STRUCTURAL MODELS FOR MOLYBDOENZYMES

钼是所有生命形式必需的微量元素，已知有30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）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”，这两种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，在严重的情况下，还会导致大脑发育迟缓和过早死亡。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。本文提出的研究是通过生物化学、生物物理和模型化合物研究的综合方案来解决亚硫酸盐氧化酶的基本性质。重点介绍了利用电子自旋回波包络调制（ESEEM）光谱探测钼环境；发展脉冲电子-电子双共振（ELDOR）光谱来探测钼-铁相互作用；并研究了控制原生亚硫酸盐氧化酶蛋白和突变亚硫酸盐氧化酶蛋白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 30 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). Fatal simultaneous deficiencies in the activities of both 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, attenuated growth of the brain and 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 by an integrated program of biochemical, biophysical and model compound studies. Emphasis will be given to the use of electron spin echo envelope modulation (ESEEM) spectroscopy to probe the molybdenum environment; to developing pulsed electron-electron double resonance (ELDOR) spectroscopy to probe the molybdenum-iron interaction; and to studying the factors that control the rates of intramolecular electron transfer between the molybdenum and iron centers in native and mutant sulfite oxidase proteins.