Photoaffinity labeling of heme proteins with chemically modified prosthetic groups

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For the analysis of heme and molybdenum cofactor insertion into apoproteins novel chemistry-based tools are essential. Of particular interest will be diazirine-functionalized heme analogs that are to be analyzed in reconstitution experiments with apoproteins, in close collaboration with our partners within the PROTRAIN research group. Key challenge is the incorporation of diazirine moieties into cofactors or proteinogenic amino acids with minimal structural alteration. Ultimately, it is intended to interrupt the reconstitution by photolysis of the diazirine moieties liberating reactive carbenes. The apoproteins will be functionalized along the insertion channel and analyzed by LTQ-Orbitrap mass spectrometry. Heme derivatives will also be applied in the search for heme binding proteins employing novel fluorescent and photoactivatable linkers. Fluorescent trifunctional linkers will be investigated as tools for the identification of protein-protein interactions via label transfer. To be able to assess the cofactor load of redox enzymes containing the molybdenum cofactor (Moco) the total synthesis of the Moco degradation products Form A and B is addressed, which feature challenging pteridin-derived architectures.