Interaction of an Engineered 3Fe-4S Cluster with a Menaquinol Binding Site of Escherichia coli DMSO Reductase

We have characterized by EPR the interaction of the Em,7 = -50 mV 4Fe-4S cluster of Escherichia coli DMSO reductase (DmsABC) with a menaquinol (MQH2) binding site. Potentiometric titrations indicate that in DmsAB(C102S)C, the Em,7 = -50 mV 4Fe-4S cluster is replaced by an Em,7 = +260 mV 3Fe-4S cluster. The Q-pool coupling assay in combination with the MQH2 analog HOQNO (2-n-heptyl-4-hydroxyquinoline-N-oxide) was used to examine the effect of the DmsB(Cl02S) mutation on physiological electron transfer through DmsABC. Forward electron transfer through the mutant (MQH2 to DmsA) is blocked in the Q-pool coupling assay, but reverse electron transfer (DmsA to MQ) is not. HOQNO elicits a significant change in the EPR line shape of the oxidized DmsAB(Cl02S)C 3Fe-4S cluster but has no effect on the line shape of the reduced 4Fe-4S clusters. We have identified a residue in DmsC involved in MQH2 oxidation. DmsC(H65), and in a double mutant, DmsAB(C102S)C(H65R), the DmsC mutation blocks the HOQNO effect on the 3Fe-4S EPR line shape, suggesting, that the DmsC(H65R) mutation either blocks HOQNO binding or blocks a conformational link between a HOQNO binding site and the DmsB(C102S) 3Fe-4S cluster. These results suggest that the MQH2 binding site of DmsC is conformationally and functionally linked to the Em,7 = -50 mV 4Fe-4S cluster of DmsB.