Reactive Intermediates in cytochrome oxidase

通过该奖项，化学部生命过程化学项目资助阿尔伯特·爱因斯坦医学院的Denis Rousseau、Syun-Ru Yeh和Gary Gerfen博士定量确定决定氧还原为水的因素如何与细胞色素c氧化酶中的质子转移相耦合。人体95%以上的氧气被细胞色素c氧化酶吸收，细胞色素c氧化酶是一种位于线粒体内膜的酶。酶将氧气还原为水会产生能量，这些能量被用来将质子转移到线粒体内膜上，形成质子梯度，进而用来产生供细胞使用的化学能。我们理解中的一个主要差距是氧还原与质子转移是如何耦合的。在这项授权中，将探索一种假定的耦合机制。该项目的结果有望对细胞色素C氧化酶的功能特性产生新的见解，从而影响生物能量学领域。这些实验将包括开发新的快速混合技术和使用最先进的光谱技术，这将成为学生和博士后研究员的杰出培训平台。以前的研究表明，过氧化氢与细胞色素c氧化酶反应产生许多与氧与细胞色素c氧化酶反应生成的中间产物相同的中间体。过氧化氢与细胞色素c氧化酶的反应机制尚不清楚。过氧化氢与血红素A3基团反应并形成自由基物种作为反应的第一步，随后自由基迁移到氨基酸残基的假设将得到验证。还将测试一个基于自由基从Tyr244转移到Tyr129的质子转移模型。PH、过氧化氢浓度和氧气浓度将会变化，以确定如何调节反应。这些测量将确定最初形成的自由基的位置以及随后形成的各种中间体的时间依赖性。最后，将确定中间体向过去通过手工混合技术检测到的中间体的完全演变。将使用快速混合技术来启动反应，将使用快速冷冻淬火方法来捕获中间体，并将设计和制造新的硅混合器；这些方法将使从微秒到分钟的时间尺度上的反应后续成为可能。电子顺磁共振波谱将用于中间体和反应产物的研究。参与该项目的学生和博士后研究员将获得生物能量学、先进技术开发和最先进的光谱学方面的教育和培训。为该项目设计的新型硅基混合器将提供给更广泛的学术界。

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Drs. Denis Rousseau, Syun-Ru Yeh and Gary Gerfen from the Albert Einstein College of Medicine to quantitatively determine the factors that determine how the reduction of oxygen to water is coupled to proton translocation in cytochrome c oxidase. Over 95% of the oxygen consumed in humans is taken up by cytochrome c oxidase, an enzyme located in the inner membrane of mitochondria. The reduction of oxygen to water by the enzyme generates energy that is harnessed to translocate protons across the inner mitochondrial membrane, forming a proton gradient, which in turn is used to generate chemical energy for use in the cell. A major gap in our understanding is how the oxygen reduction is coupled to the proton translocation. In this grant a postulated mechanism for the coupling will be explored. The results of this project are expected to yield new insights into the functional properties of cytochrome c oxidase, and thereby impact the field of Bioenergetics. The experiments will involve both the development of new rapid mixing techniques and the use of state-of-the-art spectroscopic technology, which will serve as an outstanding training platform for students and postdoctoral fellows. Previous studies have shown that the reaction of hydrogen peroxide with cytochrome c oxidase generates many of the same intermediates as those formed in the reaction of oxygen with the cytochrome c oxidase. The mechanism by which hydrogen peroxide reacts with cytochrome c oxidase is unknown. The hypothesis that hydrogen peroxide reacts with the heme a3 group and forms a radical species as the first step in the reaction and that the radical subsequently migrates to amino acid residues will be tested. A proposed model for proton translocation based on the transfer of a radical from Tyr244 to Tyr129 will be tested also. The pH, the hydrogen peroxide concentration, and the oxygen concentration will be varied to determine how the reaction is regulated. The measurements will identify where the initially formed radical is located and the time dependence of the various intermediates that are subsequently formed. Finally, the full evolution of the intermediates into those detected in the past by hand mixing techniques will be determined. Rapid mixing techniques will be used to initiate the reaction, rapid freeze quench methods will be used to trap the intermediates, and new silicon mixers will be designed and fabricated; these methods will make possible the following up of the reaction on timescales from microseconds to minutes. EPR spectroscopy will be used to study the intermediates and reaction products. Students and post-doctoral fellows who participate in the project will gain education and training in Bioenergetics, advanced technology development and state-of-the-art spectroscopy. New silicon-based mixers designed for this project will be made available to the wider academic community.