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Organohalide respiration (OHR) is a bacterial energy metabolism making use of halogenated compounds – like tetrachloroethene (PCE), a major pollutant – as terminal electron acceptors. Three models of electron transport chain exist for OHR [1], however many redox partners are not yet identified. While the catalytic enzyme is PceA (PCE reductive dehalogenase), the pceABCT gene cluster in OHR Firmicutes codes for PceC predicted to be a regulatory protein [2-3]. PceC belongs to a family of membrane-bound flavoproteins (RdhC), with a motif for a covalently bound flavin mononucleotide (FMN). Our working hypothesis is that PceC represents the missing electron transferring protein in the membrane of OHR Firmicutes between menaquinones and the terminal PceA enzyme. The predicted topology of PceC shows 6 transmembrane helices with the FMN-binding domain facing the outside of the cytoplasmic membrane. This was successfully validated by proteomic analysis of the cell surface. This orientation would be necessary if electrons are to be transferred to PceA via the FMN cofactor of PceC. So far, the production of the FMN-binding domain of PceC resulted in inclusion bodies lacking FMN. In vitro reconstitution and cofactor assembly of the PceC domain was only possible with help of a flavin transferase [4], allowing us to produce the FMN-binding domain in a soluble form. Mass spectrometry analysis confirmed the presence of covalently bound FMN to the conserved threonine of RdhC proteins. Redox properties of PceC and evolutionary aspects of the RdhC family will be also addressed. References [1] Mayer-Blackwell et al. (2016). In Organohalide-respiring bacteria, Springer. [2] Smidt et al. (2000). J Bacteriol 182:5683. [3] Maillard et al. (2005). Environ Microbiol 7:107. [4] Deka et al. (2015). MicrobiologyOpen 5:21.
Christof Holliger, Henning Paul-Julius Stahlberg, Julien Maillard, Dongchun Ni, Lorenzo Cimmino
Ardemis Anoush Boghossian, Melania Reggente, Mohammed Mouhib