Converting cytochrome b5 into cytochrome c-like protein
Converting cytochrome b5 into cytochrome c-like protein
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将细胞色素 b5 转化为细胞色素 C 样蛋白
DOI：
10.1002/cbic.200500030
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发表时间：
2005-08-01
期刊：
影响因子：
3.2
通讯作者：
Wu, HM
中科院分区：
文献类型：
--
作者：
Lin, YW;Wang, WH;Wu, HM
1356 2005 Wiley-VCH Verlag GmbH & Co. KGaA，魏因海姆DOI：10.1002/cbic. 200500030 ChemBioChem 2005，6，1356-1359血红素4-乙烯基和2-乙烯基，分离度分别为3.86和4.32（图1）。如果这两个残基被半胱氨酸取代，这些位置可能适合于共价键的形成。先前的研究表明，在细胞色素b5的57位引入半胱氨酸残基可以与血红素4-乙烯基形成硫醚键。[6]为了进一步研究血红素和蛋白质基质之间形成共价键的可能性，我们通过定点诱变用半胱氨酸取代天然cyt b5中的残基Ser 71和Asn 57。[7]从双位点突变中获得了两个具有不同颜色（红色和绿色）的主要组分，并将其命名为rb 5 N57 C/S71 C（红色）和gb 5 N57 C/S71 C（绿色）。[8]在双位点突变的细胞色素b5中，血红素辅基被共价连接到蛋白质基质上，因为血红素不能用酸化的丁-2-酮提取。[6] rb 5 N57 C/S71 C在吡啶中的血色素光谱在550 nm处显示出α-带，典型的没有双键的血红素与卟啉π-系统共轭，就像c型细胞色素一样。[4]这表明rb 5 N57 C/S71 C中血红素的两个乙烯基是饱和的，血红素基团通过两个共价键连接，因为一个共价连接在553 nm处产生α带，当血红素没有共价连接时，α带在556 nm处。[6]此外，相对于天然cytb 5（三价铁：413 nm;亚铁：423、527和556 nm），观察到两种氧化态的rb 5 N57 C/S71 C的可见光谱中的蓝移（三价铁：409 nm;亚铁：416、524和553 nm）。石田埃塔尔已经说明，这些吸收带的蓝移与天然b型和c型细胞色素之间的差异以及细胞色素B向c的突变转化一致。[9]第一章
1356 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: 10.1002/cbic. 200500030 ChemBioChem 2005, 6, 1356–1359 heme 4-vinyl and 2-vinyl groups with separations of 3.86 and 4.32, respectively (Figure 1). These positions might be suitable for covalent-bond formation if the two residues were replaced with cysteine. Previous study has shown that in cyt b5 a cysteine residue introduced at position 57 could form a thioether bond with the heme 4-vinyl group.[6] In order to further study the possibility of forming a covalent linkage between heme and the protein matrix, we replaced residues Ser71 and Asn57 in the native cyt b5 with cysteine by site-directed mutagenesis.[7] Two major components with different colors (red and green) were obtained from the double-site mutation and were named rb5 N57C/S71C (red) and gb5 N57C/S71C (green).[8] The heme prosthetic group was shown to be covalently attached to the protein matrix in the double-site mutated cyt b5, since the heme could not be extracted with acidified butan-2-one.[6]The hemochrome spectrum of rb5 N57C/S71C in pyridine exhibits an α-band at 550 nm, typical of a heme without double bonds conjugated to the porphyrin π-system, as in c-type cytochromes.[4] This demonstrates that two vinyl groups of heme in rb5 N57C/S71C were saturated and that the heme group was attached through two covalent linkages, since one covalent attachment gives the α-band at 553 nm and when heme is free from covalent attachment the α-band is at 556 nm.[6] Furthermore, blue shifts in the visible spectra of rb5 N57C/S71C in both oxidation states (ferric: 409 nm; ferrous: 416, 524, and 553nm) were observed relative to those of native cytb5 (ferric: 413nm; ferrous: 423, 527, and 556nm). Ishida etal. have illustrated that these blue shifts of absorption bands agree with the differences between natural b-and c-type cytochromes and also with the mutational conversion of cytochrome b to c.[9]