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学科主题: 生命有机化学
题名: Structure, interaction and electron transfer between cytochrome b=5, its E44A and/or E56A mutants and cytochrome c
其他题名: 细胞色素b=5,它的E44A和、或E56A突变体与细胞色素C之间的结构、相互作用和电子转换
作者: Yu-Long Sun ; Wang YH(王韵华) ; Yan MM(严曼明) ; Bin-Yun Sun ; Yi Xie ; Huang ZX(黄仲贤) ; Jiang SK(江绍开) ; Wu HM(吴厚铭)
通讯作者: 黄仲贤 ; 吴厚铭
刊名: J. Mol. Biol.
发表日期: 1999-01-01
卷: 285, 期:1, 页:347-359
收录类别: SCI
部门归属: 复旦大学化学系; 中国科学院上海有机化学研究所生命有机化学国家重点实验室
英文摘要: Site-directed mutagenesis has been used to produce variants of a tryptic fragment of bocine liver cytochrome b=5 in which Glu44 and Glu56 are mutated to alanine. The reduction potentials measured by spectroelectrochemical titration (in the presence of 1 mM(Ru(NH=3)=6^3^+, pH 7.0 and I=0.1 M) are 4.5, 6.0, 6.0 and 7.5 mV versus the standard hydrogen electrode (SHE) for the wild-type and E44A, E56A and E44/56A mutants of cytochrome b=5 and its E44/56A mutant in water solution has been achieved. Resonance assignments of side-chains have been completed successfully. The NMR results suggest that the secondary structures and global folding of the E44/56A mutant remain unchanged, but the mutation of both Glu44 and Glu56 to hydrophobic alanine may lead to the two helices containing mutated residues contracting towards the heme center. The inner mobility of the Gly42~Glu44 segment in cytochrome b=5 may be responsible for the difference of the binding mode between Glu44 and Glu56 with cytochrome c. The binding between cytochrome c and cytochrome b=5 was studied by optical difference spectra of cytochrome c and variants of cyrochrome b=5. The association constants (K=A)for the wild-type, E44A,E56A,and E44/56A mutants of cytochrome b=5 with cytochrome c, are 4.70(±0.10)×10^6M^-1, 1.88(±0.03)×10^6M^-1, 2.70(±0.13)×10^6M^-1, and 1.14(±0.05)×10^6 M^-1, respectively. This is indicative that both Glu44 and Glu56 are involved in the complex formation between cytochrome b=5 and cytochrome c. The reduction of horse heart ferricytochrome c by recombinant ferrocytochrome b=5 and its mutants has been studied. The rate constant of the electron transfer reaction between ferricytochrome c and wild-type ferrocytochrom b=5 (1.074(±0.49)×10^7 M^-1=s^-1) is higher than those of the mutant protein E44A (8.98(±0.20)×10^6 M=-1s^-1), E56A (8.76(±0.39)×10^6 M^-1s^-1), and E44/56A (8.02(±0.38)×10^6 M^-1s^-1) at 15℃, pH 7.0, I=0.35 M. The rate constants arestrongly dependent on ionic strength and temperature. These studies, by meansof a series of techniques, provide conclusive results that the interaction between cytochrome b=5 and cytochrome c is electrostatically guided, and, more importantly, that both Glu44 and Glu56 participate in the electron transfer reaction.
语种: 英语
相关网址: 查看原文
内容类型: 期刊论文
URI标识: http://ir.sioc.ac.cn/handle/331003/17243
Appears in Collections:上海有机化学研究所_期刊论文

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Recommended Citation:
Yu-Long Sun,Wang YH,Yan MM,et al. Structure, interaction and electron transfer between cytochrome b=5, its E44A and/or E56A mutants and cytochrome c[J]. J. Mol. Biol.,1999,285(1):347-359.
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