Protein Crystallography Reveals a Role for the FS0 Cluster of Escherichia coli Nitrate Reductase A (NarGHI) in Enzyme Maturation*

We have used site-directed mutagenesis, EPR spectroscopy, redox potentiometry, and protein crystallography to monitor assembly of the FS0 [4Fe-4S] cluster and molybdo-bis(pyranopterin guanine dinucleotide) cofactor (Mo-bisPGD) of the Escherichia coli nitrate reductase A (NarGHI) catalytic subunit (NarG). Cys and Ser mutants of NarG-His49 both lack catalytic activity, with only the former assembling FS0 and Mo-bisPGD. Importantly, both prosthetic groups are absent in the NarG-H49S mutant. EPR spectroscopy of the Cys mutant reveals that the Em value of the FS0 cluster is decreased by at least 500 mV, preventing its participation in electron transfer to the Mo-bisPGD cofactor. To demonstrate that decreasing the FS0 cluster Em results in decreased enzyme activity, we mutated a critical Arg residue (NarG-Arg94) in the vicinity of FS0 to a Ser residue. In this case, the Em of FS0 is decreased by 115 mV, with a concomitant decrease in enzyme turnover to ∼30% of the wild type. Analysis of the structure of the NarG-H49S mutant reveals two important aspects of NarGHI maturation: (i) apomolybdo-NarGHI is able to bind GDP moieties at their respective P and Q sites in the absence of the Mo-bisPGD cofactor, and (ii) a critical segment of residues in NarG, 49HGVNCTG55, must be correctly positioned to ensure holoenzyme maturation.

NarGHI and the other CISM archetypes comprise a mononuclear molybdenum cofactor (molybdo-bis(pyranopterin guanine dinucleotide) (Mo-bisPGD))-containing catalytic subunit, e.g. NarG; a four-cluster protein subunit, e.g. NarH; and a membrane anchor protein, e.g. NarI. Each catalytic subunit contains a [4Fe-4S] cluster in addition to Mo-bisPGD, and many of the membrane anchor subunits are also diheme cytochromes b. The five [Fe-S] clusters are referred to as FS0 -FS4, with increasing distance from Mo-bisPGD, and the entire electron transfer relay (ETR) in NarGHI, including the two hemes, spans a distance of almost 100 Å (2).
In NarGHI, Mo-bisPGD provides the site of nitrate reduction at its redox-active molybdenum atom. The molybdenum atom is coordinated by two pyranopterin guanine dinucleotide (PGD) moieties via a bis-dithiolene linkage (11)(12)(13). These PGD groups are referred to as the P-and Q-pterins, and they are proximal and distal, respectively, to the FS0 [4Fe-4S] cluster. The bis-dithiolene coordination is supplemented by one oxo group and two oxygens of NarG-Asp 222 (2). The arrangement of the P-pterin and the FS0 cluster may facilitate electron entry to the molybdenum active site via the unsaturated ring system of the P-pterin. The Q-pterin of NarG and that of the catalytic subunit (EbdA) of Aromatoleum aromaticum ethylbenzene dehydrogenase (EbdABD) (14) both have a bicyclic molybdopterin structure rather than the tricyclic pyranopterin structure found in other mononuclear molybdoenzymes studied to date (15)(16)(17).
In NarG, the molybdenum atom has midpoint potentials (E m ) of ϳ100 and 200 mV for the Mo(IV/V) and Mo(V/VI) couples, respectively (18,19). The nearby FS0 cluster has an E m of approximately Ϫ55 mV (18). During catalysis, electrons flow from FS1 with an E m value of ϩ130 mV to FS0, with this step presenting a barrier of ϳ17.9 kJ mol Ϫ1 . Thus, the FS0 E m value may play a critical role in regulating electron flow to the Mo-bisPGD cofactor.
FS0 plays a critical role in CISM enzymes because it is in direct redox equilibrium with Mo-bisPGD, either being the first [Fe-S] cluster in the ETR of the dehydrogenase enzymes (e.g. FdnGHI) or the final [Fe-S] cluster in the ETR of the reductase enzymes (e.g. NarGHI and DmsABC). FS0 is coordinated by a ferredoxin-like Cys group with the consensus sequence ( (20). Enzymes with two residues between the first two Cys residues (C A and C B ) and a Lys following the fourth Cys residue are referred to as Type I enzymes and include CISM archetypes such as FdnGHI (5) and PsrABC (8), as well as enzymes that contain a catalytic subunit but that are not archetypes of the CISM family (4). These include the FdhF subunit of the E. coli formate:hydrogen lyase complex (21) and the NapA subunit of the periplasmic nitrate reductase (NapAB) (22,23). NarG and DmsA are examples of enzymes that have a C A -C B spacing of three residues and an Arg following C D , and NarG has a His residue at the C A position. These are referred to as Type II enzymes.
The role of FS0 in NarGHI maturation has yet to be clearly established. In a ⌬mobAB strain that is unable to add GMP to the molybdenum-pyranopterin precursor (24), NarGHI lacks Mo-bisPGD but retains FS0 -FS4 and the two hemes. NarGHI is encoded by the narGHJI operon (25,26), of which only the NarG, NarH, and NarI proteins appear in the functional holoenzyme. The fourth gene product, NarJ, is a system-specific chaperone that has been suggested to hold NarGH in a cofactor-binding competent conformation (27)(28)(29). FS0 does not appear to assemble in the absence of NarJ. Li and Turner (30) have recently demonstrated an interaction between NarJ and a peptide comprising the first 50 amino acid residues of NarG, which has significant sequence similarity to a range of twin-arginine translocation (tat) pathway leader peptides (29) but lacks the critical pair of Arg residues essential for translocation to the periplasmic compartment via the tat export pathway (31)(32)(33)(34).
In this work, we have investigated the role of the NarG FS0 cluster in NarGHI maturation using a combination of site-directed mutagenesis, EPR spectroscopy, and protein crystallography. Our results demonstrate that FS0 assembly is an important prerequisite for Mo-bisPGD cofactor insertion during holoenzyme maturation.
Growth of Cells-E. coli LCB79/pVA700 was grown overnight in 5-liter batches with 1% inoculum in a B. Braun Biostat B fermenter at 30°C in the presence of 100 g ml Ϫ1 ampicillin and 100 g ml Ϫ1 streptomycin. The growth medium contained 12 g liter Ϫ1 Tryptone, 24 g liter Ϫ1 yeast extract, 5 g liter Ϫ1 NaCl, 4 ml liter Ϫ1 glycerol, and 0.1 mM ammonium molybdate. NarGHI overexpression was induced at A 600 ϭ 2.0 by the addition of 0.2 mM isopropyl-1-thio-␤-D-galactopyranoside, after which the cultures were grown for an additional 10 -11 h. Cells were harvested by centrifugation, washed in a buffer containing 100 mM MOPS and 5 mM EDTA (pH 7.0), frozen in liquid nitrogen, and stored at Ϫ70°C prior to use.

Isolation of Membrane Fractions and Purified Preparations-
Crude membrane vesicles were prepared from E. coli cells by French pressure cell lysis and differential centrifugation (37). Enriched inner membrane vesicles were prepared from these crude membranes by sucrose step centrifugation as previously described (38). All membrane preparation steps were carried out in a buffer containing 100 mM MOPS and 5 mM EDTA (pH 7.0). Excess sucrose was removed by resuspension and recentrifugation in this buffer. For EPR experiments, membranes were resuspended and recentrifuged in a buffer containing 100 mM Tricine and 5 mM EDTA (pH 8.0). Membrane samples were flash-frozen in liquid nitrogen and subsequently stored at Ϫ70°C prior to use.
Purification, Crystallization, Data Collection, and Structure Determination of NarGHI Mutants-Purified NarG-H49S, NarG-H49C, and NarG-R94S mutant enzymes were prepared by anion exchange chromatography and subsequently crystallized as previously described (2,18,39). The crystals obtained were isomorphous with the native enzyme (C222 1 , a ϭ 154.00 Å, b ϭ 241.28 Å, c ϭ 140.39 Å). Data were integrated and scaled with the HKL suite of programs (40). The structure was determined using difference Fourier techniques and was refined following rigid body refinement of the native 1.9-Å resolution model (2) with the CNS program (41). Additional cycles of manual rebuilding with Xfit (42) and refinement with CNS resulted in final models for the mutant forms of NarGHI with refinement parameters as described in Table 1.
Redox Potentiometry and EPR Spectroscopy-Redox titrations were carried out under argon at 25°C as previously described (38,43) in 100 mM Tricine and 5 mM EDTA (pH 8.0). The protein concentration used was ϳ30 mg ml Ϫ1 . The following redox mediators were used at a concentration of 50 M: quinhydrone, 2,6-dichloroindophenol, 1,2-naphthoquinone, toluylene blue, phenazine methosulfate, thionine, duroquinone, methylene blue, resorufin, indigo trisulfonate, indigo disulfonate, anthraquinone-2-sulfonic acid, phenosafranine, benzyl viologen, and methyl viologen. All samples were prepared in 3-mm internal diameter quartz EPR tubes, rapidly frozen in liquid nitrogen-chilled ethanol, and stored under liquid nitrogen until used. EPR spectra were recorded using a Bruker Elexys spectrometer equipped with a Bruker SHQE cavity and an Oxford Instruments ESR-900 flowing helium cryostat. Spectra were recorded under conditions of temperature and microwave power as described in the individual figure legends. Estimates of E m were obtained from n ϭ 1 fits of potentiometric spectral data to the Nernst equation. For studies of the Mo(V) signal of NarGHI, a Bruker ESP300E spectrometer equipped with a TE 102 microwave cavity and a Bruker liquid nitrogen-evaporating cryostat were used (Bruker ER4111 VT variable temperature unit). In this case, spectra were recorded at 150 K.
Protein Assays-Protein concentrations were assayed by the Lowry method (44), modified by the inclusion of 1% (w/v) sodium dodecyl sulfate in the incubation mixture to solubilize membrane proteins (45).

RESULTS AND DISCUSSION
NarG-His 49 and NarG-Arg 94 Are Critical for Enzyme Activity- Fig. 1 illustrates the coordination environment of the FS0 [4Fe-4S] cluster in wild-type NarGHI. The cluster is coordinated by NarG-His 49 , NarG-Cys 53 , NarG-Cys 57 , and NarG-Cys 92 . NarG-Arg 94 is sandwiched between FS0 and the proximal pterin of the Mo-bisPGD cofactor. Membranes enriched in NarGHI, NarG-H49CHI, NarG-H49SHI, and NarG-R94SHI have nitrate reductase-specific activities of 4.2, 0.1, 0.0, and 1.4, respectively (in units of mol of lapachol oxidized per min (mg of protein) Ϫ1 ). The elimination of activity in the NarG-His 49 mutants indicates that this residue plays a critical role in either holoenzyme maturation or catalytic turnover. We also used reduced benzyl viologen as electron donor in nitrate reductase assays (47,48), which donates electrons nonspecifically to NarGHI (49). Membranes enriched in NarGHI, NarG-H49CHI, NarG-H49SHI, and NarG-R94SGH exhibit nitrate reductase activities with benzyl viologen of 45, 0.4, 0.1, and 37, respectively (in units of mol of benzyl viologen oxidized per min (mg of protein) Ϫ1 ). Both assays indicate loss of activity in the NarG-His 49 mutants, but the NarG-R94S mutant retains almost wild-type levels of activity in the benzyl viologen assay and only ϳ30% of wild-type activity in the lapachol assay. The distribution of activities in the mutant mirrors that found in a mutant of the equivalent residue in E. coli DmsA (DmsA-R61S) (50). In this case, quinol:Me 2 SO oxidoreductase activity is completely eliminated, but benzyl viologen:Me 2 SO activity is retained.
FS0 Is Absent in the NarG-H49S Mutant-Previous work on the NarG-His 49 mutants indicated that the cofactor is present in the NarG-H49C mutant but absent in the NarG-H49S mutant (19). To determine how the Mo-bisPGD site is perturbed in these mutants, we subjected both to analysis by pro-tein crystallography. Fig. 2A shows the structure of the NarG-H49C mutant around the FS0 cluster and demonstrates that the Cys residue introduced by mutagenesis is clearly able to provide sulfur coordination to FS0. Also shown is the sequence of residues 49 CGVNCTG 55 , which is unresolved in the structure of the NarG-H49S mutant (see below; Fig. 2B). Significantly, this overlaps with the 51 VNCT 54 sequence that is unresolved in the structure of apomolybdo-NarGHI (Protein Data Bank code 1SIW (18)).
The NarG-H49S mutant lacks molybdenum as well as the pterin components of the cofactor but retains the GDP moieties (Fig. 2B). The structure in this region differs from that of apomolybdo-NarGHI (Protein Data Bank code 1SIW) in that it lacks FS0 but retains a single iron atom coordinated by the sulfur atoms of NarG-Cys 57 and NarG-Cys 92 . These results are consistent with correct assembly of FS0 being a prerequisite for Mo-bisPGD assembly.
Further structural analysis (Fig. 3) reveals that the unresolved sequence ( 49 CGVNCTG 55 ) of the NarG-H49S mutant is involved in a number of potentially critical interactions with the P-pyranopterin and the Q-guanine moieties. These include hydrogen bonds between the amide nitrogen of NarG-Asn 52 and dithiolene sulfurs of both pterins and a hydrogen bond between the carbonyl oxygen of NarG-Cys 53 and the N-10 secondary amine of the P-pterin. Finally, the carbonyl oxygen of the NarG-Gly 50 amide is ϳ3.4 Å from the N-7 of the purine bicycle of the guanine nucleotide attached to the Q-pterin. Based on these observations, it is likely that correct positioning of 49 CGVNCTG 55 is critical for insertion of the Mo-bisPGD cofactor during NarGHI maturation.
Analysis of the apomolybdo-NarGHI structures indicates that GDP moieties can be inserted into positions corresponding to GDP-P and GDP-Q in the NarG-H49S mutant (Fig. 2B) and into the GDP-Q position of the wild-type apomolybdoenzyme (18). These observations suggest that GDP can occupy its two binding pockets within NarG when the mature Mo-bisPGD cofactor is unavailable. The estimated intracellular concentra-tion of GDP is ϳ0.68 mM (51), a concentration that hypothetically could competitively inhibit Mo-bisPGD insertion. The final steps of cofactor insertion are orchestrated by the MobAB, MoeA, MogA, and NarJ proteins (52), and it is likely that these steps prevent inhibition by cytoplasmic GDP.
Potential Electron Transfer Pathways Are Retained in the NarG-R94S Mutant-The overall conservation of protein structure around FS0 and Mo-bisPGD in the NarG-H49C mutant prompted us to speculate that its lack of activity is due to a large shift being elicited on its E m . To test this hypothesis, we solved the structure of the NarG-R94S mutant, which should eliminate a positive charge in the vicinity of FS0, thus lowering its E m . A Lys or Arg residue is highly conserved at this position in all known CISM catalytic subunits and, in the Ralstonia eutropha NapA mutagenesis of a Lys to an Arg at this position, results in decreased enzyme activity (to ϳ23% of wild type) (53). As already indicated above, in E. coli Me 2 SO reductase (DmsABC), mutation of an Arg to a Ser at this position eliminates quinol-dependent Me 2 SO reductase activity (50). To establish that a significant shift in the FS0 E m value is responsible for the observed decrease in enzyme activity, it is important to verify that the NarG-R94S mutant introduces no gross structural changes in the vicinity of either FS0 or the Mo-bisPGD cofactor. Fig. 4A shows potential electron transfer routes from FS0 to the P-pterin. NarG-Arg 94 could contribute to electron transfer via its interaction with the amine attached to the C-8 position of the P-pterin. Other possible routes include the interaction between the carbonyl oxygen of NarG-Cys 53 and N-10 of the P-pterin. Finally, a conserved Asn (NarG-Asn 52 ) provides an interaction between its carboximide amine side chain and two dithiolene sulfurs, one from each pterin. Fig. 4B shows the structure of the NarG-R94S mutant. The side chain oxygen of the Ser variant is ϳ5.9 Å from the amine nitrogen attached to In addition to the FS0coordinating residues, the position of the 49 CGVNCTG 55 segment is shown in the NarG-H49C variant (A) that is unresolved in the structure of the NarG-H49S variant (B). FS0 and Mo-bisPGD are missing in the structure of the NarG-H49S mutant. A water molecule is coordinated by the NarG-Asp 222 residue that coordinates the molybdenum atom in the NarG-H49C and wild-type enzymes. A single iron atom is coordinated by the sulfur atoms of NarG-Cys 57 and NarG-Cys 92 . FIGURE 3. Unresolved residues in the NarG-H49S mutant. Residues comprising NarG-Ser 49 to NarG-Gly 55 are not observed in the NarG-H49S mutant. The location of these residues in the NarG-H49C mutant is shown in dark gray. The carbonyl oxygen of the NarG-Gly 50 amide is ϳ3.4 Å from the N-7 of the purine bicycle. The amine nitrogen of NarG-Asn 52 is 3.0 Å from a Q-pterin dithiolene sulfur and 3.2 Å from a P-pterin dithiolene sulfur. The carbonyl oxygen of NarG-Cys 53 is 3.6 Å from the N-10 secondary amine of the P-pterin.
C-8 of the P-pterin and 4.5 Å from the side chain sulfur of NarG-Cys 92 . This compares with 3.4 and 3.7 Å for the equivalent distances in wild-type NarGHI (Fig. 1). Although the importance of defined electron transfer conduits between ETR components has been proposed (54 -56), the presence of three potential pathways between FS0 and the Mo-bisPGD cofactor indicates that disruption of one is unlikely to significantly decrease intercenter electron transfer rates in the absence of other factors. The most likely candidate is a significant shift in the midpoint potential of FS0 that would render the FS0 3 Mo-bisPGD electron transfer step unfavorable. A similar shift could also explain the lack of enzyme activity in the NarG-H49C mutant.
EPR Analyses of the NarG Mutants-High-spin [4Fe-4S] clusters with an S ϭ 3/2 ground state exhibit EPR spectra around g ϭ 5.0 (57,58). Fig. 5 shows EPR spectra in this region of membrane samples enriched in wild-type and mutant NarGHI. The wild-type enzyme exhibits peaks at g ϭ 5.02 and g ϭ 5.55, as previously reported (18,28,59). No signals corresponding to a reduced high-spin [4Fe-4S] cluster were observed in either the NarG-H49C or the NarG-H49S variant. The NarG-R94S variant exhibited a modified high-spin spectrum with two almost merged peaks at g ϭ 5.25 and g ϭ 5.57. These features both exhibited temperature dependences similar to that of the g ϭ 5.55 feature of the wild-type spectrum (with peak signal intensities occurring at Ͻ5 K; data not shown), so it is likely that both arise from different subpopulations undergoing ⌬S ϭ 3/2 transitions. FS0 in the NarG-R94S mutant titrates with an E m of Ϫ170 mV, a decrease of 115 mV compared with its value in the wildtype enzyme (Fig. 6) (18). Given the overall lack of structural perturbation observed in the NarG-R94S mutant (Fig. 4), it is likely that the ⌬E m of Ϫ115 mV is elicited by removal of the  . EPR spectra of the reduced wild-type and mutant enzymes. EPR conditions were as follows: temperature, 9 K; microwave power, 20 milliwatts at 9.381 GHz; and modulation amplitude (peak to peak), 20 G pp at 100 KHz. Spectra are of membrane samples normalized to a protein concentration of ϳ30 mg ml Ϫ1 . WT, wild type.

FIGURE 6. Potentiometric titration of FS0 in the NarG-R94S variant.
Shown is a plot of the intensity of the FS0 signal in wild-type (WT) NarGHI (squares) and the NarG-R94S variant (triangles) versus E h . Data were fit to single n ϭ 1.0 components with E m values of Ϫ55 mV (wild-type) and Ϫ170 mV (NarG-R94S), respectively. positive charge of the Arg residue. Also, because two of the three putative electron transfer routes remain unaltered in the NarG-R94S variant, we conclude that its decreased enzyme activity results from the greater thermodynamic barrier presented by its FS0 cluster. This increases from 17.9 kJ mol Ϫ1 in the wild-type enzyme to 29.0 kJ mol Ϫ1 in the NarG-R94S mutant. Similar phenomena have been observed in other enzymes. For example, in E. coli succinate dehydrogenase (Sdh-CDAB), a mutant that decreases the E m of an intermediate ETR [4Fe-4S] (FS2) by ϳ125 mV (an SdhB-I150H mutant) also elicits a significant decrease in succinate:quinone oxidoreductase activity (60). Table 2 summarizes the effects of the mutants described herein on the Mo(IV/V/VI), FS0, and FS1 redox chemistry based on potentiometric titrations followed by EPR spectroscopy (18,61). No significant effects were elicited on the Mo(IV/V/VI) E m values in the NarG-H49C and NarG-R94S mutants. As previously reported by us (18,61), shifts in the E m values of FS0 (or the absence thereof) elicit a negative ⌬E m on FS1 ( Table 2).
The absence of an FS0 signal in the NarG-H49C mutant is surprising, because FS0 and the Mo-bisPGD cofactor are clearly visible in the structure (Fig. 2). Possible explanations for this are (i) that the cluster no longer has a high-spin ground state and (ii) that its E m is decreased to a value that prevents catalytic turnover. To address this question, we examined the potentiometric behavior of these two mutants at lower potentials. In the NarG-H49C mutant, we estimate that FS2 and FS3 have E m values of Ϫ420 and Ϫ80 mV, and in the NarG-H49S mutant, we estimate that FS2 and FS3 have potentials of Ϫ420 and Ϫ40 mV, respectively. Thus, the most likely explanation for the lack of activity in the NarG-H49C mutant is that the E m of FS0 is decreased below potentials readily accessible in potentiometric titrations (approximately Ϫ550 mV at pH 8.0). We therefore estimate that in a NarG-H49C mutant, the FS0 cluster undergoes a ⌬E m of at least Ϫ500 mV and that this is the explanation for the lack of enzyme activity in this mutant.
The increased thermodynamic barrier presented by FS0 explains the inability of the ETR to transfer electrons from quinol to nitrate. In and of itself, it does not explain the lack of activity with the soluble, nonspecific donor benzyl viologen. In the equivalent mutant of E. coli DmsABC (DmsA-R61S), quinol:Me 2 SO oxidoreductase activity is also eliminated, but activity with benzyl viologen as donor is retained (50). Comparison of the active site funnels of NarGHI and an enzyme related to DmsABC (Rhodobacter Me 2 SO reductase) reveals that the former enzyme has an unusually narrow active site funnel (4) and that this may prevent electron donation directly to the Mo-bisPGD cofactor.
Assembly of FS0 Is a Prerequisite for NarGHI Maturation-The data presented herein provide a structural facet to an emerging model for NarGHI maturation. Turner et al. (29,30) observed significant sequence similarity between the N terminus of NarG and that of the consensus tat leader. This group also demonstrated that NarJ interacts with a peptide comprising the first 50 amino acids of NarG in a tat-dependent manner (30,62). Phylogenic analyses have clearly indicated an evolutionary link between NarG and a family of enzymes that includes Rhodovulum sulfidophilum ethylbenzene dehydrogenase (EbdABC), Thauera selenatis selenate reductase (SerABC), and Ideonella dechloratans chlorate reductase (ClrABC). Each of these enzymes is directed to the periplasmic compartment by a tat leader (4,63), and as a group, they may represent a step in the evolution of NarGHI. The hypothesis that the N terminus of NarG is related to a tat leader is supported by the crystal structure of NarGHI, in which the first 40 amino acids of NarG extend away from the five domains of its core structure toward the membraneintrinsic domain of the enzyme (2). Sequence analyses (29,64) indicate that the tat recognition sequence, (S/T)RRXF(X/L)K, is replaced by the sequence 5 DRFRYFK 11 in NarG and that the first ϳ40 amino acids of NarG represent a tat pseudoleader. It is notable that NarG-Arg 6 interacts with the phosphate moiety of a phosphatidylglycerol in the structure, and it can be speculated that such an interaction occurs in other tat leaders prior to their cleavage from their respective holoenzyme precursors. Lanciano et al. (28) recently proposed a model in which FS0 and Mo-bisPGD insertion is coordinated with NarJ dissociation from the NarGH dimer during enzyme maturation. This model will require further clarification by mutagenesis of critical residues in the NarG tat pseudoleader, including NarG-Arg 6 . Crystallographic studies will also be necessary to assess the effects of a ⌬narJ mutant on the structure of the apoenzyme around the Mo-bisPGD/FS0-binding pocket. Such studies are in progress in our laboratories.
An additional unresolved question raised by the structures presented herein and elsewhere (18) is the ability of the apoenzyme to assemble GDP moieties into their respective P-and Q-binding sites. This raises the question of the possible role of NarJ in preventing GDP from competitively inhibiting holoenzyme maturation. Finally, although it is known that nucleotide addition occurs only after the proximal pterin has acquired a molybdenum atom (65), it is still not known whether the entire Mo-bisPGD cofactor is inserted into apomolybdoenzymes or whether the two halves are inserted sequentially with the loss of one of the molybdenum atoms (66). Future studies in our laboratory will resolve this issue by mutating residues surrounding the individual PGD-binding pockets.
Conclusions-Overall, we have generated structural data that indicate FS0 assembly into NarGHI is a prerequisite for Mo-bisPGD insertion. We have also demonstrated that a critical sequence of amino acids 49 HGVNCTG 55 is unresolved in the apomolybdo-NarG-H49SHI enzyme and may play a role in stabilizing Mo-bisPGD during maturation. The absence of FS0 does not prevent assembly of the holoenzyme to the cytoplasmic membrane. In NarG-H49CHI, the midpoint potential of FS0 is decreased to an extent that it is no longer able to redox-cycle in catalytic turnover. We validated the effect of decreasing FS0 E m on enzyme turnover by characterizing NarG-R94SHI, in which the potential of FS0 is decreased by 115 mV.