Consequences of removal of a molybdenum ligand (DmsA-Ser-176) of Escherichia coli dimethyl sulfoxide reductase

We have used site-directed mutagenesis and EPR spectroscopy to examine the consequences of altering the molybdenum ligand in Escherichia coli dimethyl sulfoxide (Me(2)SO) reductase (DmsABC), Mutagenesis of DmsA-Ser-176 to Ala, Cys, or His abolishes both respiratory growth on Me,SO and in vitro benzyl viologen: Me,SO oxidoreductase activity. EPR spectroscopy reveals changes in the line shape and the g(av) of the Mo(V) signals of the S176A and S176C enzymes. The midpoint potentials (E(m,7)) of the Mo(VI)/Mo(V) and Mo(V)/Mo(IV) couples in DmsABC are -15 and -175 mV. The E(m,7) the Mo(V)/Mo(IV) couple in the S176A mutant is 35 mV; however, the Mo(V) species could not be further oxidized with ferricyanide. Titration of the S176C mutant produced several overlapping Mo(V) species occurring at E(h) > -150 mV, suggesting heterogeneity in the molybdenum environment. A Mo(V) spectrum was not visible in S176H membranes poised between -435 to 350 mV or oxidized with 200 mu M ferricyanide. No differences were detected in the EPR spectra of the reduced [4Fe-4S] clusters of DmsABC and the S176A and S176H mutant enzymes; however, the S176C mutation altered the EPR line shape of one of the reduced [4Fe-4S] clusters.