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Spatiotemporal assembly and function of the modular formate hydrogenlyase complex
Finanzierung:
Deutsche Forschungsgemeinschaft (DFG) ;
Escherichia coli is a facultative anaerobic bacterium that is able to produce hydrogen through the oxidation of formate when grown on glucose and in the absence of external electron acceptors like oxygen. This reaction is catalysed by the membrane-associated formate hydrogenlyase (FHL) complex, which contains seven subunits. One of the two catalytic subunits within the cytoplasmic domain is formate dehydrogenase H (formate DH). The protein harbours a molybdenum cofactor where formate is oxidised to CO2 and electrons. The second catalytic subunit is a [NiFe]-hydrogenase that uses these electrons to reduce protons and thus generate hydrogen. The electrons are transferred between both subunits by three iron sulfur cluster proteins. The cytoplasmic domain of the FHL complex is attached to the inside of the membrane by two further integral membrane subunits.
Previous work has established that a pool of accessory proteins (called Hyp proteins) are needed for the synthesis and insertion of the NiFe(CN)2CO cofactor during maturation of the FHL complex. It is possible to purify the whole complex or individual sub-complexes, but the order of events during assembly is not understood. For example, the function of the additional accessory protein HycH remains unknown. HycH interacts with the large hydrogenase subunit HycE during assembly of the complex, but it is not a structural subunit of the FHL complex. Also, the interactions of the formate DH with the hydrogenase and the iron sulfur cluster proteins are still uncharacterized.
Understanding FHL assembly will be of great value for the biotechnological exploitation of its hydrogen-producing capability. For this reason we plan to characterise the interaction of HycH with HycE. We also want to characterize the interplay between the other subunits. Therefore, we plan to make structural investigations e.g. crystallisation and interactions studies and will determine the influence on activity and stability of the FHL complex.
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