Wu Y, Wang Y, Qian C, Lu J, Li E, Wang W, Lu J, Xie Y, Wang J, Zhu D, Huang Z, Tang W
State Key Laboratory of Coordination Chemistry, Nanjing, China.
Eur J Biochem. 2001 Mar;268(6):1620-30.
Using 1617 meaningful NOEs with 188 pseudocontact shifts, a family of 35 conformers of oxidized bovine microsomal cytochrome b5 mutant (E44/48/56A/D60A) has been obtained and is characterized by good resolution (rmsd to the mean structure are 0.047 +/- 0.007 nm and 0.095 +/- 0.008 nm for backbone and heavy atoms, respectively). The solution structure of the mutant, when compared with the X-ray structure of wild-type cytochrome b(5), has no significant changes in the whole folding and secondary structure. The binding between cytochrome b(5) and cytochrome c shows that the association constant of the mutant-cytochrome c complex is much lower than the one for wild-type complex (2.2 x 10(4) M(-1) vs. 5.1 x 10(3) M(-1)). The result suggests the four acidic residues have substantial effects on the formation of the complex between cytochrome b(5) and cytochrome c, and therefore it is concluded reasonably that the electrostatic interaction plays an important role in maintaining the stability and specificity of the complex formed. The competition between the ferricytochrome b(5) mutant and Cr(oxalate)(3) for ferricytochrome c shows that site III of cytochrome c, which is a strong binding site to wild-type cytochrome b(5), still binds to the mutant with relatively weaker strength. Our results indicate that certain bonding geometries do occur in the interaction between the present mutant and cytochrome c and these geometries, which should be quite different from the ones of the Salemme and Northrup models.
利用1617个有意义的核Overhauser效应（NOE）和188个赝接触位移，获得了氧化型牛微粒体细胞色素b5突变体（E44/48/56A/D60A）的35个构象异构体家族，其分辨率良好（主链和重原子相对于平均结构的均方根偏差分别为0.047±0.007nm和0.095±0.008nm）。与野生型细胞色素b5的X射线结构相比，该突变体的溶液结构在整体折叠和二级结构上没有显著变化。细胞色素b5与细胞色素c之间的结合表明，突变体-细胞色素c复合物的缔合常数远低于野生型复合物（2.2×10⁴M⁻¹对5.1×10³M⁻¹）。结果表明这四个酸性残基对细胞色素b5与细胞色素c之间复合物的形成有实质性影响，因此合理推断静电相互作用在维持所形成复合物的稳定性和特异性中起重要作用。高铁细胞色素b5突变体与[Cr(草酸盐)₃]³⁻对高铁细胞色素c的竞争表明，细胞色素c的位点III（对野生型细胞色素b5是强结合位点）与突变体的结合强度仍然相对较弱。我们的结果表明，在当前突变体与细胞色素c的相互作用中确实存在某些键合几何结构，并且这些几何结构应该与Salemme和Northrup模型的几何结构有很大不同。
