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

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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.