ENGINEERING A NOVEL IRON-SULFUR CLUSTER INTO THE CATALYTIC SUBUNIT OFESCHERICHIA-COLI DIMETHYLSULFOXIDE REDUCTASE

Dimethyl-sulfoxide reductase (DmsABC) is a complex [Fe-S] molynbdoenzy me that contains four [4Fe-4S] clusters visible by electron paramagnet ic resonance (EPR) spectroscopy. The enzyme contains four ferredoxin-l ike Cys groups in the electron transfer subunit, DmsB, and an addition al group of Cys residues in the catalytic subunits, DmsA. Mutagenesis of the second Cys, Cys-38, in the DmsA group to either Ser or Ala prom otes assembly of a fifth [Fe-S] cluster into the mutant enzyme. The EP R spectra, the temperature dependences, and the microwave power depend ences demonstrates that the new clusters are [3Fe-4S] clusters. The [3 Fe-4S] clusters in both of the C38S and C38A mutant enzymes are relati vely unstable in redox titrations and have midpoint potentials of appr oximately 178 and 140 mV. Mutagenesis of the DmsA Cys group to resembl e a sequence capable of binding an [4Fe-4S] cluster did not change the cluster type but reduced the amount of the cluster present in this mu tant enzyme. This report demonstrates that all four EPR detectable [Fe -S] clusters in the wild-type enzyme are ligated by DmsB. Wild-type Dm sA does not ligate an [Fe-S] cluster that is visible by EPR spectrosco py.