Research project Assembly and function of NO-reducing heme-copper oxidases

NORs are divergent members of the super-family of oxygen-reducing, proton pumping heme-copper oxidases (HCuOs), to which the mitochondrial cytochrome c oxidase (mCcO) also belongs. Despite belonging to this family, the NORs function very differently from other HCuOs in not conserving the free energy available from NO reduction by creating a proton gradient over the membrane. The closest relatives to NOR among the HCuOs are the cbb3 oxidases, which have a high oxygen affinity and are often found in pathogenic bacteria. The cbb3 oxidases can also reduce NO in contrast to the mCcO, and NORs can reduce O2. The project aims at elucidating the structure-function relationships in the bacterial NORs and cbb3 oxidases, as well as the assembly process and the specific roles of the metal ions in the active site. We expect to gain insight also into the evolution of the heme-copper oxidase family and the interplay between assembly and functional properties. Our studies involve a broad set of techniques; e.g. site-directed mutagenesis, protein purification optimisations, cryo-EM structural investigations, reconstitution into membrane mimetic systems, and time-resolved optical spectroscopy of single catalytic turnovers.