Investigation of the Environment Surrounding Iron−Sulfur Cluster 4 of Escherichia coli Dimethylsulfoxide Reductase

Iron−sulfur ([Fe-S]) clusters are common in electron transfer proteins, and their midpoint potentials (E m values) play a major role in defining the rate at which electrons are shuttled. The E m values of [Fe-S] clusters are largely dependent on the protein environment as well as solvent accessibility. The electron transfer subunit (DmsB) of Escherichia coli dimethylsulfoxide reductase contains four [4Fe-4S] clusters (FS1−FS4) with E m values between −50 and −330 mV. We have constructed an in silico model of DmsB and addressed the roles of a group of residues surrounding FS4 in electron transfer, menaquinol (MQH2) binding, and protein control of its E m. Residues Pro80, Ser81, Cys102, and Tyr104 of DmsB are located at the DmsB−DmsC interface and are critical for the binding of the MQH2 inhibitor analogue 2-n-heptyl-4-hydroxyquinoline N-oxide (HOQNO) and the transfer of electrons from MQH2 to FS4. Because the EPR spectrum of FS4 is complicated by spectral overlap and spin−spin interactions with the other [4Fe-4S] clusters of DmsB, we evaluated mutant effects on FS4 in double mutants (with a DmsB-C102S mutation) in which FS4 is assembled as a [3Fe-4S] cluster (FS4[3Fe-4S]). The DmsB-C102S/Y104D and DmsB-C102S/Y104E mutants dramatically lower the E m of FS4[3Fe-4S] from 275 to 150 mV and from 275 to 145 mV, respectively. Mutations of positively charged residues around FS4[3Fe-4S] lower its E m, but mutations of negatively charged residues have negligible effects. The E m of FS4[3Fe-4S] in the DmsB-C102S mutant is insensitive to HOQNO as well as to changes in pH from 5 to 7. The FS4[3Fe-4S] E m of the DmsB-C102S/Y104D mutant increases in the presence of HOQNO and decreasing pH. Analyses of the mutants suggest that the maximum achievable E m for FS4[3Fe-4S] of DmsB is approximately 275 mV.