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.
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ABSTRACT |
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We have used inhibitors and site-directed mutants to investigate quinol binding to the cytochrome bnr (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 (bL and bH), but have an inhibitory effect on the extent of nitrate-dependent heme reoxidation. No quinol-dependent heme bH reduction is detected in a mutant lacking heme bL (NarI-H66Y), whereas a slow but complete heme bL reduction is detected in a mutant lacking heme bH (NarI-H56R). This is consistent with physiological quinol binding and oxidation occurring at a site (QP) associated with heme bL 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 bL-associated QP site. These results suggest a model for electron transfer through NarGHI that involves quinol binding and oxidation in the vicinity of heme bL and electron transfer through heme bH to the cytoplasmically localized membrane-extrinsic catalytic NarGH dimer.
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INTRODUCTION |
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Nitrate reductase A (NarGHI)1 allows Escherichia coli to use nitrate as a terminal electron acceptor during anaerobic growth. This respiratory complex catalyzes quinol oxidation and proton release at the periplasmic side of the membrane and transfers electrons through various redox centers to the catalytic site where nitrate reduction and consumption of protons occurs (1). This leads to the generation of a proton electrochemical gradient across the cytoplasmic membrane (1). NarGHI comprises a catalytic subunit (NarG) containing a non-covalently bound molybdenum cofactor (2), an electron transfer subunit (NarH) containing multiple [Fe-S] clusters (3, 4), and a membrane anchor subunit (NarI) that is believed to be the location of quinol binding and oxidation (5). NarI (cytochrome bnr) is a diheme b-type cytochrome (6, 7), the polypeptide of which is predicted to traverse the membrane bilayer five times (6). Such a transmembrane topology is supported by recent studies of site-directed mutants of NarI that identified the histidine axial ligands of the two b-type hemes on helices II (His56 and His66) and V (His187 and His205) (7). In NarI, the low potential heme (heme bL, Em,7 = +10 mV) appears to be near the periplasmic surface of the membrane, whereas the high potential heme (heme bH, Em,7 = +120 mV) appears to be near the cytoplasmic surface. These positions relative to the membrane surfaces were predicted from the disposition of the coordinating histidines within the transmembrane helices and corroborated by EPR and optical studies of wild-type and site-directed NarI mutants (7). Such topological characteristics enable the NarI protein to interact directly with the membrane-embedded quinols and facilitate the transfer of electrons from the periplasmic to the cytoplasmic site of the membrane.
To better understand the interaction of quinols with NarGHI, and more specifically with NarI, it is necessary to define functional domains and locate binding sites of inhibitors at key positions on the electron transfer pathway from quinol to nitrate. Specific inhibitors of cytochrome b-containing respiratory enzymes have been important tools for delineating the electron transfer mechanism of ubiquinol:cytochrome c oxidoreductase (cytochrome bc1, complex III) (8-10). It is noteworthy that the cytochrome b mutations of the bc1 complex that affect the inhibitor binding sites are all concentrated in four regions delineating the two quinol binding sites Qo and Qi located on the positive (outer) and negative (inner) sides, respectively, of the cytoplasmic membrane in prokaryotes or the inner mitochondrial membrane in eukaryotes (11). The recently determined crystal structure of the bc1 complex from bovine heart mitochondria clearly shows that both antimycin A and myxothiazol-binding sites partly overlap the quinone-binding site at the Qi site and the Qo site, respectively (12). 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) is the most general inhibitor of the quinone-reacting cytochromes b, and its structure is reminiscent of physiological quinones. Such similarity suggests that its binding pocket may overlap quinone-interacting sites in the vicinity of the b-cytochromes. HOQNO has been shown to inhibit the quinol:nitrate oxidoreductase activity from various organisms (13-15), as well as nitrate-dependent proton extrusion into the periplasm (1). HOQNO does not inhibit the benzyl viologen:nitrate oxidoreductase activity (5), suggesting that its binding site is associated with NarI (this subunit is not essential for benzyl viologen:nitrate oxidoreductase activity). Emerging structural data on NarI (7) combined with information obtained from the effects of specific inhibitors would be of importance in delineating the electron transfer and proton release mechanism of NarGHI.
Using optical and EPR spectroscopies of both wild-type and site-directed mutants of NarI, we report herein spectral shifts caused by several inhibitors of quinol oxidation. Our results suggest that physiological quinol oxidation occurs at a stigmatellin/HOQNO binding site (QP) which is located close to the low potential heme (heme bL), near the periplasmic side of the membrane.
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EXPERIMENTAL PROCEDURES |
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Bacterial Strains and Plasmids-- The E. coli strains and plasmids used in this study are listed in Table I. Plasmids pVA700 and pCD7 carry the narGHJI operon and the narI gene, respectively, under the control of the tac promoter (ptac).
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Growth of Bacteria and Preparation of E. coli Membranes
Vesicles--
For studies of proton translocation, enzyme activity,
and heme reduction/oxidation, cells were grown microaerobically at
37 °C as described previously (16). For EPR studies, cells were
grown microaerobically overnight at 30 °C. Cultures were harvested
when the A600 reached 1.0. Membrane vesicles
were prepared by French pressure lysis in 100 mM MOPS
buffer (pH 7.0) containing 5 mM EDTA and
phenylmethanesulfonyl fluoride (0.2 mM) (16). Membranes were frozen in liquid N2 and stored at 70 °C until
use.
Enzyme Assays-- The benzyl viologen:nitrate oxidoreductase activities were assayed by a method modified from Jones and Garland (17, 18). Menadiol:nitrate and duroquinol:nitrate oxidoreductase activities were measured as described by Guigliarelli et al. (16). The I50 values for inhibitors were defined as the concentration required to reduce the quinol-nitrate reductase activity by 50%. These values were deduced from the inhibitor titration curves of enzyme activity.
Quinacrine Fluorescence Quenching-- Assays were performed as described (1) with a Jobin-Yvon spectrofluorometer model JY3D, using N2-saturated buffers. The reaction was initiated with 50 µM nitrate.
Kinetics of Heme Reduction and Reoxidation-- Heme reduction and reoxidation were followed using a DW2A Chance AMINCO spectrophotometer. This instrument had a dual wavelength configuration, and heme reduction and reoxidation were followed using a wavelength of 560 nm with a reference wavelength of 575 nm. Reactions were observed at 25 °C in potassium phosphate buffer (K2HPO4/KH2PO4 50 mM, pH 7.5) with quinol analogs (menadiol or duroquinol) as electron donors (125 µM) and nitrate as electron acceptor (250 µM). Reaction conditions were as indicated in the individual figure legends.
Inhibitor-induced Optical Shift Measurements-- For these experiments, the DW2A spectrophotometer was used in a dual-beam configuration. Difference spectra were recorded of membranes plus inhibitor minus membranes without inhibitor. Inhibitors were added from stock solutions made with ethanol as solvent, and equivalent volumes of this solvent were added to cuvettes that did not have inhibitor added. Membrane vesicles were first reduced by adding a few grains of sodium dithionite, and the base line was recorded. Inhibitors or ethanol were then added to the cuvettes. Experiments were carried out using a 50 mM MOPS buffer at pH 7.0.
EPR Spectroscopy and Redox Titrations-- EPR spectra were recorded using a Bruker ESP300 spectrometer equipped with an Oxford Instrument ESR-900 flowing helium cryostat. Instrument conditions and temperatures were as described in the individual figure legends.
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RESULTS |
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Inhibitor Effects on the Quinol:Nitrate Reductase Activities-- Quinol:nitrate oxidoreductase activities, using menadiol as electron donor, were measured in the presence of various potent inhibitors of electron transfer in the mitochondrial bc1 complex. It appears that of all the compounds tested, the most effective are HOQNO (I50 = 6 µM) and stigmatellin (I50 = 0.25 µM) whose structures are strongly reminiscent of quinones. DCMU and antimycin A are poor inhibitors and have measurable effects only at high concentrations (I50 > 40 µM). Finally, myxothiazol, atrazin, funiculosin, UHDBT and DBMIB appeared to have no effect on the quinol:nitrate oxidoreductase activity. Fig. 1 shows the concentration dependence of the HOQNO and stigmatellin inhibition of menadiol:nitrate reductase activity. The major part of the quinol activity is inhibited in a hyperbolic fashion, the remaining part of the activity constituting about 5 to 20% of the overall activity.
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Effects of Inhibitors on the Nitrate Reductase Mediated Proton
Translocation--
Quinacrine hydrochloride distributes according to
the transmembrane pH and its accumulation within the membrane
vesicles, in response to an inward translocation of protons, gives rise to a quenching of its fluorescence (19, 20). This phenomenon has been
used herein to study the effects of the inhibitors on the nitrate
reductase-dependent proton translocation in membrane preparations. These experiments were performed on membrane vesicles with overexpressed wild-type holoenzyme that possess an inside-out orientation with respect to the original cells.
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Effect of Inhibitors on the Reduction and Reoxidation of the NarI Hemes-- The inhibitory effect of HOQNO, stigmatellin and, to a lesser extent DCMU, on the nitrate reductase activity was further assessed by observing NarI heme reduction by menadiol and subsequent reoxidation by nitrate. This was done to investigate which step is modified by the inhibitors in the overall steady-state electron transfer from quinol to nitrate. The results are shown in Fig. 3A. While quinol-dependent heme reduction is only slightly affected by the inhibitors, the extent of the nitrate-dependent heme reoxidation appears to be significantly decreased by HOQNO and stigmatellin (Fig. 3A), and to a lesser extent by DCMU (data not shown). Adding HOQNO and stigmatellin at the same time to the assays does not lead to further modification of the behavior of the hemes (data not shown), suggesting that both inhibitors act at the same level in the electron transfer pathway. This contrasts with what is observed in the mitochondrial cytochrome bc1 complex in which HOQNO and stigmatellin bind at separate sites on the opposite sides of the mitochondrial inner membrane (10).
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Effect of Inhibitors on the Optical Spectrum of the NarI
Hemes--
It is generally accepted that in the presence of effective
inhibitors, the most affected heme is that closest to the inhibitor binding site. We therefore examined the effect of inhibitors on the
optical spectrum of the NarI hemes. As expected, no shifts are observed
in the heme -bands of reduced NarGHI in the presence of saturating
amounts of myxothiazol, funiculosin, DBMIB, atrazin or UHDBT (data not
shown). With the wild-type strain LCB2048/pVA700, the difference
spectra observed after adding saturating amounts of HOQNO or
stigmatellin to the reduced membrane vesicles are similar in shape
(Fig. 4, traces 1 and
4). These S-shaped curves have maxima and minima at 555.5 nm
and 550.5 nm, respectively. The difference spectrum obtained after
adding antimycin A or DCMU are similar in shape but much lower in
amplitude in comparison to those observed with HOQNO and stigmatellin
(data not shown).
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EPR Spectral Shifts Caused by the Inhibitors in the Wild-type and NarI Mutant Enzymes-- Fig. 5 shows the effects of some of the inhibitors used herein on the EPR lineshapes of oxidized hemes bL and bH in the presence of HOQNO (B), stigmatellin (C), DCMU (D), and antimycin A (E). HOQNO elicits a change in the Gz of heme bL from approximately 3.36 to 3.50, whereas stigmatellin appears to have the opposite effect on this heme, moving its Gz from 3.36 to approximately 3.31. DCMU and antimycin A appear to have no detectable effect on the EPR lineshape of heme bL. None of the inhibitors tested appeared to have any effect on the Gz of heme bH at approximately 3.76.
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DISCUSSION |
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The sequence of NarI is remarkably conserved among the membrane-bound nitrate reductases from various organisms, and is proposed to have five transmembrane helices (I-V) with a periplasmic N terminus and a cytoplasmic C terminus (6, 23). Additional evidence for this model comes from the identification of the histidine ligands to the two hemes of this subunit (7). Both hemes bL and bH are ligated by histidines located on helices II and V (7), and heme bH is located near the cytoplasmic surface and heme bL is located near the periplasmic surface. It has previously been shown that NarGHI releases protons into the periplasm during enzyme turnover (1), raising the question of the exact location within the enzyme where proton release takes place. We have shown herein that the periplasmically localized heme bL is closely associated with a quinol binding site (QP), and this is consistent with a role for this quinol site-heme motif in proton release during enzyme turnover.
We have used inhibitors to study the location of quinol-binding site(s) within NarGHI in much the same way as has been reported for the mitochondrial cytochrome bc1 complex (24-27). In the latter enzyme in those cases where effects can be measured, the heme which is closest to the site of inhibitor binding is generally the most affected. Using a similar approach with NarGHI, we have investigated both the effects of inhibitors on the spectral properties of the hemes of NarI and their effects on heme reduction by menadiol and subsequent reoxidation by nitrate. Based on the lineshape perturbations in both EPR and optical experiments, it is clear that HOQNO and stigmatellin are able to bind at a site (QP) in close proximity to heme bL. However, HOQNO or stigmatellin binding at this location does not greatly affect the reducibility of both hemes by menadiol (see below).
In contrast to the effect of the inhibitors on heme reduction, HOQNO and stigmatellin inhibit the extent of nitrate-dependent heme reoxidation (Fig. 3A). Possible explanations for this phenomenon include the following:
HOQNO and Stigmatellin Bound to the QP Site Affects the Em,7 of Heme bL-- Inhibitor binding in the vicinity of heme bL could significantly raise the Em,7 of the heme, resulting in it becoming non-nitrate oxidizable. HOQNO and stigmatellin have been shown to cause significant shifts in the Em,7s of Q-site associated prosthetic groups in other respiratory chain enzymes. For example, in Bacillus subtilis succinate:menaquinone oxidoreductase (SQR) addition of HOQNO causes a negative shift of the Em,7 of the Q-site associated heme bL (28). In the cytochrome bc1 complex, stigmatellin induces a large positive shift in the Em,7 of the Rieske center ([2Fe-2S] cluster), but has little effect on the Em,7 of the heme bL (25). Experiments are in progress in our laboratories to determine the effects of stigmatellin and HOQNO on the Em,7s of the two hemes of NarI.
A Second Quinol Binding Site (Qnr) Could Exist between Heme bH of NarI and the [Fe-S] Clusters of NarH-- Inhibitor binding at this site would inhibit nitrate-dependent electron flow from NarI. The presence of such a site would be consistent with the effect of HOQNO on the EPR signal of a semiquinone radical species that has been observed in a previous study of mutants of NarH that lack the highest potential [4Fe-4S] cluster of this subunit (29). However, the elimination of the semiquinone radical species by HOQNO could be due to inhibition of electron transfer from the QP site (see preceding paragraph) rather than by displacement of the semiquinone species. In the B. subtilis SQR it has also been suggested that there are two quinol binding sites (30), one associated with heme bL and one located near heme bH and the S3 [3Fe-4S] cluster. Although there is evidence for the presence of a Qnr site in NarGHI (29, 31), we believe that the simplest explanation for the data presented herein is that there is a single dissociable quinol binding site (QP) and that the Qnr site is non-dissociable (29, 31).
A proposed mechanism of HOQNO-inhibition of B. subtilis SQR has been suggested by Smirnova et al. (28) in which HOQNO acts as a semiquinone anion analog that displaces the physiological Q ![]() |
ACKNOWLEDGEMENT |
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We thank Dr. Wolfgang Nitschke for helpful discussions.
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FOOTNOTES |
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* This work was funded by the CNRS.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a fellowship from the Ministère de l'Education Supérieure et de la Recherche.
Supported by a NATO Collaborative Research Grant (awarded to
J. H. W.).
Supported by a travel grant from the Alberta Heritage
Foundation for Medical Research and to whom correspondence should be addressed. Tel.: 33 4 91164431; Fax: 33 4 91718914; E-mail:
blasco{at}ibsm.cnrs-mrs.fr.
1 The abbreviations used are: DBMIB, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone; DCMU, 3-(3, 4-dichlorophenyl)-1,1-dimethylurea; EPR, electron paramagnetic resonance; HOQNO, 2-n-heptyl-4-hydroxyquinoline-N-oxide; MOPS, 4-morpholinepropane-sulfonic acid; NarGH; soluble catalytic dimer of nitrate reductase A; NarGHI, nitrate reductase A holoenzyme; NarI, cytochrome subunit of NarGHI (cytochrome bnr); SQR, succinate:quinone oxidoreductase; UHDBT, 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole.
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REFERENCES |
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