COPPER-OXYGEN INTERMEDIATES IN CATECHOL OXIDASE

邻苯二酚氧化酶是植物中引起布朗宁的酶，它催化邻二酚氧化成醌，同时催化O2还原成水。 儿茶酚氧化酶含有3型双核铜中心，类似于酪氨酸酶和血蓝蛋白。 金属酶对氧的活化是生物学上的一个重要研究课题，但由于金属-氧物种的瞬时性，中间体的性质一直难以表征。 本研究旨在测量邻苯二酚氧化酶的反应动力学，并确定O2结合，O-O键活化和裂解，底物结合的中间体。 为了实现这一点，反应将在几个稳定和良好的表征步骤中保持平衡，然后将使用一种新的电子转移技术来触发反应。 在该方法中，使用可光活化的Ru系留的底物类似物实现进入或离开活性位点的电子转移（ET）。 ET通过Ru和活性中心配体之间的共价键快速发生（106 s-1），从而提供了一种调节中间体产生的方法。

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.