Metal Selectivity of Manganese Superoxide Dismutase

描述(由申请人提供)： 这项建议的总体目标是了解真核细胞中锰超氧化物歧化酶(SOD2)的体内金属离子选择性。Sod2是线粒体的一种关键抗氧化酶。当锰结合到催化部位时，酶是活性的，但在体内与其他金属(如铁)的相互作用尚未被探索。我们之前已经证明，在mtm1Delta突变菌株中，积累高线粒体铁的酵母中SOD2活性受到损害。我们现在将测试铁是否可以在体内抑制SOD2的活性，以及Mtm1p是否有助于对正确金属的选择性。我们将(目标1)确定铁是否能占据SOD2的金属结合部位并在体内灭活该酶。SOD2的活性和纯化到均一的酶的金属含量将作为细胞内锰和铁有效性的函数进行评估。其次(目标2)，我们将测试mtm1突变体的高线粒体铁是否通过遗传和营养控制细胞内铁水平而导致SOD2活性的丧失。在目标3中，将利用转录全基因组微阵列分析来确定Mtmlp丢失后复杂的细胞反应以及导致SOD2失活的途径。总之，这些研究旨在促进我们对真核细胞SOD2如何在体内获得其锰辅助因子的理解。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to understand the in vivo metal ion selectivity for eukaryotic manganese superoxide dismutase (SOD2). SOD2 is a critical anti-oxidant enzyme of the mitochondria. The enzyme is active when manganese is bound to the catalytic site, but in vivo interactions with other metals such as iron have not been explored. We have previously shown that SOD2 activity is impaired in mtm1delta mutant strains of yeast that accumulate high mitochondrial iron. We will now test whether iron can inhibit SOD2 activity in vivo and whether Mtm1p facilitates selectivity for the correct metal. We shall (Aim 1) determine whether iron can occupy the metal binding site of SOD2 and inactivate the enzyme in vivo. The activity of SOD2 and metal content of the enzyme purified to homogeneity will be assessed as a function of intracellular manganese and iron availability. Secondly (Aim 2), we shall test whether the high mitochondrial iron ofmtml mutants is responsible for loss of SOD2 activity by genetically and nutritionally controlling intracellular iron levels. In Aim 3, transcriptional genome-wide microarray analysis will be utilized to determine the complex cellular response to loss of Mtmlp and pathways that lead to inactivation of SOD2. Together, these studies are designed to advance our understanding of how eukaryotic SOD2 obtains its manganese co-factor in vivo.