Interaction of 2-n-heptyl-4-hydroxyquinoline-N-oxide with dimethyl sulfoxide reductase of Escherichia coli.

We have studied the interaction of the menaquinol analog 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) with dimethyl sulfoxide reductase (DmsABC) and the effect of a mutation in the DmsC subunit (DmsABCH65R) using fluorescence titration and stopped-flow methods. The titration data show that the HOQNO fluorescence is quenched when HOQNO binds to DmsABC. The binding stoichiometry is determined to be about 1:1. The mutant DmsABCH65R blocks HOQNO binding to the protein. It is therefore proposed that there is one high-affinity HOQNO binding site per DmsABC molecule located in the DmsC subunit. Stopped-flow kinetic studies show that the interaction can be described by a two-step equilibrium model, a fast bimolecular step followed by a slow unimolecular step. The quenching of HOQNO fluorescence occurs in the bimolecular step. The rates for the forward and reverse reaction of the first equilibrium are determined to be k1 = (3.9 +/- 0.3) x 10(5) M-1 s-1 and k2 = 0. 10 +/- 0.01 s-1, respectively. The dissociation constant for the first equilibrium, Kd1 = k2/k1, is calculated to be about 260 nM. The upper limit of the overall dissociation constant is estimated to be 6 nM.