COPPER-OXYGEN INTERMEDIATES IN CATECHOL OXIDASE

邻苯二酚氧化酶是导致植物褐变的一种酶，它催化邻苯二酚氧化为其对苯二酚，同时将O2还原为水。儿茶酚氧化酶含有一个类似酪氨酸酶和血蓝蛋白的三型双核铜中心。金属酶激活氧的性质具有很大的生物学意义，但由于金属-氧物种的瞬变性质，中间体一直很难表征。本研究旨在测定和鉴定邻苯二酚氧化酶反应中O2结合、O-O键活化和断裂以及底物结合的中间产物。为了实现这一点，反应将稳定在几个稳定的和充分表征的步骤，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，进出活性中心的电子转移(ET)是使用可光激活的Ru系留底物类似物来实现的。ET通过Ru和活性中心配体之间的共价键迅速发生(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.