Protein radicals in O2 reduction by Cytochrome Oxidase

描述（申请人提供）：研究金属酶细胞色素c氧化酶的作用机制。细胞色素氧化酶通过在双核血红素a3/CuB活性位点催化氧还原成水，维持线粒体内一系列呼吸酶的电子流动。细胞色素氧化酶利用在这一过程中释放的能量进行质子跨膜泵送，从而直接促进化学渗透质子梯度的形成。细胞色素氧化酶最近加入了一类新的金属自由基酶。独特的组氨酸-酪氨酸交联位点被氧化成自由基。自由基位点作为CuB配体的结构位置强烈表明其在双氧还原和质子泵送中的作用。由于活性位点的结构，自由基既没有直接观察到，也没有使用传统的光谱技术进行表征。我们打算通过使用两种新的和独特的仪器来克服这一限制，这两种仪器连接了三个光谱域-电子吸收，红外振动和电子顺磁-从而大大提高了分辨率和灵敏度。我们最近的研究结果表明，即使在大的蛋白质中，芳香族自由基也可以通过其紫外吸收来检测和表征。采用新的方法，我们将研究关键的自由基模型，即酪氨酸、色氨酸、组氨酸和组氨酸-酪氨酸位点模型，重点研究它们的紫外吸收和振动特性。该结果将应用于细胞色素氧化酶中难以捉摸的自由基的检测。由于质子在细胞色素氧化酶的能量守恒中起着中心作用，因此在催化循环的关键阶段，自由基位点的质子化状态将被特别研究。本提案中描述的技术和预期结果将直接适用于广泛的医学相关酶。紫外光谱技术在自由基检测中的应用将为快速发展的金属酶家族中瞬态自由基的早期检测和动力学研究提供一种简单易行的工具。

DESCRIPTION (provided by applicant): The mechanism by which metalloenzyme cytochrome c oxidase operates will be studied. Cytochrome oxidase sustains electron flow through a cascade of respiratory enzymes in mitochondrion by catalyzing reduction of oxygen into water at the binuclear heme a3/CuB active site. Cytochrome oxidase harnesses the energy released in this process and utilizes it for transmembrane pumping of protons thus directly contributing to the buildup of chemiosmotic proton gradient. Cytochrome oxidase recently joined a new class of metalloradical enzymes.The unique histidine-tyrosine cross-linked site is oxidized to a radical. Structural position of the radical site as a ligand to CuB strongly suggests its role in both the dioxygen reduction and proton pumping. Due to the structure of the active site, radical species have neither been directly observed nor characterized using traditional spectroscopic techniques. We intend to overcome this limitation by using two new and unique instruments, which bridge three spectroscopic domains - electronic absorption, infra-red vibrational and electron paramagnetic - thus dramatically improving resolution and sensitivity. Our recent results show that aromatic radicals can be detected and characterized by their UV absorption even in large proteins. Using new methodology we will investigate key models radicals, namely tyrosine, tryptophan, histidine and the model for histidine-tyrosine site, with the emphasis on their UV absorption and vibrational properties. The results will be applied to detection of elusive radicals in cytochrome oxidase. Protonation states of radical sites at critical stages of the catalytic cycle will be particularly investigated because of the central role protons play in energy conservation by cytochrome oxidase. The techniques described in this proposal and anticipated results will be directly applicable for a broad range of medically relevant enzymes. Application of UV spectroscopy for radical detection will provide a simple and accessible tool for an early detection and kinetic studies of transient radicals in the rapidly growing family of metalloradical enzymes.