INHIBITOR BINDING WITHIN THE NARI SUBUNIT (CYTOCHROME B(NR)) ESCHERICHIA-COLI NITRATE REDUCTASE-A

We have used inhibitors and site-directed mutants to investigate quino l binding to the cytochrome b(nr) (NarI) of Escherichia coli nitrate r eductase (NarGHI). Both stigmatellin and 2-n-heptyl-4-hydroxyquinoline -N-oxide (HOQNO) inhibit menadiol:nitrate oxidoreductase activity with I-50 values of 0.25 and 6 mu M, respectively, and prevent the generat ion 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 inh ibitory 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 consis tent with physiological quinol binding and oxidation occurring at a si te (Q(P)) associated with heme b(L) which is located toward the peripl asmic side of NarI. Optical and EPR spectroscopies performed in the pr esence of stigmatellin or HOQNO provide further evidence that these in hibitors bind at a heme b(L)-associated Q(P) site. These results sugge st a model for electron transfer through NarGHI that involves quinol b inding and oxidation in the vicinity of heme b(L) and electron transfe r through heme b(H) to the cytoplasmically localized membrane-extrinsi c catalytic NarGH dimer.