Structures & Redox Chemistry in Sulfinic Acid Reduction

描述（由申请人提供）：亚磺酸还原酶，硫氧还蛋白（Srx），最近被鉴定为酵母中负责还原几种氧化失活的过氧化物酶（Prx）中亚磺酸部分（Cys-SO2-）的酶。这一发现打破了Prx酶家族过氧化不可逆的教条。此外，ATP-和Mg 2 +-依赖性修复或“逆还原”的过氧化Prx的可能调节这些酶的作用，作为过氧化氢介导的细胞内信号转导的调节剂。基于对各种人Prx的体内氧化态的分析，Srx对不同Prx同种型的修复似乎以不同的速率进行，尽管它们的整体序列同一性程度高。我们最近对人Srx与磷酸盐或ADP复合物的结构测定揭示了一种新的蛋白质折叠和一种新的核苷酸结合基序。其他初步数据已经证实了酵母Srx行动的拟议计划的某些方面，虽然在我们的研究结果中有几个惊喜（分离的二硫键，而不是硫代亚磺酸酯连接的中间体，和还原的Srx：Prx复合物的谷胱甘肽，而不是硫氧还蛋白）表明，需要进一步的调查。 该提案的目标是：确定与辅因子和人Prx复合的人Srx的晶体结构（目标1）;并进行稳态和部分转换实验，结合定点诱变，以阐明中间体的性质和反应速率，并表征对催化至关重要的残基（目标2和3）。这些调查将大大有助于我们了解亚磺酸还原酶作用的分子起源和参与这一过程的新型硫化学。

DESCRIPTION (provided by applicant): The sulfinic acid reductase, sulfiredoxin (Srx), was recently identified as the enzyme in yeast responsible for the reduction of the sulfinic acid moiety (Cys-SO2-) within several oxidatively-inactivated peroxiredoxins (Prxs). This discovery shattered the dogma of the irreversibility of overoxidation for the Prx enzyme family. Moreover, the ATP- and Mg2+-dependent repair or "retroreduction" of the overoxidized Prxs may modulate the role of these enzymes as regulators of hydrogen peroxide-mediated intracellular signaling. Based on the analysis of the in vivo oxidation state for a variety of human Prxs, repair of different Prx isoforms by Srx appears to proceed at different rates, in spite of their high overall degree of sequence identity. Our recent structure determinations of human Srx in complex with either phosphate or ADP have revealed a new protein fold and a novel nucleotide binding motif. Other preliminary data have confirmed some aspects of the proposed scheme for yeast Srx action, although several surprises in our results (the isolation of a disulfide-bonded rather than thiosulfinate-linked intermediate, and the reduction of the Srx:Prx complex by glutathione rather than thioredoxin) have suggested that further investigation is required. The goals of this proposal are: to determine the crystal structures of human Srx in complex with cofactors and human Prxs (Aim 1); and to carry out steady-state and partial turnover experiments coupled with site-directed mutagenesis to elucidate the nature and reaction rates of intermediates, and to characterize residues critical to catalysis (Aims 2 & 3). These investigations will contribute significantly to our understanding of the molecular origins of sulfinic acid reductase action and the novel sulfur chemistry involved in this process.