COPPER-OXYGEN INTERMEDIATES IN CATECHOL OXIDASE

儿茶酚氧化酶是一种导致植物褐变的酶，它催化邻二酚氧化为醌，同时将O2还原为水。儿茶酚氧化酶含有类似酪氨酸酶和血青素的3型双核铜中心。金属酶对氧的活化具有重要的生物学意义，但由于金属-氧的瞬态性质，中间体一直难以表征。本研究旨在测量动力学并鉴定儿茶酚氧化酶反应中O2结合、O-O键激活和裂解以及底物结合的中间产物。为了实现这一目标，反应将在几个稳定且具有良好特征的步骤中保持平衡，然后将使用一种新的电子转移技术来触发反应。在这种方法中，电子转移（ET）进入或离开活性位点是使用光活化的Ru系固底物类似物实现的。ET通过Ru和活性位点配体之间的共价键快速发生（106 s-1），从而为调节中间体的产生提供了一种途径。

Catechol oxidase, the enzyme that causes browning in plants, catalyzes the oxidation of ortho-diphenols to their quinones with concomitant reduction of O2 to water. Catechol oxidase contains a type 3, dinuclear copper center similar to tyrosinase and hemocyanin. The nature of oxygen activation by metalloenzymes is of great biological interest, but intermediates have been difficult to characterize due to the transient nature of the metal-oxygen species. This study seeks to measure the kinetics and identify intermediates of O2 binding, O-O bond activation and cleavage, and substrate binding in the reaction of catechol oxidase. To accomplish this, the reaction will be poised at several stable and well-characterized steps, and then a novel technique of electron transfer will be will be used to trigger reactions. In this method, electron transfer (ET) either into or out of the active site is achieved using photo-activatible Ru- tethered substrate analogs. ET occurs rapidly (106 s-1) through the covalent link between Ru and the active site ligand, and thus offers a way to regulate the production of intermediates.