Accumulation on the Cytoplasmic Membrane of the Precursor to Dimethyl Sulfoxide Reductase in Molybdenum Cofactor-Deficient Mutants of Rhodobacter sphaeroides f. sp. denitrificans

The role of the molybdenum cofactor (Mo cofactor) in the translocationof dimethyl sulfoxide (DMSO) reductase to the periplasmic spacewas studied in vivo by isolating chlorate-resistant mutantsof Rhodobacter sphaeroides f. sp. denitrificans. More than 50%of the chlorate-resistant mutants isolated were defective inthe biosynthesis of the Mo cofactor and all of these mutantsaccumulated the precursor form of the enzyme. About 45% of themutants contained the same level of Mo cofactor as the parentstrain and exhibited normal levels of DMSO reductase and nitratereductase activities when chlorate was absent from the medium,but the activities of these enzymes were depressed when chloratewas present. Much of the accumulated precursor form of the enzymein a Mo cofactor-deficient mutant was bound to the cytoplasmicmembrane and was sensitive to treatment with proteinase K fromthe periplasmic side of the membrane, an indication that theprecursor was exposed on the periplasmic surface of the membrane.The precursor accumulated on the membrane of the parent strainwhen molybdate was removed from the medium or upon additionof tungstate and this precursor was also sensitive to the treatmentwith proteinase K from the periplasmic side. These results suggestthat the Mo cofactor is necessary for proteolytic processingof the precursor to the mature enzyme on the periplasmic sideof the membrane, whereas binding of the precursor to the membraneand translocation across it can occur in the absence of thecofactor. Almost all of the Mo cofactor available for directreconstitution in vitro of nitrate reductase activity from thenit-l mutant of Neurospora crassa was present in the cytoplasmicfractions. (Received December 11, 1991; Accepted March 25, 1992)