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首页> 外文期刊>Biochemistry >Redesign of the proton-pumping machinery of cytochrome c oxidase: proton pumping does not require Glu(I-286).
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Redesign of the proton-pumping machinery of cytochrome c oxidase: proton pumping does not require Glu(I-286).

机译：重新设计细胞色素C氧化酶的质子泵送机械：质子泵送不需要Glu（I-286）。

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摘要

One of the putative proton-transfer pathways leading from solution toward the binuclear center in many cytochrome c oxidases is the D-pathway, so-called because it starts with a highly conserved aspartate [D(I-132)] residue. Another highly conserved amino acid residue in this pathway, glutamate(I-286), has been indicated to play a central role in the proton-pumping machinery of mitochondrial-type enzymes, a role that requires a movement of the side chain between two distinct positions. In the present work we have relocated the glutamate to the opposite side of the proton-transfer pathway by constructing the double mutant EA(I-286)/IE(I-112). This places the side chain in about the same position in space as in the original enzyme, but does not allow for the same type of movement. The results show that the introduction of the second-site mutation, IE(I-112), in the EA(I-286) mutant enzyme results in an increase of the enzyme activity by a factor of >10. In addition, the double mutant enzyme pumps approximately 0.4 proton per electron. This observation restricts the number of possible mechanisms for the operation of the redox-driven proton pump. The proton-pumping machinery evidently does require the presence of a protonatable/polar residue at a specific location in space, presumably to stabilize an intact water chain. However, this residue does not necessarily have to be at a strictly conserved location in the amino acid sequence. In addition, the results indicate that E(I-286) is not the "proton gate" of cytochrome c oxidase controlling the flow of pumped protons from one to the other side of the membrane.
机译：在许多细胞色素C氧化酶中从溶液引向双核中心的推定质子转移途径之一是D途径，之所以称为D途径，是因为它以高度保守的天冬氨酸[D（I-132）]残基开始。已表明该途径中另一个高度保守的氨基酸残基谷氨酸（I-286）在线粒体型酶的质子泵送机制中起着核心作用，这种作用需要侧链在两个不同的分子之间移动职位。在本工作中，我们通过构建双突变体EA（I-286）/ IE（I-112）将谷氨酸重新定位于质子转移途径的另一侧。这将侧链在空间上的位置与原始酶中的位置大致相同，但不允许相同类型的运动。结果表明，在EA（I-286）突变酶中引入第二位点突变IE（I-112）导致酶活性增加了> 10倍。此外，双突变酶每个电子泵浦约0.4质子。该观察结果限制了氧化还原驱动质子泵运行的可能机制的数量。质子泵送机器显然确实需要在空间中的特定位置存在可质子化/极性残留物，以稳定完整的水链。但是，该残基不必一定在氨基酸序列中严格保守的位置。另外，结果表明，E（I-286）不是细胞色素c氧化酶的“质子门”，它控制着泵送质子从膜的一侧流向另一侧。

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