Inhibitor Binding within the NarI Subunit (Cytochromebnr) of Escherichia coli Nitrate Reductase A

We have used inhibitors and site-directed mutants to investigate quinol binding to the cytochromebnr (NarI) of Escherichia colinitrate reductase (NarGHI). Both stigmatellin and 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) inhibit menadiol:nitrate oxidoreductase activity withI50 values of 0.25 and 6 μm, respectively, and prevent the generation of a NarGHI-dependent proton electrochemical potential across the cytoplasmic membrane. These inhibitors have little effect on the rate of reduction of the two hemes of NarI (bLand bH), but have an inhibitory effect on the extent of nitrate-dependent heme reoxidation. No quinol-dependent heme bH reduction is detected in a mutant lacking heme bL(NarI-H66Y), whereas a slow but complete hemebL reduction is detected in a mutant lacking heme bH (NarI-H56R). This is consistent with physiological quinol binding and oxidation occurring at a site (QP) associated with heme bL which is located toward the periplasmic side of NarI. Optical and EPR spectroscopies performed in the presence of stigmatellin or HOQNO provide further evidence that these inhibitors bind at a hemebL-associated QP site. These results suggest a model for electron transfer through NarGHI that involves quinol binding and oxidation in the vicinity of hemebL and electron transfer through hemebH to the cytoplasmically localized membrane-extrinsic catalytic NarGH dimer.