Inhibitor Binding within the NarI Subunit (Cytochromeb nr) of Escherichia coli Nitrate Reductase A

Magalon, Axel; Rothery, Richard A.; Lemesle-Meunier, Danielle; Frixon, Chantal; Weiner, Joël H.; Blasco, Francis

doi:10.1074/jbc.273.18.10851

Cited by 32 publications

(93 citation statements)

References 37 publications

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“…It has been shown that the single point mutation of His-66 to tyrosine (H66Y) prevents the insertion of the heme b D as well as the binding of quinol analogs and inhibitors. Consequently, no quinol-dependent heme reduction is detected (7,9). However, in the NarGHI-PCP complex structure we present here the 13.2-Å edge-toedge distance between the heme b P and the bound PCP could still support electron transfer even in the absence of heme b D (32).…”

Section: Pcp Is a Potentmentioning

confidence: 71%

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

Bertero

Rothery

Boroumand

et al. 2005

Journal of Biological Chemistry

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118

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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...

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Section: Pcp Is a Potentmentioning

confidence: 71%

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

Bertero

Rothery

Boroumand

et al. 2005

Journal of Biological Chemistry

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118

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“…First, it has been previously shown that the EPR spectrum of heme b D is exquisitely sensitive to the binding of the Q-site inhibitors 2-n-heptyl 4-hydroxyquinoline-N-oxide (HOQNO), pentachlorophenol (PCP), and stigma-tellin. 10,41 Herein, we show that binding of HOQNO collapses the heterogeneity in the heme b D EPR signal (Figure 3). Second, the heterogeneity is dependent upon growth conditions (Figure 2A), as are the membrane concentrations of menaquinone and ubiquinone.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2014

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The membrane subunit (NarI) of Escherichia coli nitrate reductase A (NarGHI) contains two btype hemes, both of which are the highly anisotropic low-spin type. Heme b D is distal to NarGH and constitutes part of the quinone binding and oxidation site (Q-site) through the axially coordinating histidine-66 residue and one of the heme b D propionate groups. Bound quinone participates in hydrogen bonds with both the imidazole of His66 and the heme propionate, rendering the EPR spectrum of the heme b D sensitive to Q-site occupancy. As such, we hypothesize that the heterogeneity in the heme b D EPR signal arises from the differential occupancy of the Q-site. In agreement with this, the heterogeneity is dependent upon growth conditions but is still apparent when NarGHI is expressed in a strain lacking cardiolipin. Furthermore, this heterogeneity is sensitive to Q-site variants, NarI-G65A and NarI-K86A, and is collapsible by the binding of inhibitors. We found that the two main g z components of heme b D exhibit differences in reduction potential and pH dependence, which we posit is due to differential Q-site occupancy. Specifically, in a quinone-bound state, heme b D exhibits an E m,8 of −35 mV and a pH dependence of −40 mV pH −1 . In the quinone-free state, however, heme b D titrates with an E m,8 of +25 mV and a pH dependence of −59 mV pH −1 . We hypothesize that quinone binding modulates the electrochemical properties of heme b D as well as its EPR properties.Nitrate reductase A (NarGHI) from Escherichia coli is a membrane-bound quinol:nitrate oxidoreductase that is expressed under anaerobic conditions in the presence of nitrate. 1 It The authors declare no competing financial interest. Supporting InformationResults of the cytochrome bd and bo 3 deletions on NarGHI EPR heme spectra as well as anaerobic growth curves for NarGHI, NarGHI-K86A, NarGHI-G65A, and the background strain, LCB79. This material is available free of charge via the Internet at http:// pubs.acs.org. CIHR Author Manuscript CIHR Author Manuscript CIHR Author Manuscriptfunctions as a terminal reductase, coupling quinol oxidation to nitrate reduction, and contributes to the generation of a proton electrochemical potential across the cytoplasmic membrane. 2 NarGHI comprises a catalytic subunit (NarG, 140 kDa), an electron-transfer subunit (NarH, 58 kDa), and a membrane anchor subunit (NarI, 26 kDa). NarG contains a molybdo-bis(pyranopterin guanine dinucleotide) (Mo-bisPGD) cofactor that is the site of nitrate reduction as well as a single tetranuclear iron-sulfur ([4Fe-4S]) cluster known as FS0. NarH contains three [4Fe-4S] clusters (FS1-FS3) and one trinuclear iron-sulfur cluster ([3Fe-4S], FS4). NarI anchors the NarGH subunits to the inside of the cytoplasmic membrane and contains two hemes b that are proximal (b P ) and distal (b D ) to the NarGH subunits, respectively. Overall, these subunits provide a molecular scaffold for an electrontransfer relay connecting the site of quinol oxidation adjacent to heme b D in NarI (the Qsite) with the...

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“…Instead, a peak at g ϭ 2.92 is clearly resolved. This peak has been well characterized in NarI-enriched membranes and attributed to the b P heme in a relaxed NarI conformation occurring upon the absence of the NarGH dimer (16,17,28). The quantitation of the EPR signals associated to the b D heme shows that the proportion of this center is significantly higher than those of Moco and FS0 in ⌬1-41NarGHI (Table 2).…”

Section: Uncoupling Redox Cofactor Incorporation From the Membrane-anmentioning

confidence: 97%

Biogenesis of a Respiratory Complex Is Orchestrated by a Single Accessory Protein

Lanciano

Vergnes

Grimaldi

et al. 2007

Journal of Biological Chemistry

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The biogenesis of respiratory complexes is a multistep process that requires finely tuned coordination of subunit assembly, metal cofactor insertion, and membrane-anchoring events. The dissimilatory nitrate reductase of the bacterial anaerobic respiratory chain is a membrane-bound heterotrimeric complex nitrate reductase A (NarGHI) carrying no less than eight redox centers. Here, we identified different stable folding assembly intermediates of the nitrate reductase complex and analyzed their redox cofactor contents using electron paramagnetic resonance spectroscopy. Upon the absence of the accessory protein NarJ, a global defect in metal incorporation was revealed. In addition to the molybdenum cofactor, we show that NarJ is required for specific insertion of the proximal iron-sulfur cluster (FS0) within the soluble nitrate reductase (NarGH) catalytic dimer. Further, we establish that NarJ ensures complete maturation of the b-type cytochrome subunit NarI by a proper timing for membrane anchoring of the NarGH complex. Our findings demonstrate that NarJ has a multifunctional role by orchestrating both the maturation and the assembly steps.All biological systems require the biogenesis of functional respiratory or photosynthetic complexes for their viability. In bacteria, bioenergetic electron transfer chains are associated to the inner membrane. Biogenesis of these complexes is an intricate process that requires several steps such as the synthesis of the different subunits, their assembly, the incorporation of various types of metal or organic cofactors, and the anchoring of the complex to the membrane. In the case of exported metalloproteins, the assembly and cofactor incorporation steps need to be accomplished prior to translocation of the inner membrane via the twin arginine translocase apparatus (1-3). Importantly, accessory proteins are often involved in biogenesis of metalloproteins (4 -9). Although it is most likely that all these events occur in a coordinate fashion to yield a final functional multimeric metalloprotein, information about how this coordination is performed is scarce.The well studied and characterized Escherichia coli dissimilatory quinol-nitrate oxidoreductase of the anaerobic respiratory chain, referred to as the nitrate reductase A (NarGHI) 4 (10, 11), can be considered as a suitable model for deciphering the biogenesis pathway of multimeric metalloproteins. NarGHI is a non-exported membrane-bound respiratory complex composed of three subunits that bind eight redox centers: (i) a catalytic subunit (NarG) containing a molybdenum-bis-molybdopterin guanine dinucleotide cofactor (Moco) and a proximal [4Fe-4S] cluster (FS0) (12, 13), (ii) an electron transfer subunit (NarH) carrying one [3Fe-4S] cluster (FS4) and three [4Fe-4S] clusters (FS1 to FS3) (14, 15), and (iii) a quinol-oxidizing membrane-bound subunit (NarI) containing two b-type hemes (b D and b P ) (11,16,17). The NarJ protein encoded by the narGHJI operon plays an essential role in nitrate reductase activity, enabling Moco insertion...

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Inhibitor Binding within the NarI Subunit (Cytochromeb nr) of Escherichia coli Nitrate Reductase A

Cited by 32 publications

References 37 publications

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

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

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

Biogenesis of a Respiratory Complex Is Orchestrated by a Single Accessory Protein

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