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J Biol Chem, Vol. 273, Issue 18, 10851-10856, May 1, 1998

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

Axel Magalon, Richard A. Rothery^¶, Danielle Lemesle-Meunier^**, Chantal Frixon, Joel H. Weiner^¶, and Francis Blasco

From the  Laboratoire de Chimie Bactérienne, IBSM, CNRS, 31 chemin Joseph Aiguier, 13402 Marseille cedex 20, France, the ^** Laboratoire de Bioénergétique et Ingénierie des Protéines, IBSM, CNRS, 31 chemin Joseph Aiguier, 13402 Marseille, France, and the ^¶ Medical Research Council Group in the Molecular Biology of Membranes, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7 Canada.

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.

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