Abstract
The crystal structure of Escherichia coli nitrate reductase A (NarGHI) in complex with pentachlorophenol has been determined to 2.0 A of resolution. We have shown that pentachlorophenol is a potent inhibitor of quinol:nitrate oxidoreductase activity and that it also perturbs the EPR spectrum of one of the hemes located in the membrane anchoring subunit (NarI). This new structural information together with site-directed mutagenesis data, biochemical analyses, and molecular modeling provide the first molecular characterization of a quinol binding and oxidation site (Q-site) in NarGHI. A possible proton conduction pathway linked to electron transfer reactions has also been defined, providing fundamental atomic details of ubiquinol oxidation by NarGHI at the bacterial membrane.
Highlights
Escherichia coli, when grown anaerobically in the presence of nitrate, synthesizes the membrane-bound quinol:nitrate oxidoreductase NarGHI1 [1, 2]
NarGHI catalyzes electron transfer from a quinol binding site located in NarI through the redox cofactors aligned as an “electric wire” through the complex to the molybdo-bis(molybdopterin guanine dinucleotide cofactor in NarG, where nitrate is reduced to nitrite
Studies of quinol binding to NarGHI have been expedited by the use of the menaquinone analog HOQNO (Fig. 1d), which has been demonstrated to inhibit enzyme activity by binding to a single site in close proximity to heme bD within NarI [6, 7]
Summary
Escherichia coli, when grown anaerobically in the presence of nitrate, synthesizes the membrane-bound quinol:nitrate oxidoreductase NarGHI1 [1, 2] This enzyme has been the subject of intense biochemical, biophysical, and structural studies. NarGHI catalyzes electron transfer from a quinol binding site located in NarI through the redox cofactors aligned as an “electric wire” through the complex (bD 3 bP 3 FS4 3 FS3 3 FS2 3 FS1 3 FS0) to the molybdo-bis(molybdopterin guanine dinucleotide cofactor in NarG, where nitrate is reduced to nitrite. We have solved the crystal structure at 2.0 Å of resolution of NarGHI in complex with the quinol binding inhibitor pentachlorophenol (PCP), which is structurally related to the physiological quinol substrates of the enzyme This structure shows the existence of a periplasmically oriented Q-site in NarI (termed QD in reference to its close proximity to the heme bD), confirming previous spectrophotometric and kinetic results (6 – 9).
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