Protein radicals in O2 reduction by Cytochrome Oxidase

描述(申请人提供)：将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催化双核血红素A3/CUB活性部位的氧还原到水中来维持线粒体呼吸酶级联的电子流动。细胞色素氧化酶利用在这一过程中释放的能量，并将其用于跨膜泵送质子，从而直接促进化学渗透质子梯度的建立。细胞色素氧化酶最近加入了一类新的金属酶。独特的组氨酸-酪氨酸交联点被氧化为自由基。作为CUB配体的自由基位点的结构位置强烈地表明它在氧气还原和质子泵中的作用。由于活性中心的结构，自由基物种既不能直接观察到，也不能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-从而显著提高了分辨率和灵敏度。我们最近的结果表明，即使在大的蛋白质中，芳香族自由基也可以通过它们的紫外光吸收来检测和表征。使用新的方法，我们将研究关键模型自由基，即酪氨酸、色氨酸、组氨酸和组氨酸-酪氨酸位点模型，重点是它们的紫外光吸收和振动性质。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所起的中心作用，在催化循环的关键阶段将特别研究自由基位置的质子化状态。这项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。将紫外光谱应用于自由基检测，将为快速发展的金属酶家族中的瞬时自由基的早期检测和动力学研究提供一种简单易用的工具。

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.