STRUCTURAL MODELS FOR MOLYBDOENZYMES

钼是所有生命形式必需的微量元素，已知有50多种钼酶催化氧化还原反应，这些反应对碳、氮和硫的代谢至关重要。在人体中，钼酶在亚硫酸盐氧化成硫酸盐（亚硫酸盐氧化酶）和嘌呤代谢的某些方面（黄嘌呤氧化酶，黄嘌呤脱氢酶）中起着生理上至关重要的作用。在儿童中，由于先天缺乏“钼辅助因子”，这些酶的活性同时存在致命的缺陷。亚硫酸盐氧化酶蛋白本身的点缺陷也可产生亚硫酸盐氧化酶缺乏症的严重神经症状。这些症状包括晶状体脱位、智力迟钝，严重者还会过早死亡。克隆和表达人亚硫酸盐氧化酶的方法的发展揭示了几种不同的临床点突变，导致分离的亚硫酸盐氧化酶缺乏。高度同源的鸡肝亚硫酸盐氧化酶的x射线结构为解释人类酶的致死性点突变提供了分子基础。本文通过生物物理、生化和模型化合物的综合研究，探讨了亚硫酸盐氧化酶和其他钼酸酶的基本性质。重点是利用变频脉冲电子顺磁共振（EPR）技术，特别是电子自旋回波包络调制（ESEEM）和脉冲电子核双共振（ENDOR）光谱，详细探测野生型和突变型酶中钼活性位点的环境。在富含17-O的水中，对催化过程中钼中心氧原子的迁移进行脉冲EPR研究。利用蛋白质薄膜伏安法研究了钼中心的电子转移反应，并利用闪光光解和理论建模研究了控制原生和突变亚硫酸盐氧化酶中钼和铁中心之间分子内电子转移速率的因素。

Molybdenum is an essential trace element for all forms of life, and over 50 molybdoenzymes are known that catalyze oxidation-reduction reactions that are essential in the metabolism of carbon, nitrogen and sulfur. In humans molybdoenzymes play physiologically vital roles in the oxidation of sulfite to sulfate (sulfite oxidase) and in certain aspects of purine metabolism (xanthine oxidase, xanthine dehydrogenase). Fatal simultaneous deficiencies in the activities of these enzymes due to an inborn deficiency of a "molybdenum cofactor" have been well documented in children. Point defects in the sulfite oxidase protein itself can also produce the severe neurological symptoms of sulfite oxidase deficiency. These symptoms include dislocated ocular lenses, mental retardation, and, in severe cases, early death. Development of methods to clone and express human sulfite oxidase has revealed several different clinical point mutations that result in isolated sulfite oxidase deficiency. The X-ray structure of the highly homologous chicken liver sulfite oxidase provides a molecular basis for interpreting the fatal point mutations of the human enzyme. The research proposed here addresses the fundamental properties of sulfite oxidase and other molybdoenzymes by an integrated program of biophysical, biochemical and model compound studies. Emphasis will be given to the use of variable frequency pulsed electron paramagnetic resonance (EPR) techniques, especially electron spin echo envelope modulation (ESEEM) and pulsed electron-nuclear double resonance (ENDOR) spectroscopies, to probe in detail the surroundings of the molybdenum active site in wild-type and mutant enzymes. The trafficking of oxygen atoms at the molybdenum center during catalysis will be followed by pulsed EPR studies in water enriched in 17-O. The electron transfer reactions at the molybdenum center will be investigated by protein film voltammetry, and the factors that control the rates of intramolecular electron transfer between the molybdenum and iron center in native and mutant sulfite oxidase will be investigated by flash photolysis and theoretical modeling.