From the Medical Research Council Group in the
Molecular Biology of Membranes, Department of Biochemistry, University
of Alberta, Edmonton, Alberta T6G 2H7, Canada, the
§ Laboratoire de Chimie Bactérienne, CNRS, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 9, France, and the
¶ Laboratoire de Bioénergétique et
Ingénierie des Protéines, CNRS, 31 chemin Joseph
Aiguier, 13402 Marseille Cedex 9, France
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ABSTRACT |
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We have studied the effect of a mobAB
mutation and tungstate on molybdo-molybdopterin-guanine dinucleotide
(Mo-MGD) insertion into Escherichia coli nitrate reductase
(NarGHI). Preparation of fluorescent oxidized derivatives of MGD (Form
A and Form B) indicates that in a mobAB mutant there is
essentially no detectable cofactor present in either the membrane-bound
(NarGHI) or purified soluble (NarGH) forms of the enzyme. Electron
paramagnetic resonance characterization of membrane-bound
cofactor-deficient NarGHI suggests that it has altered electrochemistry
with respect to the dithionite reducibility of the [Fe-S] clusters of
NarH. Potentiometric titrations of membrane-bound NarGHI indicate that
the NarH [Fe-S] clusters have midpoint potentials at pH 8.0 (Em,8.0 values) of +180 mV ([3Fe-4S]
cluster), +130, 55, and
420 mV ([4Fe-4S] clusters) in a wild-type
background and +180, +80,
35, and
420 mV in a mobAB
mutant background. These data support the following conclusions: (i) a
model for Mo-MGD biosynthesis and assembly into NarGHI in which both
metal chelation and nucleotide addition to molybdopterin precede
cofactor insertion; and (ii) the absence of Mo-MGD significantly affects Em,8.0 of the highest potential
[4Fe-4S] cluster.
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INTRODUCTION |
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Escherichia coli, when grown anaerobically with nitrate as respiratory oxidant, develops a respiratory chain terminated by a membrane-bound quinol:nitrate oxidoreductase (NarGHI)1 (1-3). This enzyme is a heterotrimeric complex iron-sulfur molybdoenzyme comprising a molybdenum cofactor-containing catalytic subunit (NarG; 139 kDa), an [Fe-S] cluster-containing electron transfer subunit (NarH; 58 kDa), and a heme-containing membrane anchor subunit (NarI; 26 kDa). NarGHI is an excellent example of a family of bacterial iron-sulfur molybdoenzymes, which includes E. coli Me2SO reductase (DmsABC (4)), formate dehydrogenase N (FdnGHI (5)), and Wolinella succinogenes polysulfide reductase (PsrABC (6)). Sequence comparisons between NarG and a large number of bacterial molybdoenzymes indicate that this subunit binds the molybdenum cofactor and is the site of nitrate reduction (3, 4, 7).
The structures of a number of bacterial molybdoenzymes have recently
been determined. These include Me2SO reductases from Rhodobacter sphaeroides (8) and Rhodobacter
capsulatus (9), and formate dehydrogenase H from E. coli (10). In each case, there is a molybdo-bis(molybdopterin
guanine dinucleotide) (Mo-bisMGD) at the active site. Given the
significant sequence similarity between these enzymes and that of the
catalytic subunit of NarGHI (NarG), it is likely that NarG also
contains a Mo-bisMGD cofactor at its active site. However, because the
cofactor has not been unequivocally identified as a Mo-bisMGD, it will
be referred to herein as Mo-MGD. One of the final steps in the
biosynthesis of Mo-MGD (11) is the addition of a guanine nucleotide to
Mo-molybdopterin (Mo-MPT) to form Mo-MGD (12). In a mobAB
mutant, this step does not take place. The mob locus
comprises two open reading frames, mobA and mobB.
The gene product of mobA, MobA, has been shown to be
responsible for nucleotide addition (13), whereas the gene product of
mobB, MobB, enhances nucleotide addition but is not
essential for it (14). The effect of mobAB mutants on
E. coli NarGHI and DmsABC has been studied in detail (15,
16). Purified soluble NarGH dimer prepared from a mobAB
strain can be incubated in the presence of MobA and GTP, generating
enzyme that is able to reduce nitrate with benzyl viologen
(BV) as electron donor (13). This reconstitution presumably
occurs as a result of the conversion of Mo-MPT to Mo-MGD. However, its
extent corresponds to only 5-10% of the expected level. In these
reconstitution experiments, it has been suggested that the source of
Mo-MPT is the purified NarGH dimer (13, 15). These results contrast
with those reported for DmsABC, in which no MPT or molybdenum was
detected in the enzyme studied in a mobAB background (16).
However, in this latter case, the enzyme studied was the membrane-bound
heterotrimer rather than the soluble DmsAB dimer. It would therefore be
interesting to determine the effect of the mobAB mutation on
the membrane-bound NarGHI holoenzyme.
The operon encoding NarGHI (narGHJI) has a fourth open reading frame (narJ) whose product is not part of the holoenzyme (2, 17). NarJ is a system-specific chaperone that is produced in substoichiometric amounts compared with the other subunits encoded by the operon (18). It appears to hold cofactor-deficient NarGH in a cofactor-insertion competent conformation in E. coli strains that are unable to synthesize cofactor (19). The NarGH dimer appears to be unstable and is devoid of Mo-MGD in a narJ mutant background.
The [Fe-S] clusters of NarGHI have been investigated in considerable detail using a combination of site-directed mutagenesis of cluster-ligating Cys residues and EPR spectroscopy (20-24). These studies have provided convincing evidence for the presence of one [3Fe-4S] cluster and three [4Fe-4S] clusters located in the NarH subunit of purified preparations of the NarGH dimer. These clusters appear to be ligated by four ferredoxin-like Cys groups (I-IV), and the presence of these groups identifies NarH as being a member of an emerging family of four cluster proteins. This family includes E. coli DmsB (of DmsABC), FdnH (of the formate dehydrogenase, FdnGHI), HycB (of formate-hydrogen lyase), and NarY (of the alternative nitrate reductase, NarZYV), as well as many other proteins from other organisms (3, 4). It has recently been suggested on the basis of sequence alignments that the enzyme contains a fifth cluster, a [4Fe-4S] cluster, ligated by a cluster of Cys residues located toward the N terminus of NarG (3, 25). In the structure of formate dehydrogenase H (FdhF) (10), an N-terminal Cys group ligates a [4Fe-4S] cluster that is in close proximity to one of the pterins of the Mo-bisMGD cofactor. In the Thiosphaera pantotropha periplasmic nitrate reductase (NapA), a cluster was detected by EPR spectroscopy (10) which can also be assigned to the N-terminal Cys group. In both these enzymes, a role for the cluster can be envisioned in electron transfer to or from the Mo-MGD cofactor. Although many of the bacterial molybdoenzymes have an N-terminal Cys group in their catalytic subunit, there are important subclasses with different spacings between the putative cluster ligands (26). In both NarGHI and DmsABC, there is no evidence for the presence of a cluster within the catalytic subunit (22, 26), and the enzymes both belong to a specific subclass.
In this paper, we have investigated the effect of a mobAB mutation on the assembly of Mo-MGD into the soluble NarGH dimer and the membrane-bound NarGHI holoenzyme. We have compared the effect of the mobAB mutation with the effect of the molybdenum antagonist tungsten and have investigated the effect of the absence of Mo-MGD on the midpoint potentials of the NarH [Fe-S] clusters.
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MATERIALS AND METHODS |
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Bacterial Strains and Plasmids
E. coli LCB79 (araD139
(lacIPOZYA-argF) rpsL thi
79(nar-lac) StrepR) (27) is a wild-type
with respect to mobAB and was used to express fully
functional NarGHI. E. coli TP1000 (F
lacU169 araD139 rpsL150 relA1 ptsF rbsR flbB
(mobAB)
StrepR) (17) is a mobAB deletion mutant. NarGHI
was overexpressed from pVA700 (23) transformed into either LCB79 or
TP1000. For quantification of the molybdenum content of wild-type
NarGHI, E. coli LCB2048 (thi-1 thr-1 leu-6 lacY1
supE44 rpsL175
nar25(narG-narH)
(nar'U-narZ')
KanR SpecR
StrepR (28)) transformed with pVA700 was used.
Growth of Cells and Preparation of Membrane Vesicles
Cells were grown microaerobically in 2-liter batch cultures of
Terrific Broth (29) at 30 °C in the presence of 100 µg
ml1 streptomycin and ampicillin. Where appropriate, the
growth medium was supplemented with 15 mM sodium tungstate.
A 10% innoculum was used, and NarGHI overexpression was achieved by
addition of 0.2 mM
isopropyl-1-thio-
-D-galactopyranoside (IPTG). Following addition of the innoculum and the IPTG, cells were grown overnight with
gentle shaking at 30 °C. Cells were harvested by centrifugation, washed in 100 mM MOPS and 5 mM EDTA (pH7).
Membrane vesicles were prepared as described previously (30).
Preparation of Purified NarGH Dimer
Purified NarGH was prepared from wild-type and mobAB genetic backgrounds as described previously (20). Lyophilized NarGH was resuspended in 100 mM Tris and 5 mM EDTA (pH 8.3).
Preparation of Fluorescent MPT Derivatives
Membrane Vesicles-- The presence of MPT in membrane vesicles was assayed by acid denaturation followed by I2 and KI oxidation to produce an extract that contained the Form A fluorescent derivative of MPT that will be referred herein to as a "Form A extract" (12). 20 mg of membrane protein was used as starting material. Prior to the recording of fluorescence spectra using a Perkin-Elmer LS-50B luminescence spectrometer, small aliquots (100-200 µl) of the acid-denatured iodine-oxidized extract were added to 3 ml of 1 M NH4OH in a fluorescence cuvette.
Purified NarGH-- The presence of MPT in purified NarGH was assayed by preparation of an extract containing the Form B fluorescent derivative of MPT that will be referred to herein as a "Form B extract" (31, 32). Protein samples containing 2 mg of NarGH were diluted with 1 ml of 100 mM Tris (pH 7.5) and were incubated at 100 °C for 25 min. Insoluble material was removed by centrifugation at 13,000 rpm for 25 min. Aliquots of the supernatant fraction (200 µl) were added to 3 ml of 1 M NH4OH in a fluorescence cuvette. Excitation and emission spectra were recorded as described in the legend to Fig. 1.
Quantitation of Molybdenum
The amount of molybdenum present in membrane fractions was determined by wet ashing the samples and performing inductively coupled plasma emission spectroscopy (ICPES). The molybdenum content of membrane vesicles from TP1000/pVA700 (mobAB mutant) and LCB2048/pVA700 (wild-type) was analyzed by ICPES. The NarGHI content of the membranes used in the ICPES studies was determined by rocket immunoelectrophoresis as described previously (20).
Protein Assays
Protein concentrations were assayed by the Lowry method, modified by the inclusion of 1% (w/v) sodium dodecyl sulfate in the incubation mixture to solubilize membrane proteins (33).
Preparation of EPR Samples
Membrane vesicles were suspended at a protein concentration of
30 mg ml1 in 100 mM MOPS and 5 mM
EDTA (pH 7.0). Dithionite-reduced (5 mM) samples were
incubated under argon at 23 °C for 5 min. Oxidized samples were
prepared by incubating membranes in the presence of 0.2 mM
potassium ferricyanide for 2 min. EPR spectra were recorded using a
Bruker Spectrospin ESP-300 spectrometer equipped with an Oxford
Instruments ESR-900 flowing helium cryostat. Instrument conditions and
temperatures were as described in the individual figure legends.
Redox Potentiometry
Redox titrations were carried out under argon at pH 8.0 in 100 mM Tricine and 5 mM EDTA as described previously (34). Membranes prepared in 100 mM MOPS and 5 mM EDTA (pH 7) (see above) were pelleted by ultracentrifugation and resuspended in the pH 8.0 buffer. The following redox mediators were used at a concentration of 50 µM: quinhydrone, 2,6-dichlorophenolindophenol, 1,2-naphthoquinone, toluylene blue, phenazine methosulfate, thionine, duroquinone, methylene blue, resorufin, indigotrisulfonate, indigodisulfonate, anthraquinone-2-sulfonic acid, phenosafranine, benzyl viologen, and methyl viologen. All samples were prepared in 3-mm internal diameter quartz EPR tubes, were rapidly frozen in liquid nitrogen-chilled ethanol, and were stored under liquid nitrogen until use.
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RESULTS |
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Effect of the mobAB Mutation on the Presence of Mo-MGD in NarGHI-- We have previously shown that in a mobAB mutant, no detectable Mo-MPT is assembled into the DmsABC holoenzyme (16). This result contrasts with reports that the NarGH dimer purified from a mobAB mutant contains Mo-MPT (13, 15). To reconcile these results, we subjected membrane-bound NarGHI holoenzyme to acid denaturation followed by I2 and KI oxidation to produce the fluorescent Form A derivative of MPT (12). As a negative control for these experiments, we also studied Form A extracts from membranes from tungstate-grown cells. Fig. 1A, a and d, shows excitation and emission spectra of a Form A extract from NarGHI overexpressed from a wild-type strain. Noticeable in these spectra are intense peaks at approximately 390 and 445 nm in the excitation and emission spectra, respectively, consistent with the fluorescent species present being the Form A derivative of Mo-MPT (12). Fig. 1A, b and e, show equivalent spectra obtained from membranes prepared from wild-type cells grown in the presence of 15 mM tungstate. Noticeable in these spectra are the absence of intense peaks attributable to the Form A derivative of Mo-MGD. Fig. 1A, c and f, show equivalent spectra from an extract from membranes containing overexpressed NarGHI from mobAB mutant cells. EPR spectroscopy (see below) demonstrates that normal levels of overexpressed NarGHI are accumulated into the membranes in cells grown in the presence of tungstate and in mobAB mutant cells, suggesting that the mobAB mutation and tungstate have a similar effect on the cofactor composition of NarGHI, viz. elimination of detectable membrane-bound Mo-MPT or Mo-MGD. To determine if there is a correlation between pterin and Mo content, we subjected membrane samples containing overexpressed wild-type and mobAB mutant NarGHI to ICPES analysis. Wild-type enzyme contains approximately 0.90 mol of Mo/NarGHI heterotrimer, whereas the mobAB mutant enzyme contains only 0.01 mol of Mo/NarGHI heterotrimer. These results are in agreement with previous studies of DmsABC (16).
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Effect of the Absence of Cofactor on EPR Spectra of the NarH
[Fe-S] Clusters--
To investigate the functional relationship
between Mo-MGD cofactor of NarG and the NarH [Fe-S] clusters, we
recorded EPR spectra at 12K of dithionite-reduced and
ferricyanide-oxidized membrane samples containing wild-type and
mobAB mutant NarGHI. Fig.
2a shows the spectrum of
dithionite-reduced wild-type NarGHI in membranes at pH 7.0. We have
previously demonstrated that in the absence of redox-mediators, the
Em,8.3 = 400 mV cluster remains oxidized in
the presence of excess dithionite (24), and the spectrum in Fig.
2a corresponds to this partially reduced form of the enzyme. Fig. 2b shows an equivalent spectrum of mobAB
mutant NarGHI. This spectrum corresponds to that of the fully reduced
enzyme reported by Guigliarelli et al. (22). The spectrum of
NarGHI in membranes from tungstate-grown cells is essentially identical
to that of mobAB mutant NarGHI (data not shown), indicating
that the cofactor-deficient form of the enzyme may have significantly
altered [Fe-S] cluster midpoint potentials. Fig. 2, c and
d, shows spectra of ferricyanide-oxidized wild-type and
mobAB mutant NarGHI in membranes, showing the spectrum of
the [3Fe-4S] cluster of NarH. Only minor differences are apparent between the two spectra, indicating that the EPR lineshape of the
[3Fe-4S] cluster is not significantly altered by the absence of
Mo-MGD.
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Effect of the Absence of Cofactor on the Redox Potentiometry of the NarH [Fe-S] Clusters-- Three observations prompted us to examine the effect of the absence of Mo-MGD on the redox potentiometry of the NarH [Fe-S] clusters: (i) the dithionite-reducibility of the lowest potential [4Fe-4S] cluster in cofactor-deficient NarGHI (Fig. 2); (ii) the presence of a [4Fe-4S] cluster in close proximity to the Mo-bisMGD of FdhF of E. coli (10); and (iii) the presence of a [4Fe-4S] cluster in NapA of T. pantotropha (25).
Fig. 3 shows representative EPR spectra of redox-poised samples at pH 8.0 for NarGHI-enriched membranes from a wild-type strain. Between +332 and +243 mV, the spectrum is dominated by the oxidized [3Fe-4S] cluster. The EPR spectrum of this cluster in membrane-bound NarGHI has a slightly axial line shape, with a peak at g = 2.02 (gz), and a peak-trough at g = 1.99-1.96 (gxy). Between +243 and +129 mV, the spectrum of the [3Fe-4S] cluster diminishes and is substantially replaced by that of the highest potential [4Fe-4S] cluster of NarGHI. As reported by Guigliarelli et al. (22), the spectrum of this species appears to arise from two conformations, characterized by features at approximately g = 2.05, 1.95, 1.87 and g = 2.01, 1.89, 1.87, respectively (22). The spectrum is also complicated by an overlapping Mo(V) signal at approximately g = 1.98.
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Midpoint Potentials of the Mo(IV/V) and Mo(V/VI) Couples of Wild-type NarGHI at pH 8.0-- Fig. 6 shows a plot of the intensity of the g = 1.98 peak of the Mo(V) spectrum of NarGHI from LCB79/pVA700 membranes versus Eh. The EPR spectrum of the Mo(V) signal at 75K (data not shown) is essentially identical to the "high pH" form previously reported (22, 35, 36), and the potentiometric data can be fitted to two Em,8.0 values of +190 mV for the Mo(V/VI) couple and +95 mV for the Mo(IV/V), in agreement with previously reported values (22). A more detailed study of the electrochemistry of the Mo-MGD cofactor of NarGHI and the effects of pH and NarG site-directed mutants will appear elsewhere.2
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DISCUSSION |
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We have demonstrated herein that the effects of the mobAB mutation of E. coli TP1000 and growth of the wild-type strain (LCB79/pVA700) in the presence of excess tungstate are essentially identical, resulting in the absence of significant quantities of Mo-MGD (or Mo-MPT) in membranes enriched in NarGHI. This result is entirely consistent with the effects of a mobAB mutation and tungstate on a similar E. coli anaerobic reductase, DmsABC (16). Furthermore, we have shown that the absence of cofactor has a significant effect on the midpoint potentials of the two highest-potential [Fe-S] clusters of NarH. In the mobAB mutant and tungstate-grown enzymes, all of the [Fe-S] clusters appear to be reducible by dithionite at pH 7.0, whereas in the wild-type enzyme, the lowest potential [4Fe-4S] cluster remains oxidized in the presence of excess dithionite (24). These results have important implications for (i) the biosynthetic pathway and mechanism of insertion of Mo-MGD, (ii) the functional relationship between the NarH [Fe-S] clusters and the Mo-MGD cofactor of NarG, and (iii) the pathway of electron transfer through NarGHI.
The absence of cofactor in NarGH and NarGHI from the mobAB
mutant strain has to be reconciled with previous studies that have demonstrated reconstitution of BV:nitrate oxidoreductase
activity by addition of MobA and GTP to NarGH isolated from a
mobAB mutant background. In these studies, it was assumed
that NarGH was the source of Mo-MPT (13, 15). The data presented herein
and previous mobAB mutant reconstitution results can be
reconciled if the observed turnover numbers in the reconstitution
experiments correspond to a small fraction of the turnover number of
the Mo-MGD-containing enzyme derived from a wild-type strain. It is
also possible that the occupancy of Mo-MPT in NarGHI and NarGH from a
mobAB mutant strain is different (cf. Fig. 1,
A and B), with there being more cofactor present
in the soluble dimer, as suggested by the increased relative
fluorescence of samples derived from the mobAB mutant soluble dimer compared with those derived from NarGHI in
mobAB mutant membranes. The suggestion that there is a low
occupancy of Mo-MGD in the mobAB mutant enzyme appears to be
the simplest explanation that can reconcile the lack of detectable
cofactor and the observed reconstitution of activity. Recent findings
on the function of NarJ suggest that it functions to hold
cofactor-deficient NarGH in a cofactor-binding conformation (19). NarJ
is found to bind specifically to cofactor-deficient NarGH from a
mobAB mutant (data not shown), and it is possible that the
previously observed reconstitution in mobAB mutant NarGH
arose from Mo-MPT provided by a Mo-MPT carrier protein associated with
a NarGH·NarJ complex.
The absence of Mo-MPT from the mobAB mutant NarGHI holoenzyme and NarGH dimer has implications for the final stages of cofactor biosynthesis and insertion into E. coli molybdoenzymes. Fig. 7 shows a model for this process incorporating the results presented herein. MPT is converted to Mo-MPT by the addition of molybdenum. Molybdenum is bound and transported into the cytoplasm of E. coli by the products of the modABCD operon (37, 38) and subsequently chelated by MPT. The presence of tungsten (as tungstate) prevents both molybdenum ligation by MPT and insertion of the organic component of the cofactor into NarGH. Previous studies have suggested that in the presence of tungstate, cofactor accumulates in the cytoplasm either as an "empty" MPT or as a tungsten-ligated form (39). Neither of these two forms appear to be able to assemble into NarGHI (Fig. 1). In contrast to the conclusions drawn from earlier studies, our results with a mobAB mutant indicate that nucleotide attachment and metal chelation must precede cofactor insertion into NarGH. Since both the soluble (NarGH) (20, 22) and membrane-bound (NarGHI) (24, 40) forms of the enzyme can readily be studied and appear to contain Mo-MGD in a wild-type background, cofactor insertion most likely occurs prior to attachment of the NarGH dimer to the membrane anchor subunit (NarI). In the presence of tungstate or in a mobAB mutant background, it is clear that attachment of cofactor-deficient NarGH to the membrane-bound NarI is still able to occur, thus bypassing the blockage in Mo-MGD biosynthesis and insertion. Overall, the final stages of cofactor biosynthesis and insertion into NarGHI appear to be very similar to those proposed for its biosynthesis and insertion into DmsABC (16), except that in the latter case no system-specific chaperone has so far been identified.
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We have evaluated the midpoint potentials of the NarH [Fe-S] clusters
in the NarGHI holoenzyme at pH 8.0 in Tricine buffer. In previous
studies of the soluble NarGH dimer (20-23), the midpoint potentials of
the [Fe-S] clusters of NarH in the soluble NarGH dimer have been
determined to be +80 mV ([4Fe-4S] cluster), +60 mV ([3Fe-4S]
cluster), 200 mV, and
400 mV ([4Fe-4S] clusters) in a Tris buffer
at pH 8.3. Clearly, the data presented herein (Fig. 5) suggest that the
Em of the three highest-potential clusters appear to be significantly higher in membrane preparations of NarGHI
compared with the soluble NarGH dimer. Three possible explanations exist for these differences: (i) the conformation of the NarH subunit
is altered by the presence of the NarI membrane anchor subunit,
resulting in higher Em for the three highest
potential clusters; (ii) a lack of solvent accessibility to the
clusters in the membrane-bound enzyme causes the higher
Em values; and (iii) freezing-induced pH changes
occur in samples prepared in the Tris buffer, as has been previously
demonstrated by Williams-Smith et al. (41). To generate the
potentiometric data presented herein, a zwitterionic buffer was used
(Tricine) instead of the anionic Tris buffer used in previous
potentiometric studies of the NarGH dimer [Fe-S] clusters. Freezing
of Tris buffers can result in an apparent
pH of approximately +2.3
(41), which could result in an alkalinization of the Tris buffer
previously used in studies of NarGH to a pH of approximately 9.6.
The effect of the mobAB mutation on the
Em,8.0 of the highest-potential [4Fe-4S]
cluster suggests that this cluster may be located in close proximity to
the Mo-MGD cofactor of NarGHI. The absence of Mo-MGD causes a
Em,8.0 of
50 mV for this cluster. This
cluster is ligated by the first three Cys residues of Cys group I of
NarH (21) and the last Cys residue of group IV (23), and its absence in
NarH mutants dramatically inhibits, but does not abolish, electron
transfer through NarGHI to nitrate. It is therefore possible that it
functions in a similar manner to the [4Fe-4S] cluster of FdhF (10),
acting as the direct electron donor to the Mo-MGD. The effect of the
absence of cofactor on the Em,8.0 of the highest
potential [4Fe-4S] cluster is somewhat surprising in light of its
effect on the dithionite-reducibility of the lowest potential
[4Fe-4S] cluster (Em,8.0 =
420 mV). We anticipated that loss of cofactor would raise the
Em,8.0 of this low potential center. A previous
study (24) has demonstrated that the presence of redox mediators such
as those routinely used in potentiometric titrations allows the
Em,8.0 =
420 mV cluster to become accessible
to reducing equivalents derived from dithionite. However, it appears
from the results presented herein that the dithionite accessibility of
the Em,8.0 =
420 mV cluster is related to the
absence of Mo-MGD, suggesting that this might be achieved via the empty
cofactor-binding pocket. This would place the
Em,8.0 =
420 mV cluster in the vicinity of
this pocket in close proximity to the highest potential [4Fe-4S]
cluster. This suggestion is in agreement with the model for [Fe-S]
cluster ligation proposed by Guigliarelli et al. (23) in
which the Cys groups I and IV provide ligands to the highest and lowest
potential [4Fe-4S] clusters, respectively. It is also in agreement
with the broadening of the features of the spectrum of the highest
potential cluster upon reduction of the lowest potential cluster, which
suggests that these two clusters might form an eight-iron
ferredoxin-like pair within NarGHI (23).
The observed shift in the Em,8.0 of the low
potential component of the [3Fe-4S] cluster from 100 mV in the
wild-type enzyme to 55 mV in the mobAB mutant and
tungstate-grown enzyme is probably a consequence of the proximity of
the [3Fe-4S] cluster to the highest potential [4Fe-4S] cluster in
the enzyme. This phenomenon may be a manifestation of the negative
redox cooperativity observed between the highest potential [4Fe-4S]
cluster and the [3Fe-4S] cluster in previous studies of the NarGH
dimer (22). This is supported by the similar magnitudes of the observed
shifts for the [4Fe-4S] cluster and subpopulation of the [3Fe-4S]
cluster (Em, 8.0 =
50 mV for the [4Fe-4S]
cluster and
Em, 8.0 =
45 mV for the
[3Fe-4S] cluster).
Our investigation of the [Fe-S] cluster Em,8.0
values of the wild-type and mobAB mutant NarGHI in
situ in membrane vesicles was prompted by the dithionite
reducibility of the Em,8.0 = 420 mV cluster in
cofactor-deficient NarGHI in the absence of redox mediators at pH 7.0. Potentiometric studies were carried out at pH 8.0, as this is close to
the pH used in previous EPR determinations of the NarH [Fe-S] cluster
Em. Although differences in the
Em,8.0 values were observed between the
wild-type and cofactor-deficient enzymes, it should be noted that the
EPR spectra of wild-type and mobAB mutant NarGHI in their
various redox states are nearly identical (cf. Figs. 3 and
4), indicating that neither the structure of the [Fe-S] centers nor
their relative arrangement is modified by the absence of Mo-MGD.
Overall, the data presented herein indicate that the final stages of Mo-MGD biosynthesis and insertion into NarGHI are similar to those proposed for DmsABC (16), except that DmsABC appears to accept Mo-MGD without the aid of a NarJ-like chaperone. The absence of Mo-MGD from NarGHI has a significant effect on the Em,8.0 of the highest potential [4Fe-4S] cluster and on a subpopulation of the [3Fe-4S] cluster. These results represent an important step in delineating (i) the pathway of Mo-MGD cofactor biosynthesis and insertion and (ii) the electron transfer pathway to the cofactor through the [Fe-S] clusters of NarH.
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ACKNOWLEDGEMENT |
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We are grateful to Dr. J. C. Germanique for providing help with the molybdenum quantification by ICPES (Europole Méditerranéen de l'Arbois, Aix les Milles, France).
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FOOTNOTES |
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* This work was supported by a grant from the Medical Research Council of Canada (PG11440) (to J. H. W.), by the Center National de la Recherche Scientifique, by a travel grant from the Alberta Heritage Foundation for Medical Research (to G. G. and F. B.), and by a NATO Collaborative Research Grant (to J. H. W.).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.
To whom correspondence should be addressed: Dept. of
Biochemistry, 474 Medical Sciences Bldg., University of Alberta,
Edmonton, Alberta T6G 2H7. Tel.: 403-492-2761; Fax:
403-492-0886.
1
The abbreviations used are: NarGHI, nitrate
reductase holoenzyme; BV, reduced benzyl viologen; DmsABC,
Me2SO reductase; EPR, electron paramagnetic resonance;
ICPES, inductively coupled plasma emission spectroscopy; IPTG,
isopropyl-
-D-1-thiogalactopyranoside; MGD, molybdopterin
guanine dinucleotide; Mo-bisMGD, molybdo-bis(molybdopterin guanine
dinucleotide); Mo-MGD, molybdo-MGD; MPT, molybdopterin; Mo-MPT,
molybdo-MPT; NarGH, nitrate reductase soluble dimer; FdnGHI, formate
dehydrogenase N; FdhF, formate dehydrogenase H; MOPS, 4-morpholinepropanesulfonic acid; Tricine, N-[2-
hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
2 A. Magalon, M. Asso, B. Guigliarelli, R. A. Rothery, G. Giordano, P. Bertrand, and F. Blasco, submitted for publication.
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