CONVERSION OF CYTOCHROME B(562) TO C-TYPE CYTOCHROMES
CONVERSION OF CYTOCHROME B(562) TO C-TYPE CYTOCHROMES
复制标题
DOI：
10.1021/bi00046a027
复制
发表时间：
1995-11-21
期刊：
影响因子：
2.9
通讯作者：
FEARNLEY, IM
中科院分区：
文献类型：
--
作者：
BARKER, PD;NEROU, EP;FEARNLEY, IM
来自大肠杆菌周质的细胞色素 b(562) 是细胞色素家族中唯一的成员，该家族共享 4-α-螺旋束结构基序，但不具有共价结合的血红素。我们将半胱氨酸残基引入到细胞色素 b(562) 的氨基酸序列中，其位置与该家族其他成员中的氨基酸序列同源，从而生成了几乎所有 c 型细胞色素中普遍存在的血红素结合肽 (-C-X-Y-C-H-)。由此产生的含有单半胱氨酸的突变体 R98C 和 Y101C 以及结合这两种突变的双突变体已被表达到大肠杆菌的周质中。每个突变的脱辅基蛋白和全蛋白产物都已被分离，并且所有突变体都产生具有共价连接的血红素的多个物种。离子交换色谱、光谱、SDS 凝胶电泳和电喷雾质谱的结果鉴定出那些似乎是细胞色素 b(562) 全蛋白的物种，与血红素具有硫醚共价连接，这是它们与野生型蛋白质之间化学成分的唯一差异。 H-1-NMR 实验的结果证明了这些蛋白质中存在预期的 C 型共价键，并表明血红素袋的结构并未受到共价修饰的显着干扰。与野生型蛋白质相比，这些蛋白质都具有扰动的光谱，这与修饰一致，但仍然具有六配位、低自旋细胞色素的特征，其中 Met-His 连接到两种氧化态的血红素铁。
Cytochrome b(562) from the periplasm of Escherichia coli is the only member of a family of cytochromes sharing the 4-alpha-helical bundle structural motif that does not have a covalently bound heme. We have introduced cysteine residues into the amino acid sequence of cytochrome b(562) in positions homologous to those found in the other members of the family, generating the ubiquitous heme-binding peptide (-C-X-Y-C-H-) found in virtually all c-type cytochromes. The resulting single-cysteine-containing mutants, R98C and Y101C, together with the double mutant combining both of these mutations have been expressed into the periplasm of E. coli. The apo- and holoprotein products of each mutation have been isolated, and all the mutants produce multiple species with covalently attached heme. Results from ion exchange chromatography, optical spectroscopy, SDS gel electrophoresis, and electrospray mass spectrometry identified those species that appear to be cytochrome b(562) holoprotein with thioether covalent Linkages to the heme as the only difference in chemical composition between them and the wild-type protein. Results from H-1-NMR experiments prove the existence of the expected c-type covalent bonds in each of these proteins and show that the structure of the heme pocket is not significantly perturbed by the covalent modification(s). These proteins all have perturbed optical spectra, compared with those of the wild-type protein, that are consistent with the modifications but are still characteristic of six-coordinate, low-spin cytochromes with Met-His ligation to the heme iron in both oxidation states.