A Proteomic View at the Biochemistry of Syntrophic Butyrate Oxidation in <em>Syntrophomonas wolfei</em>

<div><p>In syntrophic conversion of butyrate to methane and CO₂, butyrate is oxidized to acetate by secondary fermenting bacteria such as <i>Syntrophomonas wolfei</i> in close cooperation with methanogenic partner organisms, e.g., <i>Methanospirillum hungatei</i>. This process involves an energetically unfavourable shift of electrons from the level of butyryl-CoA oxidation to the substantially lower redox potential of proton and/or CO₂ reduction, in order to transfer these electrons to the methanogenic partner <i>via</i> hydrogen and/or formate.</p> <p>In the present study, all prominent membrane-bound and soluble proteins expressed in <i>S. wolfei</i> specifically during syntrophic growth with butyrate, in comparison to pure-culture growth with crotonate, were examined by one- and two-dimensional gel electrophoresis, and identified by peptide fingerprinting-mass spectrometry. A membrane-bound, externally oriented, quinone-linked formate dehydrogenase complex was expressed at high level specifically during syntrophic butyrate oxidation, comprising a selenocystein-linked catalytic subunit with a membrane-translocation pathway signal (TAT), a membrane-bound iron-sulfur subunit, and a membrane-bound cytochrome. Soluble hydrogenases were expressed at high levels specifically during growth with crotonate. The results were confirmed by native protein gel electrophoresis, by formate dehydrogenase and hydrogenase-activity staining, and by analysis of formate dehydrogenase and hydrogenase activities in intact cells and cell extracts. Furthermore, constitutive expression of a membrane-bound, internally oriented iron-sulfur oxidoreductase (DUF224) was confirmed, together with expression of soluble electron-transfer flavoproteins (EtfAB) and two previously identified butyryl-CoA dehydrogenases.</p> <p>Our findings allow to depict an electron flow scheme for syntrophic butyrate oxidation in <i>S. wolfei</i>. Electrons derived from butyryl-CoA are transferred through a membrane-bound EtfAB:quinone oxidoreductase (DUF224) to a menaquinone cycle and further <i>via</i> a <i>b</i>-type cytochrome to an externally oriented formate dehydrogenase. Hence, an ATP hydrolysis-driven proton-motive force across the cytoplasmatic membrane would provide the energy input for the electron potential shift necessary for formate formation.</p>