Structure and assembly of the bacterial module for the respiration of nitrous oxide

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The project comprises the investigation of the anaerobic respiratory module of Pseudomonas stutzeri utilizing the greenhouse gas nitrous oxide. Structural insights are being sought for an energy-requiring multimeric complex, including an ATPase which is necessary for the assembly of nitrous oxide reductase. The soluble components, i.e. the cytoplasmic ATPase and a periplasmic metal insertase will be overexpressed, purified, biochemically characterized and subjected to crystallization studies. The project involves binding studies with the ATPase, site-directed mutagenesis of the ATPase and insertase to identify essential residues and gain functional insights. The latter is also sought by establishing a vesicular in vitro system based on isolated ATPase, insertase, and the overexpressed membrane-bound transducer or carrier protein. The project comprises the isolation of nitrous oxide reductase from different sources with the objective of obtaining its structure. Structural investigation of the catalytic site of nitrous oxide reductase by means of mutagenesis and spectroscopic characterization of the mutant proteins will be done. We aim also at obtaining structural information on the respiratory nitrate reductase from a pseudomonad as an example of a respiratory complex that catalyzes proton translocation by a redox loop mechanism.