Function and assembly of heme-copper oxidases in aerobic respiratory chains

所有细胞都参与了能量转换的过程，在这个过程中，食物被分解，一些能量被收获。然后，这些能量被用来为生化合成、养分运输和细胞移动提供燃料。有一组酶催化能量转移的过程，对这些酶及其作用机制的研究构成了生物能量学领域的很大一部分。对于使用氧气进行呼吸的生物体来说，很大一部分能量转换来自于将氧气转化为水的过程。该反应在需氧物种中由血红素-铜氧化酶家族的一种成员催化。血红素-铜氧化酶家族的成员有一个氧反应位点，它包括一个紧密分布的血红素部分和一个铜离子，它们在与氧的反应中高度合作。这项建议的研究旨在了解血红素-铜氧化酶家族将氧气转化为水的分子机制，以及从这一过程中获取能量的过程。我们使用光谱和动力学方法以及重组DNA方法来制造突变酶，以揭开这些机制的物理细节到原子细节。这项提议的第二个方面是了解这些催化剂是如何组合在一起的，这些催化剂本质上是分子机器。血红素-铜氧化酶的组装过程本质上是复杂的，因为它涉及几个蛋白质亚基的组装和四个活性金属中心的同时插入。对这一过程的监管对其成功至关重要。我们特别感兴趣的是铜离子在血红素-铜氧化酶络合物中的传递和结合。这个过程涉及一种特定的铜结合蛋白，它可以陪伴金属离子进入其在血红素-铜酶中的作用部位。我们的研究将使我们深入了解能量转移的功能方面，以及这些系统是如何组装的。我们方案的一个中心方面是培训高素质的人员，使他们能够应用生物物理方法来理解生化机制，并将他们的发现放在生物系统的背景下。

All cells engage in a process of energy conversion in which food is broken down and some energy is harvested. This energy is then used to fuel biochemical synthesis, nutrient transport and cellular mobility. There are a set of enzymes that act to catalyze the processes of energy transfer and the study of these enzymes and their mechanisms of action make up a large part of the field of bioenergetics. For organisms that use oxygen for respiration a large fraction of energy conversion derives from the process of the conversion of oxygen to water. This reaction is catalyzed in aerobic species by a member of the heme-copper oxidase family of enzymes. Members of the heme-copper oxidase family are distinguished by an oxygen reaction site that includes a heme moiety and a copper ion that are closely spaced and highly cooperative n their reaction with oxygen. The research in this proposal is aimed at understanding the molecular mechanism used by the heme-copper oxidase family of enzyme to convert oxygen to water, and the harvesting of energy from this process. We use a combination of spectroscopic and kinetic approaches along with recombinant DNA methods to make mutant enzymes to unravel the physical details of these mechanisms to atomic detail. A second aspect of this proposal is to understand how these catalysts, which are essentially molecular machines, are put together. The process of assembly for heme-copper oxidases is intrinsically complex as it involves the assembly of several protein subunits and the simultaneous insertion of four reactive metal centers. The regulation of this process is important for its success. We are interested particularly in the delivery and incorporation of copper ions into the heme-copper oxidase complex. This process involves a specific copper binding protein that acts to chaperone the metal ion into its site of action in the heme-copper enzyme. Our studies will give insight into the functional aspects of energy transfer and how these systems are assembled. A central aspect of our progam is training highly qualified personnel in the application of biophysical approaches to understaniding biochemical mechanisms and to place their findings into the context of a biological system.