Inhibitor binding within the NarI subunit (Cytochrome b(nr)) of Escherichia coli nitrate reductase A

Abstract

We have used inhibitors and site-directed mutants to investigate quinol binding to the cytochrome b(nr) (NarI) of Escherichia coli nitrate reductase (NarGHI). Both stigmatellin and 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) inhibit menadiol:nitrate oxidoreductase activity with I50 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 (b(L) and b(H)), but have an inhibitory effect on the extent of nitrate- dependent heme reoxidation. No quinol-dependent heme b(H) reduction is detected in a mutant lacking heme b(L) (NarI-H66Y), whereas a slow but complete heme b(L) reduction is detected in a mutant lacking heme b(H) (NarI- H56R). This is consistent with physiological quinol binding and oxidation occurring at a site (Q(p)) associated with heme b(L) 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 heme b(L)-associated Q(p) site. These results suggest a model for electron transfer through NarGHI that involves quinol binding and oxidation in the vicinity of heme b(L) and electron transfer through heme b(H) to the cytoplasmically localized membrane-extrinsic catalytic NarGH dimer.