Reactive Intermediates in cytochrome oxidase

有了这个奖项，化学部门的生命过程化学项目正在资助博士。来自阿尔伯特爱因斯坦医学院的Denis Rousseau， Syun-Ru Yeh和Gary Gerfen定量地确定了决定细胞色素c氧化酶中氧还原为水如何与质子易位相结合的因素。人体95%以上的氧气被细胞色素c氧化酶吸收，这种酶位于线粒体的内膜上。酶将氧还原为水，产生能量，利用能量将质子转移到线粒体内膜上，形成质子梯度，质子梯度反过来被用来产生细胞内使用的化学能。我们在理解上的一个主要空白是氧还原是如何与质子易位耦合的。在本赠款中，将探讨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。这些实验将涉及新的快速混合技术的开发和最先进的光谱技术的使用，这将成为学生和博士后研究人员的优秀培训平台。以往的研究表明，过氧化氢与细胞色素c氧化酶反应产生的中间体与氧与细胞色素c氧化酶反应产生的中间体有许多相同。过氧化氢与细胞色素c氧化酶反应的机制尚不清楚。过氧化氢与血红素a3基团反应并形成自由基是反应的第一步，自由基随后迁移到氨基酸残基的假设将被验证。一个基于自由基从Tyr244转移到Tyr129的质子易位模型也将被测试。pH值、过氧化氢浓度和氧气浓度的变化将决定如何调节反应。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，将确定中间体是否完全演变为过去通过手工混合技术检测到的中间体。快速混合技术将用于启动反应，快速冷冻淬火方法将用于捕获中间体，并将设计和制造新的硅混合器；这些方法可以在从微秒到分钟的时间尺度上跟踪反应。EPR光谱将用于研究中间体和反应产物。参与该项目的学生和博士后将获得生物能源学、先进技术开发和最新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.