In vitro reconstitution reveals major differences between human and bacterial cytochrome c synthases.
In vitro reconstitution reveals major differences between human and bacterial cytochrome c synthases.
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DOI：
10.7554/elife.64891
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发表时间：
2021-05-11
期刊：
影响因子：
7.7
通讯作者：
Kranz RG
中科院分区：
文献类型：
--
作者：
Sutherland MC;Mendez DL;Babbitt SE;Tillman DE;Melnikov O;Tran NL;Prizant NT;Collier AL;Kranz RG
细胞色素c是线粒体和细菌中普遍存在的一种血红素蛋白，它们都具有CXXCH(CysXxxXxxCysHis)基序和共价连接的血红素。我们描述了首次使用纯化的线粒体(HCCS)和细菌(CcsBA)细胞色素c合成酶在体外重建细胞色素c的生物发生。我们使用细胞色素c和含有CXXCH的多肽类似物作为底物，研究识别决定因素、硫醚结合以及随后细胞色素c的释放和折叠。多肽类似物揭示了HCCS和CcsBA之间非常不同的识别要求。对于HCCS，需要一个最小的16聚体多肽，由CXXCH和相邻的α螺旋1组成，但这两个硫醇都不是识别的关键。对于细菌CcsBA，硫醇和组氨酸都是必需的，但不是α螺旋1。血红素结合的多肽类似物不从HCCS活性部位释放；因此，折叠在释放机制中是重要的。多肽类似物作为细胞色素c生物合成的抑制物，为靶向控制铺平了道路。从微小的细菌到最高的树木，地球上的大多数生命都携带一种名为细胞色素c的蛋白质，这种蛋白质有助于创造能量为细胞提供动力。细胞色素c之所以能做到这一点，要归功于它的血红素，这是一种能够实现产生能量过程所需的化学反应的分子。尽管动物和细菌都依赖于细胞色素c，但它们用来将血红素连接到细胞色素上的酶有所不同。发现这种细胞色素c合成酶如何工作的变化将有助于找到在细菌中使该酶失活的化合物，但在人类中则不会。然而，在活细胞中研究细胞色素c合成酶是具有挑战性的。为了绕过这个问题，Sutherland，Mendez，Babbitt等人。在试管中成功重组人和细菌的细胞色素c合成酶。这使他们能够详细检查酶识别哪些结构，以确定将血红素连接到目标上的位置。实验表明，人类和细菌合酶实际上依赖于细胞色素c的不同部分来定位自己。不同的短化合物也可以阻断人类或细菌的酶。人类和细菌细胞色素c合成酶之间的差异可能导致新的抗生素，使细胞色素失活，杀死细菌，同时保护患者。下一步是确定特定干扰细菌中细胞色素c合成酶的分子，并可能在临床试验中进行测试。
Cytochromes c are ubiquitous heme proteins in mitochondria and bacteria, all possessing a CXXCH (CysXxxXxxCysHis) motif with covalently attached heme. We describe the first in vitro reconstitution of cytochrome c biogenesis using purified mitochondrial (HCCS) and bacterial (CcsBA) cytochrome c synthases. We employ apocytochrome c and peptide analogs containing CXXCH as substrates, examining recognition determinants, thioether attachment, and subsequent release and folding of cytochrome c. Peptide analogs reveal very different recognition requirements between HCCS and CcsBA. For HCCS, a minimal 16-mer peptide is required, comprised of CXXCH and adjacent alpha helix 1, yet neither thiol is critical for recognition. For bacterial CcsBA, both thiols and histidine are required, but not alpha helix 1. Heme attached peptide analogs are not released from the HCCS active site; thus, folding is important in the release mechanism. Peptide analogs behave as inhibitors of cytochrome c biogenesis, paving the way for targeted control. From tiny bacteria to the tallest trees, most life on Earth carries a protein called cytochrome c, which helps to create the energy that powers up cells. Cytochrome c does so thanks to its heme, a molecule that enables the chemical reactions required for the energy-creating process. Despite both relying on cytochrome c, animals and bacteria differ in the enzyme they use to attach the heme to the cytochrome. Spotting variations in how this ‘cytochrome c synthase’ works would help to find compounds that deactivate the enzyme in bacteria, but not in humans. However, studying cytochrome c synthase in living cells is challenging. To bypass this issue, Sutherland, Mendez, Babbitt et al. successfully reconstituted cytochrome c synthases from humans and bacteria in test tubes. This allowed them to examine in detail which structures the enzymes recognize to spot where to attach the heme onto their target. The experiments revealed that human and bacterial synthases actually rely on different parts of the cytochrome c to orient themselves. Different short compounds could also block either the human or bacterial enzyme. Variations between human and bacterial cytochrome c synthase could lead to new antibiotics which deactivate the cytochrome and kill bacteria while sparing patients. The next step is to identify molecules that specifically interfere with cytochrome c synthase in bacteria, and could be tested in clinical trials.