDINATION BY PROTEIN ENGINEERING IN MYOGLOBIN - BISIMIDAZOLE LIGATION IN THE HIS(64)-]VAL/VAL(68)-]HIS DOUBLE MUTANT
ALTERATION OF AXIAL COORDINATION BY PROTEIN ENGINEERING IN MYOGLOBIN - BISIMIDAZOLE LIGATION IN THE HIS(64)-]VAL/VAL(68)-]HIS DOUBLE MUTANT
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DOI：
10.1074/jbc.270.27.15993
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发表时间：
1995-07-07
影响因子：
4.8
通讯作者：
GEORGE, GN
中科院分区：
文献类型：
--
作者：
DOU, Y;ADMIRAAL, SJ;GEORGE, GN
猪和人肌红蛋白已被改造成能逆转远端组氨酸和缬氨酸的位置(即His(64)(E7) -> Val和Val（68)(E11) -> His）。测定了人和猪H64V/V68H肌红蛋白的光谱和配体结合特性，并用x射线晶体学测定了猪H64V/V68H双突变体的结构，分辨率为2.07埃。晶体结构表明，His（68）的N-epsilon位于距血红素铁2.3埃的位置，形成六配位结构，His（68）的咪唑面相对于血红素法线倾斜；此外，它与His（93）的结果不平行，与我们之前的建议一致(Qin， J, La Mar， G. N., Dou， Y,, admiral， S. J, and Ikeda-Saito， M. (1994) J. Biol。化学，269,1083-1090)。在低温下，血红素铁处于低自旋状态，表现出高度各向异性的EPR谱(g(1) = 3.34, g(2) = 2.0, g(3) < 1)，与肌红蛋白的咪唑配合物有很大的不同。低自旋铁态的平均铁氮距离为2.01埃，H64V/V68H肌红蛋白的铁态表现出与b型细胞色素相似的光谱，与x射线晶体学测定的六配位双咪唑血红蛋白结构一致，双突变使野生型蛋白的铁/铁偶中点电位从+54 mV降至-128 mV。亚铁H64V/V68H肌红蛋白结合CO和NO形成稳定的复合物，但它与O-2的反应导致快速自氧化为铁。所有这些结果表明，His（64）和Val（68）在野生型肌红蛋白中的三维位置与侧链的化学性质在促进可逆O-2结合和抑制自氧化方面同样重要。
Pig and human myoglobin have been engineered to reverse the positions of the distal histidine and valine (i.e. His(64)(E7) --> Val and Val(68)(E11) --> His). Spectroscopic and ligand binding properties have been measured for human and pig H64V/V68H myoglobin, and the structure of the pig H64V/V68H double mutant has been determined to 2.07-Angstrom resolution by x-ray crystallography. The crystal structure shows that the N-epsilon of His(68) is located 2.3 Angstrom away from the heme iron, resulting in the formation of a hexacoordinate species, The imidazole plane of His(68) is tilted relative to the heme normal; moreover it is not parallel to that of His(93), in agreement with our previous proposal (Qin, J,, La Mar, G. N., Dou, Y,, Admiraal, S. J., and Ikeda-Saito, M. (1994) J. Biol. Chem. 269, 1083-1090). At cryogenic temperatures, the heme iron is in a low spin state, which exhibits a highly anisotropic EPR spectrum (g(1) = 3.34, g(2) = 2.0, and g(3) < 1), quite different from that of the imidazole complex of metmyoglobin. The mean iron-nitrogen distance is 2.01 Angstrom for the low spin ferric state as determined by x-ray spectroscopy, The ferrous form of H64V/V68H myoglobin shows an optical spectrum that is similar to that of b-type cytochromes and consistent with the hexacoordinate bisimidazole hemin structure determined by the x-ray crystallography, The double mutation lowers the ferric/ferrous couple midpoint potential from +54 mV of the wild-type protein to -128 mV. Ferrous H64V/V68H myoglobin binds CO and NO to form stable complexes, but its reaction with O-2 results in a rapid autooxidation to the ferric species. All of these results demonstrate that the three-dimensional positions of His(64) and Val(68) in the wild-type myoglobin are as important as the chemical nature of the side chains in facilitating reversible O-2 binding and inhibiting autooxidation.