Structural and Biochemical Characterization of a Quinol Binding Site of Escherichia coli Nitrate Reductase A

Abstract

The crystal structure of Escherichia coli nitrate reductase A (NarGHI) in complex with pentachlorophenol has been determined to 2.0 Å 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.Escherichia coli, when grown anaerobically in the presence of nitrate, synthesizes the membrane-bound quinol:nitrate oxidoreductase NarGHI 1 (1, 2). This enzyme has been the subject of intense biochemical, biophysical, and structural studies. The protein complex contains three subunits with characteristic redox prosthetic groups: NarG (140 kDa), the catalytic subunit with a molybdo-bis(molybdopterin guanine dinucleotide) cofactor and an [Fe-S] cluster (FS0); NarH (58 kDa), the electron transfer subunit with four [Fe-S] clusters (FS1, FS2, FS3, and FS4); NarI (26 kDa), the integral membrane subunit with two b-type hemes, termed b P and b D to indicate their proximal (b P ) and distal (b D ) positions to the catalytic site. NarG and NarH form a soluble cytoplasmically localized catalytic domain anchored to the membrane by NarI. NarGHI catalyzes electron transfer from a quinol binding site located in NarI through the redox cofactors aligned as an "electric wire" through the complex (b D 3 b P 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.NarGHI often forms a respiratory chain with the formate dehydrogenase FdnGHI via the lipid soluble quinol pool. Electron transfer from formate to nitrate is coupled to proton translocation across the cytoplasmic membrane generating proton motive force by a redox loop mechanism (3). In the redox loop mechanism proton translocation is the net result of the topographically segregated reduction of quinone and oxidation of quinol on opposite sites of the membrane. The high resolution structures of both respiratory complexes, FdnGHI and NarGHI, have been recently solved (1, 4). Crystallographic analysis of FdnGHI has shown the presence of a quinone reduction site oriented toward the cytoplasm (4). Existing biochemical and biophysical evidence indicates that the quinol binding and oxidation functionality of NarGHI is provided by the NarI subunit (5-7), but no high resolution structural information has been made available to date.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 subs...

Determination of the Proton Environment of High Stability Menasemiquinone Intermediate in Escherichia coli Nitrate Reductase A by Pulsed EPR

“…most likely His-66 N ␦ . The similar 14 N HYSCORE pattern observed for both radicals suggests that the H-bond involved in binding USQ D has similar characteristics to that detected in the present study. This provides further support for its involvement in ubisemiquinone stabilization at the Q D site.…”

“…The non-zero isotropic hyperfine coupling of this nitrogen suggests that the interaction occurs via a hydrogen bond, allowing electron spin density to be transferred from the radical to the interacting nucleus. Interestingly, these experiments did not support a direct H-bond between MSQ D (or USQ D ) and Lys-86, a residue in the Q D site that was previously shown to be essential for quinol oxidation and menasemiquinone detection (11,14). Indeed, no evidence for the transfer of a measurable spin density on any other nuclei than that mentioned above was found.…”

“…This step likely involves Lys-86, a residue that is located at the protein surface, at the entrance of the Q D cavity, and that is required for correct binding of quinol analogues and for semiquinone detection at the Q D site of NarGHI (11,13,14). Thus, we speculate that Lys-86 could be a direct hydrogen bond donor to the quinol molecule, facilitating proton abstraction at the quinol O4 oxygen and, concomitantly, the first electron transfer step.…”

“…The use of ESEEM and HYSCORE (hyperfine sublevel correlation) spectroscopies on either the wild-type enzyme or the enzyme uniformly enriched with 15 N nuclei provided direct evidence for nitrogen ligation to MSQ D and USQ D . On the basis of the direct determination of the quadrupolar parameters of the corresponding interacting 14 N by S-band (ϳ3 GHz) HYSCORE experiments, we assigned the latter to an N ␦ imidazole nitrogen and proposed it to arise from the heme b D axial ligand His-66 (13). The non-zero isotropic hyperfine coupling of this nitrogen suggests that the interaction occurs via a hydrogen bond, allowing electron spin density to be transferred from the radical to the interacting nucleus.…”

Q-Site Occupancy Defines Heme Heterogeneity in Escherichia coli Nitrate Reductase A (NarGHI)

“…10 Fermenter growths were conducted in 5 or 10 L batches in either fermenter broth or glycerol-peptone-fumarate (GPF) (anaerobic growth) 21 at pH 7.0 using 2 M NaOH and 1 M HCl for pH adjustments. 11 The GPF was supplemented with 4 mM KNO 3 11 When the OD 600 reached 0.5 (Anaerobic, GPF culture) or 2.0 (Semianaerobic, FB cultures), 1 mM isopropyl-1-thio-β-Dgalactopyranoside (IPTG) was added to induce NarGHI expression from pVA700. 11 Stir speed, postinduction incubation temperature, and aeration rates are detailed in the legend of Figure 2.…”