Design and synthesis of de novo cytochromes c.
Design and synthesis of de novo cytochromes c.
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DOI：
10.1021/bi049546e
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发表时间：
2004-07
期刊：
影响因子：
2.9
通讯作者：
Manabu Ishida;N. Dohmae;Y. Shiro;T. Oku;T. Iizuka;Y. Isogai
中科院分区：
文献类型：
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作者：
Manabu Ishida;N. Dohmae;Y. Shiro;T. Oku;T. Iizuka;Y. Isogai
天然C型细胞色素的特征在于共有Cys-X-X-Cys-His血红素结合基序（其中X是任何氨基酸），通过该基序，血红素通过将两个半胱氨酸残基的巯基添加到血红素的乙烯基上而共价连接到蛋白质上。在这项工作中，共有序列被用于血红素结合位点的设计的四螺旋束，并与组氨酸残基或甲硫氨酸残基定位在第六个协调位点的脱辅基蛋白的合成和反应与铁原卟啉IX（原血红素）在温和的还原条件下在体外。这些多肽通过单个硫醚键结合每个螺旋-环-螺旋单体中的一个血红素，并如所设计的那样以全息形式形成四螺旋束二聚体。他们表现出可见的吸收光谱特征的c型细胞色素，其中的吸收带转移到较低的波长相比，b型血红素结合中间体相同的蛋白质。出乎意料的是，设计的细胞色素c与双组氨酸协调血红素铁表现出氧化还原电位类似的b型中间体，其中没有硫醚键。此外，细胞色素c与His和Met残基作为轴向配体表现出的氧化还原电位仅增加了15-30 mV相比，与双-His的协调。这些结果表明，高度积极的氧化还原电位的天然细胞色素c不仅是由于血红素共价结构，包括蛋氨酸连接，但也由于非共价和周围的血红素疏水环境。血红素与天然细胞色素c中多肽的共价连接可能有助于其更高的氧化还原电位，通过降低氧化形式相对于还原形式的热力学稳定性而不损失血红素。
Natural c-type cytochromes are characterized by the consensus Cys-X-X-Cys-His heme-binding motif (where X is any amino acid) by which the heme is covalently attached to protein by the addition of the sulfhydryl groups of two cysteine residues to the vinyl groups of the heme. In this work, the consensus sequence was used for the heme-binding site of a designed four-helix bundle, and the apoproteins with either a histidine residue or a methionine residue positioned at the sixth coordination site were synthesized and reacted with iron protoporphyrin IX (protoheme) under mild reducing conditions in vitro. These polypeptides bound one heme per helix-loop-helix monomer via a single thioether bond and formed four-helix bundle dimers in the holo forms as designed. They exhibited visible absorption spectra characteristic of c-type cytochromes, in which the absorption bands shifted to lower wavelengths in comparison with the b-type heme binding intermediates of the same proteins. Unexpectedly, the designed cytochromes c with bis-His-coordinated heme iron exhibited oxidation-reduction potentials similar to those of their b-type intermediates, which have no thioether bond. Furthermore, the cytochrome c with His and Met residues as the axial ligands exhibited redox potentials increased by only 15-30 mV in comparison with the cytochrome with the bis-His coordination. These results indicate that highly positive redox potentials of natural cytochromes c are not only due to the heme covalent structure, including the Met ligation, but also due to noncovalent and hydrophobic environments surrounding the heme. The covalent attachment of heme to the polypeptide in natural cytochromes c may contribute to their higher redox potentials by reducing the thermodynamic stability of the oxidized forms relatively against that of the reduced forms without the loss of heme.