A novel pathway of sulfur oxidation: The heterodisulfide reductase-like system

在该项目的第一阶段，我们收集了明确的遗传证据表明，杂二硫化物样酶复合物（sHdr）与特定的脂肪酸结合蛋白（LbpA）一起构成了一种新的金属酶系统，该系统在细菌细胞质中将硫烷硫氧化为亚硫酸盐。虽然第一次深入了解的结构和功能的sHdr复合物获得通过结晶的sHdrA亚基和阐明其电子传导辅基的氧化还原电位，许多问题仍然存在。因此，该项目的第二阶段致力于充分阐明sHdr系统的遗传学，调节和生物化学，包括其辅基的生物物理特性，澄清反应机制和电子流以及相互作用伙伴。我们将讨论以下主要问题：1。 功能活跃的sHdr复合体的确切组成是什么？2. 存在哪些辅基，它们的性质是什么，它们如何协同工作？3. 我们能否描述sHdr-LbpA系统的反应机制？4. sHdr复合物的形成是如何在化学有机营养生物如Hyphomicrobium acidificans的转录水平上调节的？在这些生物体中，sHdr复合体的功能是如何与碳和能量代谢的中心途径交织在一起的？以上问题的答案将通过不同实验方法的组合来收集。将使用蓝色非变性PAGE和免疫检测在模式生物细胞匀浆和/或膜中研究sHdr复合物组成。sHdr复合物将从化能无机自养源生物中纯化。纯复合物的详细的生物化学，生物物理和结构表征将提供进一步的见解。将在存在通过项目433613342获得的holo-LbpA蛋白的情况下研究复合物的活性。遗传研究包括基因失活和基因互补在遗传可及的H。这不仅有助于阐明sHdr基因的转录调控，而且还有助于进一步鉴定sHdr途径的重要组成部分。生长试验表明，H.不同底物上的嗜热链球菌野生型、缺失和互补突变体或其组合对于理解这种广泛分布的α-变形杆菌中碳代谢和无机电子供体氧化的相互作用以及能够进行类似混合代谢模式的许多其他细菌中的相互作用至关重要。

In the first period of the project we collected unambiguous genetic evidence that a heterodisulfide-like enzyme complex (sHdr) in conjunction with a specific lipoate-binding protein (LbpA) constitutes a novel metallo-enzyme system that oxidizes sulfane sulfur to sulfite in the bacterial cytoplasm. Although first insights into the structure and function of the sHdr complex were obtained through crystallization of the sHdrA subunit and elucidation of the redox potentials of its electron-conducting prosthetic groups, many questions remain. The second period of the project is therefore dedicated to full elucidation of the genetics, regulation, and biochemistry of the sHdr system including biophysical properties of its prosthetic groups, clarification of the reaction mechanism and electron flow as well as interaction partners. We will address the following major questions:1. What is the exact composition of the functionally active sHdr complex? 2. Which prosthetic groups are present, what are their properties and how do they work together?3. Can we delineate a reaction mechanism for the sHdr-LbpA system? 4. How is sHdr complex formation regulated on the transcriptional level in chemoorganotrophs like Hyphomicrobium denitrificans? How is sHdr complex function intertwined with central pathways of carbon and energy metabolism in these organisms?Answers to the above questions will be gathered by a combination of different experimental approaches. sHdr complex composition will be studied in model organism cell homogenates and/or membranes using Blue native PAGE and immunodetection. The sHdr complex will be purified from chemolithoautotrophic source organisms. Detailed biochemical, biophysical and structural characterization of the pure complex will provide further insights. The activity of the complex will be studied in the presence of holo-LbpA proteins available through project 433613342. Genetic studies including gene inactivation and complementation in the genetically accessible H. denitrificans will not only contribute to clarify transcriptional regulation of shdr genes but also identify further important components of the sHdr pathway. Growth experiments with H. denitrificans wildtype, deletion and complementation mutants on different substrates or combinations thereof are crucial for understanding the interplay of carbon metabolism and oxidation of inorganic electrons donors in this wide-spread Alphaproteobacterium as well as in the many other bacteria capable of similar mixed metabolic modes.