BACKGROUND For decades, semiquinone intermediates have been suggested to play an essential role in catalysis by one of the most enigmatic proton-pumping enzymes, respiratory complex I, and different mechanisms have been proposed on their basis. However, the difficulty in investigating complex I semiquinones, due to the many different enzymes embedded in the inner mitochondrial membrane, has resulted in an ambiguous picture and no consensus. RESULTS In this paper, we re-examine the highly debated origin of semiquinone species in mitochondrial membranes using a novel approach. Our combination of a semi-artificial chimeric respiratory chain with pulse EPR spectroscopy (HYSCORE) has enabled us to conclude, unambiguously and for the first time, that the majority of the semiquinones observed in mitochondrial membranes originate from complex III. We also identify a minor contribution from complex II. CONCLUSIONS We are unable to attribute any semiquinone signals unambiguously to complex I and, reconciling our observations with much of the previous literature, conclude that they are likely to have been misattributed to it. We note that, for this earlier work, the tools we have relied on here to deconvolute overlapping EPR signals were not available. Proposals for the mechanism of complex I based on the EPR signals of semiquinone species observed in mitochondrial membranes should thus be treated with caution until future work has succeeded in isolating any complex I semiquinone EPR spectroscopic signatures present.

中文翻译：

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使用嵌合呼吸链和 EPR 光谱来确定先前分配给线粒体复合物 I 的半醌物种的起源。


背景几十年来，半醌中间体被认为在最神秘的质子泵酶之一——呼吸复合物 I 的催化中发挥着重要作用，并且基于它们提出了不同的机制。然而，由于线粒体内膜中嵌入了许多不同的酶，研究复合物 I 半醌很困难，导致图像模糊且没有达成共识。结果在本文中，我们使用一种新方法重新审视了备受争议的线粒体膜中半醌物种的起源。我们将半人工嵌合呼吸链与脉冲 EPR 光谱 (HYSCORE) 相结合，使我们首次明确得出结论，在线粒体膜中观察到的大多数半醌源自复合物 III。我们还确定了复合体 II 的一个微小贡献。结论 我们无法将任何半醌信号明确地归因于复合物 I，并且将我们的观察结果与之前的许多文献进行协调，得出结论，它们很可能被错误地归因于它。我们注意到，对于这项早期工作，我们所依赖的对重叠 EPR 信号进行解卷积的工具不可用。因此，在未来的工作成功分离出任何存在的复合物 I 半醌 EPR 光谱特征之前，应谨慎对待基于在线粒体膜中观察到的半醌种类的 EPR 信号的复合物 I 机制的建议。