Cape Jonathan L, Bowman Michael K, Kramer David M
Institute of Biological Chemistry, Washington State University, 289 Clark Hall, Pullman, WA 99164-6314, USA.
Proc Natl Acad Sci U S A. 2007 May 8;104(19):7887-92. doi: 10.1073/pnas.0702621104. Epub 2007 Apr 30.
The cytochrome bc1 and related complexes are essential energy-conserving components of mitochondrial and bacterial electron transport chains. They orchestrate a complex sequence of electron and proton transfer reactions resulting in the oxidation of quinol, the reduction of a mobile electron carrier, and the translocation of protons across the membrane to store energy in an electrochemical proton gradient. The enzyme can also catalyze substantial rates of superoxide production, with deleterious physiological consequences. Progress on understanding these processes has been hindered by the lack of observable enzymatic intermediates. We report the first direct detection of a semiquinone radical generated by the Q(o) site using continuous wave and pulsed EPR spectroscopy. The radical is a ubisemiquinone anion and is sensitive to both specific inhibitors and mutations within the Q(o) site as well as O2, suggesting that it is the elusive intermediate responsible for superoxide production. Paramagnetic interactions show that the new semiquinone species is buried in the protein, probably in or near the Q(o) site but not strongly interacting with the 2Fe2S cluster. The semiquinone is substoichiometric, even with conditions optimized for its accumulation, consistent with recently proposed models where the semiquinone is destabilized to limit superoxide production. The discovery of this intermediate provides a critical tool to directly probe the elusive chemistry that takes place within the Q(o) site.
细胞色素bc1及相关复合物是线粒体和细菌电子传递链中至关重要的能量守恒组件。它们精心编排了一系列复杂的电子和质子转移反应，导致醌醇氧化、一种可移动电子载体还原，以及质子跨膜转运，从而将能量存储在电化学质子梯度中。该酶还能催化产生大量超氧化物，产生有害的生理后果。由于缺乏可观测的酶促中间体，对这些过程的理解进展一直受阻。我们报告了首次使用连续波和脉冲电子顺磁共振光谱法直接检测到由Q(o)位点产生的半醌自由基。该自由基是一个泛半醌阴离子，对Q(o)位点内的特定抑制剂和突变以及O2均敏感，这表明它就是导致超氧化物产生的难以捉摸的中间体。顺磁相互作用表明，新的半醌物种埋藏在蛋白质中，可能在Q(o)位点内或附近，但与2Fe2S簇没有强烈相互作用。即使在为其积累而优化的条件下，半醌的化学计量比也不足，这与最近提出的模型一致，即半醌不稳定以限制超氧化物的产生。这一中间体的发现为直接探究Q(o)位点内难以捉摸的化学反应提供了关键工具。
