Comparative membrane metalloproteomics of respiring anaerobes

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Energy conservation in anaerobically respiring microorganisms highly depends on metal-containing multimeric membrane-bound protein complexes allowing the oxidation of electron donors, the structured conduction of electrons and the reduction of terminal electron acceptors. Although many respiratory complexes have been studied in detail in selected organisms, the overall distribution of metalloproteins, metal-containing cofactors and metal ions in respiring anaerobes is unknown. Still, a significant number of metalloprotein complexes encoded in the genomes of anaerobes and predicted to be located in the membrane have no affiliated function. The Mo-containing FeS cluster proteins and the 11-subunit complex I homologues, both abundantly encoded in prokaryotic genomes, are among them. We propose to study the occurrence and distribution of membrane-bound metalloprotein complexes in diverse prokaryotes by ICP-MS (inductively coupled plasma mass spectrometry) and LC-MS/MS (liquid chromatography mass spectrometry) to quantify metals and protein subunits, respectively. The study will consist of three work packages: First, the comparative analysis of metals and proteins in membrane preparations of whole cells, i.e. shotgun membrane metalloproteomics; second, the comparative analysis of metals and proteins in metalloprotein complexes after separation of the complexes by native gel electrophoresis; and third, the mass spectrometric analysis of metal-containing organic cofactors associated with metalloproteins. We will focus on protein complexes containing the metals Fe, Ni, Co and Mo and the metal-containing cofactors F430, cobalamin and Mo-pterins, all involved in key reactions of anaerobic energy-conserving processes.The project is connected to a series of other projects proposed within the SPP, both with regard to their topic and methodologically. By investigating bacteria, in which specific metalloproteins are investigated by other groups of the SPP, we can integrate results of these groups into our work to assemble an overall picture of metalloproteomics and at the same time provide supplementary information to these groups. Other cooperative topics are Mo-cofactor and cofactor F430 analysis as well as the detection of heterologously overexpressed and assembled metalloproteins in E. coli. Finally, methodological cooperation in metal mass spectrometry, protein mass spectrometry and secondary ion mass spectrometry (nanoSIMS, ToF-SIMS) is projected.