Generation, characterization and crystallization of a cytochrome c(1)-subunit IV fused cytochrome bc(1) complex from Rhodobacter sphaeroides.

Generation, characterization and crystallization of a cytochrome c(1)-subunit IV fused cytochrome bc(1) complex from Rhodobacter sphaeroides.
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球形红杆菌细胞色素 c(1)-IV 亚基融合细胞色素 bc(1) 复合物的生成、表征和结晶。

DOI:
10.1016/j.bbabio.2011.10.005
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发表时间:
2012
期刊:
Biochimica et biophysica acta
影响因子:
--
通讯作者:
Yu,Linda
Yu,Linda
中科院分区:
--
文献类型:
--
作者:
Su,Ting;Esser,Lothar;Xia,Di;Yu,Chang-An;Yu,Linda

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细胞色素bc 1复合物催化电子从泛醇转移到细胞色素c(或细胞色素c2)的反应,并将该反应与质子跨膜转运偶联。Rhodobacter sphaeroides bc 1复合物的结晶导致晶体中只含有三个核心亚基。为了减轻结晶过程中亚基IV从三亚基核心复合物中解离的问题,我们最近设计了一种R。其中亚基IV的N-末端与细胞色素c1的C-末端融合,在两个融合亚基之间具有14-甘氨酸接头,并且在亚基IV的C-末端具有6-组氨酸标签(c1- 14 Gly-IV-6 His)。与野生型酶相比,纯化的融合突变体复合物显示出更高的电子转移活性、更高的结构稳定性和更少的超氧化物生成。初步的结晶尝试与这种突变体复合物产生晶体含有四个亚基和衍射X射线到5.5nm分辨率。
Cytochrome bc1complex catalyzes the reaction of electron transfer from ubiquinol to cytochrome c (or cytochrome c2) and couples this reaction to proton translocation across the membrane. Crystallization of the Rhodobacter sphaeroides bc1complex resulted in crystals containing only three core subunits. To mitigate the problem of subunit IV being dissociated from the three-subunit core complex during crystallization, we recently engineered an R. sphaeroides mutant in which the N-terminus of subunit IV was fused to the C-terminus of cytochrome c1with a 14-glycine linker between the two fusing subunits, and a 6-histidine tag at the C-terminus of subunit IV (c1-14Gly-IV-6His). The purified fusion mutant complex shows higher electron transfer activity, more structural stability, and less superoxide generation as compared to the wild-type enzyme. Preliminary crystallization attempts with this mutant complex yielded crystals containing four subunits and diffracting X-rays to 5.5Å resolution.
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