DataSheet1_Electron Transfer Coupled to Conformational Dynamics in Cell Respiration.docx

Cellular respiration is a fundamental process required for energy production in many organisms. The terminal electron transfer complex in mitochondrial and many bacterial respiratory chains is cytochrome c oxidase (CcO). This converts the energy released in the cytochrome c/oxygen redox reaction into a transmembrane proton electrochemical gradient that is used subsequently to power ATP synthesis. Despite detailed knowledge of electron and proton transfer paths, a central question remains as to whether the coupling between electron and proton transfer in mammalian mitochondrial forms of CcO is mechanistically equivalent to its bacterial counterparts. Here, we focus on the conserved span between H376 and G384 of transmembrane helix (TMH) X of subunit I. This conformationally-dynamic section has been suggested to link the redox activity with the putative H pathway of proton transfer in mammalian CcO. The two helix X mutants, Val380Met (V380M) and Gly384Asp (G384D), generated in the genetically-tractable yeast CcO, resulted in a respiratory-deficient phenotype caused by the inhibition of intra-protein electron transfer and CcO turnover. Molecular aspects of these variants were studied by long timescale atomistic molecular dynamics simulations performed on wild-type and mutant bovine and yeast CcOs. We identified redox- and mutation-state dependent conformational changes in this span of TMH X of bovine and yeast CcOs which strongly suggests that this dynamic module plays a key role in optimizing intra-protein electron transfers.

细胞呼吸（Cellular respiration）是众多生物体能量产生所必需的核心生理过程。线粒体与多数细菌呼吸链中的末端电子传递复合物为细胞色素c氧化酶（cytochrome c oxidase，缩写CcO），其可将细胞色素c/氧气氧化还原反应释放的能量转化为跨膜质子电化学梯度，后续该梯度将用于驱动ATP合成。尽管学界对电子与质子传递通路已有较为详尽的认知，但仍存在一个核心科学问题：哺乳动物线粒体来源的CcO中，电子传递与质子传递之间的偶联机制，是否与其细菌同源体等价。本研究聚焦于亚基I的跨膜螺旋X（transmembrane helix X，缩写TMH X）中H376与G384之间的保守区段。这一构象动态区域曾被提出，可将氧化还原活性与哺乳动物CcO中假定的质子传递H通路相连接。我们在具备遗传可操作性的酵母CcO中构建了两种螺旋X突变体：Val380Met（V380M）与Gly384Asp（G384D），二者均因蛋白内电子传递受到抑制以及CcO周转效率降低，引发了呼吸缺陷表型。针对这些突变体的分子特征，我们通过对野生型与突变型牛、酵母CcO开展长时标全原子分子动力学模拟进行了研究。我们在牛与酵母CcO的TMH X该区段中，鉴定出了依赖于氧化还原状态与突变状态的构象变化，这强烈提示该动态模块在优化蛋白内电子传递过程中发挥关键作用。