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• W2029198060 abstract "We have studied the effect of a mobABmutation 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 mobABmutant 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. We have studied the effect of a mobABmutation 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 mobABmutant 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. Escherichia coli, when grown anaerobically with nitrate as respiratory oxidant, develops a respiratory chain terminated by a membrane-bound quinol:nitrate oxidoreductase (NarGHI) 1The 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-β-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. (1Blasco F. Iobbi C. Giordano G. Chippaux M. Bonnefoy V. Mol. Gen. Genet. 1989; 218: 249-256Crossref PubMed Scopus (152) Google Scholar, 2Blasco F. Pommier J. Augier V. Chippaux M. Giordano G. Mol. Microbiol. 1992; 6: 221-230Crossref PubMed Scopus (73) Google Scholar, 3Berks B.C. Ferguson S.J. Moir J.W.B. Richardson D.J. Biochim. Biophys. Acta. 1995; 1232: 97-123Crossref PubMed Scopus (507) Google Scholar). 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 (4Weiner J.H. Rothery R.A. Sambasivarao D. Trieber C.A. Biochim. Biophys. Acta. 1992; 1102: 1-18Crossref PubMed Scopus (119) Google Scholar)), formate dehydrogenase N (FdnGHI (5Berg B.L. Li J. Heider J. Stewart V. J. Biol. Chem. 1991; 266: 22380-22385Abstract Full Text PDF PubMed Google Scholar)), and Wolinella succinogenes polysulfide reductase (PsrABC (6Krafft T. Bokranz M. Klimmek O. Schröder I. Fahrenholz F. Kojro E. Kröger A. Eur. J. Biochem. 1992; 206: 5456-5463Crossref Scopus (91) Google Scholar)). 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 (3Berks B.C. Ferguson S.J. Moir J.W.B. Richardson D.J. Biochim. Biophys. Acta. 1995; 1232: 97-123Crossref PubMed Scopus (507) Google Scholar, 4Weiner J.H. Rothery R.A. Sambasivarao D. Trieber C.A. Biochim. Biophys. Acta. 1992; 1102: 1-18Crossref PubMed Scopus (119) Google Scholar, 7Berks B.C. Richardson D.J. Reilly A. Willis A.C. Ferguson S.J. Biochem. J. 1995; 309: 983-992Crossref PubMed Scopus (128) Google Scholar). The structures of a number of bacterial molybdoenzymes have recently been determined. These include Me2SO reductases fromRhodobacter sphaeroides (8Schindelin H. Kisker C. Hilton J. Rajagopalan K.V. Rees D.C. Science. 1996; 272: 1615-1621Crossref PubMed Scopus (442) Google Scholar) and Rhodobacter capsulatus (9Schneider F. Löwe J. Huber R. Schindelin H. Kisker C. Knäben J. J. Mol. Biol. 1996; 263: 53-69Crossref PubMed Scopus (256) Google Scholar), and formate dehydrogenase H from E. coli (10Boyington J.C. Gladyshev V.N. Khangulov S.V. Stadtman T.C. Sun P.D. Science. 1997; 275: 1305-1308Crossref PubMed Scopus (497) Google Scholar). 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 (11Rajagopalan K.V. Neidhardt F.C. 2nd. Ed. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. ASM Press, Washington, D. C.1996: 674-679Google Scholar) is the addition of a guanine nucleotide to Mo-molybdopterin (Mo-MPT) to form Mo-MGD (12Johnson J.L. Indermaur L.W. Rajagopalan K.V. J. Biol. Chem. 1991; 266: 12140-12145Abstract Full Text PDF PubMed Google Scholar). In a mobABmutant, 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 (13Palmer T. Vasishta A. Whitty P.W. Boxer D. Eur. J. Biochem. 1994; 222: 687-692Crossref PubMed Scopus (48) Google Scholar), whereas the gene product of mobB, MobB, enhances nucleotide addition but is not essential for it (14Eaves D.J. Palmer T. Boxer D. Eur. J. Biochem. 1997; 246: 690-697Crossref PubMed Scopus (26) Google Scholar). The effect of mobAB mutants onE. coli NarGHI and DmsABC has been studied in detail (15Santini C. Iobbi-Nivol C. Romane C. Boxer D.H. Giordano G. J. Bacteriol. 1992; 174: 7934-7940Crossref PubMed Google Scholar,16Rothery R.A. Simala Grant J.L. Johnson J.L. Rajagopalan K.V. Weiner J.H. J. Bacteriol. 1995; 177: 2057-2063Crossref PubMed Google Scholar). Purified soluble NarGH dimer prepared from a mobABstrain 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 (13Palmer T. Vasishta A. Whitty P.W. Boxer D. Eur. J. Biochem. 1994; 222: 687-692Crossref PubMed Scopus (48) Google Scholar). 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 (13Palmer T. Vasishta A. Whitty P.W. Boxer D. Eur. J. Biochem. 1994; 222: 687-692Crossref PubMed Scopus (48) Google Scholar, 15Santini C. Iobbi-Nivol C. Romane C. Boxer D.H. Giordano G. J. Bacteriol. 1992; 174: 7934-7940Crossref PubMed Google Scholar). These results contrast with those reported for DmsABC, in which no MPT or molybdenum was detected in the enzyme studied in a mobAB background (16Rothery R.A. Simala Grant J.L. Johnson J.L. Rajagopalan K.V. Weiner J.H. J. Bacteriol. 1995; 177: 2057-2063Crossref PubMed Google Scholar). 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 (2Blasco F. Pommier J. Augier V. Chippaux M. Giordano G. Mol. Microbiol. 1992; 6: 221-230Crossref PubMed Scopus (73) Google Scholar, 17Palmer T. Santini C. Iobbi-Nivol C. Eaves D.J. Boxer D.H. Giordano G. Mol. Microbiol. 1996; 20: 875-884Crossref PubMed Scopus (146) Google Scholar). NarJ is a system-specific chaperone that is produced in substoichiometric amounts compared with the other subunits encoded by the operon (18Liu X. DeMoss J.A. J. Biol. Chem. 1997; 272: 24266-24271Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). It appears to hold cofactor-deficient NarGH in a cofactor-insertion competent conformation in E. colistrains that are unable to synthesize cofactor (19Blasco F. Dos-Santos J.P. Magalon A. Frixon C. Guigliarelli B. Santini C.L. Giordano G. Mol. Microbiol. 1998; (in press)Google Scholar). The NarGH dimer appears to be unstable and is devoid of Mo-MGD in a narJmutant 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 (20Augier V. Guigliarelli B. Asso M. Bertrand P. Frixon C. Giordano G. Chippaux M. Blasco F. Biochemistry. 1993; 32: 2013-2023Crossref PubMed Scopus (54) Google Scholar, 21Augier V. Asso M. Guigliarelli B. More C. Bertrand P. Santini C. Blasco F. Chippaux M. Giordano G. Biochemistry. 1993; 32: 5099-5108Crossref PubMed Scopus (40) Google Scholar, 22Guigliarelli B. Asso M. More C. Augier V. Blasco F. Pommier J. Giordano G. Bertrand P. Eur. J. Biochem. 1992; 207: 61-68Crossref PubMed Scopus (93) Google Scholar, 23Guigliarelli B. Magalon A. Asso M. Bertrand P. Frixon C. Giordano G. Blasco F. Biochemistry. 1996; 35: 4828-4836Crossref PubMed Scopus (89) Google Scholar, 24Magalon A. Rothery R.A. Giordano G. Blasco F. Weiner J.H. J. Bacteriol. 1997; 179: 5037-5045Crossref PubMed Google Scholar). 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 (3Berks B.C. Ferguson S.J. Moir J.W.B. Richardson D.J. Biochim. Biophys. Acta. 1995; 1232: 97-123Crossref PubMed Scopus (507) Google Scholar, 4Weiner J.H. Rothery R.A. Sambasivarao D. Trieber C.A. Biochim. Biophys. Acta. 1992; 1102: 1-18Crossref PubMed Scopus (119) Google Scholar). 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 (3Berks B.C. Ferguson S.J. Moir J.W.B. Richardson D.J. Biochim. Biophys. Acta. 1995; 1232: 97-123Crossref PubMed Scopus (507) Google Scholar, 25Breton J. Berks B.C. Reilly A. Thomson A.J. Ferguson S.J. Richardson D.J. FEBS Lett. 1994; 345: 76-80Crossref PubMed Scopus (56) Google Scholar). In the structure of formate dehydrogenase H (FdhF) (10Boyington J.C. Gladyshev V.N. Khangulov S.V. Stadtman T.C. Sun P.D. Science. 1997; 275: 1305-1308Crossref PubMed Scopus (497) Google Scholar), 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 pantotrophaperiplasmic nitrate reductase (NapA), a cluster was detected by EPR spectroscopy (10Boyington J.C. Gladyshev V.N. Khangulov S.V. Stadtman T.C. Sun P.D. Science. 1997; 275: 1305-1308Crossref PubMed Scopus (497) Google Scholar) 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 (26Trieber C.A. Rothery R.A. Weiner J.H. J. Biol. Chem. 1996; 271: 4620-4626Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). In both NarGHI and DmsABC, there is no evidence for the presence of a cluster within the catalytic subunit (22Guigliarelli B. Asso M. More C. Augier V. Blasco F. Pommier J. Giordano G. Bertrand P. Eur. J. Biochem. 1992; 207: 61-68Crossref PubMed Scopus (93) Google Scholar, 26Trieber C.A. Rothery R.A. Weiner J.H. J. Biol. Chem. 1996; 271: 4620-4626Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), and the enzymes both belong to a specific subclass. In this paper, we have investigated the effect of a mobABmutation 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. E. coli LCB79 (araD139Δ(lacIPOZYA-argF) rpsL thiφ79(nar-lac) StrepR) (27Pascal M.C. Burini J.F. Ratouchniak J. Chippaux M. Mol. Gen. Genet. 1982; 188: 103-106Crossref PubMed Scopus (45) Google Scholar) is a wild-type with respect to mobAB and was used to express fully functional NarGHI. E. coli TP1000 (F− ΔlacU169 araD139 rpsL150 relA1 ptsF rbsR flbB Δ(mobAB)StrepR) (17Palmer T. Santini C. Iobbi-Nivol C. Eaves D.J. Boxer D.H. Giordano G. Mol. Microbiol. 1996; 20: 875-884Crossref PubMed Scopus (146) Google Scholar) is a mobAB deletion mutant. NarGHI was overexpressed from pVA700 (23Guigliarelli B. Magalon A. Asso M. Bertrand P. Frixon C. Giordano G. Blasco F. Biochemistry. 1996; 35: 4828-4836Crossref PubMed Scopus (89) Google Scholar) 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 Δnar25(narG-narH) Δ(nar′U-narZ′) Ω KanR SpecRStrepR (28Blasco F. Nunzi F. Pommier J. Brasseur R. Chippaux M. Giordano G. Mol. Microbiol. 1992; 6: 209-219Crossref PubMed Scopus (39) Google Scholar)) transformed with pVA700 was used. Cells were grown microaerobically in 2-liter batch cultures of Terrific Broth (29Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.1989Google Scholar) at 30 °C in the presence of 100 μg ml−1 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 mmisopropyl-1-thio-β-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 (30Rothery R.A. Weiner J.H. Biochemistry. 1991; 30: 8296-8305Crossref PubMed Scopus (90) Google Scholar). Purified NarGH was prepared from wild-type and mobABgenetic backgrounds as described previously (20Augier V. Guigliarelli B. Asso M. Bertrand P. Frixon C. Giordano G. Chippaux M. Blasco F. Biochemistry. 1993; 32: 2013-2023Crossref PubMed Scopus (54) Google Scholar). Lyophilized NarGH was resuspended in 100 mm Tris and 5 mm EDTA (pH 8.3). 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” (12Johnson J.L. Indermaur L.W. Rajagopalan K.V. J. Biol. Chem. 1991; 266: 12140-12145Abstract Full Text PDF PubMed Google Scholar). 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. 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” (31Johnson J.L. Rajagopalan K.V. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 6856-6860Crossref PubMed Scopus (273) Google Scholar, 32Johnson J.L. Hainline B.E. Rajagopalan K.V. Arison B.H. J. Biol. Chem. 1984; 259: 5414-5422Abstract Full Text PDF PubMed Google Scholar). 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 mNH4OH in a fluorescence cuvette. Excitation and emission spectra were recorded as described in the legend to Fig. 1. 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 (20Augier V. Guigliarelli B. Asso M. Bertrand P. Frixon C. Giordano G. Chippaux M. Blasco F. Biochemistry. 1993; 32: 2013-2023Crossref PubMed Scopus (54) Google Scholar). 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 (33Markwell M.A.D. Haas S.M. Bieber L.L. Tolbert N.E. Anal. Biochem. 1978; 87: 206-210Crossref PubMed Scopus (5347) Google Scholar). Membrane vesicles were suspended at a protein concentration of 30 mg ml−1 in 100 mm MOPS and 5 mmEDTA (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 mmpotassium 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 titrations were carried out under argon at pH 8.0 in 100 mm Tricine and 5 mm EDTA as described previously (34Rothery R.A. Weiner J.H. Biochemistry. 1996; 35: 3247-3257Crossref PubMed Scopus (61) Google Scholar). 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. We have previously shown that in a mobABmutant, no detectable Mo-MPT is assembled into the DmsABC holoenzyme (16Rothery R.A. Simala Grant J.L. Johnson J.L. Rajagopalan K.V. Weiner J.H. J. Bacteriol. 1995; 177: 2057-2063Crossref PubMed Google Scholar). This result contrasts with reports that the NarGH dimer purified from a mobAB mutant contains Mo-MPT (13Palmer T. Vasishta A. Whitty P.W. Boxer D. Eur. J. Biochem. 1994; 222: 687-692Crossref PubMed Scopus (48) Google Scholar, 15Santini C. Iobbi-Nivol C. Romane C. Boxer D.H. Giordano G. J. Bacteriol. 1992; 174: 7934-7940Crossref PubMed Google Scholar). 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 (12Johnson J.L. Indermaur L.W. Rajagopalan K.V. J. Biol. Chem. 1991; 266: 12140-12145Abstract Full Text PDF PubMed Google Scholar). As a negative control for these experiments, we also studied Form A extracts from membranes from tungstate-grown cells. Fig. 1 A, 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 (12Johnson J.L. Indermaur L.W. Rajagopalan K.V. J. Biol. Chem. 1991; 266: 12140-12145Abstract Full Text PDF PubMed Google Scholar). Fig. 1 A, 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. 1 A, 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 (16Rothery R.A. Simala Grant J.L. Johnson J.L. Rajagopalan K.V. Weiner J.H. J. Bacteriol. 1995; 177: 2057-2063Crossref PubMed Google Scholar). Previous work has demonstrated reconstitution of BV⨥:nitrate oxidoreductase activity of the purified NarGH dimer from amobAB mutant strain in which the only source of Mo-MPT has been suggested to be the purified enzyme (13Palmer T. Vasishta A. Whitty P.W. Boxer D. Eur. J. Biochem. 1994; 222: 687-692Crossref PubMed Scopus (48) Google Scholar, 15Santini C. Iobbi-Nivol C. Romane C. Boxer D.H. Giordano G. J. Bacteriol. 1992; 174: 7934-7940Crossref PubMed Google Scholar). The lack of fluorescent derivatives obtained from the mobAB mutant membranes prompted us to reinvestigate the Mo-MPT content of NarGH purified from a mobAB mutant strain. Fig. 1 Bshows fluorescent spectra of Form B extracts from wild-type (Fig. 1 B, a and c) and mobABmutant (Fig. 1 B, b and d) NarGH. In spectra of the wild-type sample, intense peaks were observed in the excitation spectrum at approximately 290 and 410 nm. In the emission spectrum, an intense peak was observed at 480 nm. These wavelengths are consistent with the species released from the wild-type NarGH being the Form B derivative of Mo-MGD. In fluorescence spectra of the mobAB mutant NarGH extract (Fig. 1 B, b and d), the features of the fluorescence spectra are diminished, with a diffuse peak in the excitation spectrum at 365 nm and a small peak at approximately 475 nm in the emission spectrum. These results suggest that, in a mobAB mutant, Mo-MPT is either absent from NarGH or is present at very low occupancy (see “Discussion”). 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. 2 a 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 (24Magalon A. Rothery R.A. Giordano G. Blasco F. Weiner J.H. J. Bacteriol. 1997; 179: 5037-5045Crossref PubMed Google Scholar), and the spectrum in Fig. 2 a corresponds to this partially reduced form of the enzyme. Fig. 2 b shows an equivalent spectrum of mobABmutant NarGHI. This spectrum corresponds to that of the fully reduced enzyme reported by Guigliarelli et al. (22Guigliarelli B. Asso M. More C. Augier V. Blasco F. Pommier J. Giordano G. Bertrand P. Eur. J. Biochem. 1992; 207: 61-68Crossref PubMed Scopus (93) Google Scholar). 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. 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 (10Boyington J.C. Gladyshev V.N. Khangulov S.V. Stadtman T.C. Sun P.D. Science. 1997; 275: 1305-1308Crossref PubMed Scopus (497) Google Scholar); and (iii) the presence of a [4Fe-4S] cluster in NapA of T. pantotropha (25Breton J. Berks B.C. Reilly A. Thomson A.J. Ferguson S.J. Richardson D.J. FEBS Lett. 1994; 345: 76-80Crossref PubMed Scopus (56) Google Scholar). 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 atg = 2.02 (gz), and a peak-trough atg = 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. (22Guigliarelli B. Asso M. More C. Augier V. Blasco F. Pommier J. Giordano G. Bertrand P. Eur. J. Biochem. 1992; 207: 61-68Crossref PubMed Scopus (93) Google Scholar), 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 (22Guigliarelli B. Asso M. More C. Augier V. Blasco F. Pommier J. Giordano G. Bertrand P. Eur. J. Biochem. 1992; 207: 61-68Crossref PubMed Scopus (93) Google Scholar). The spectrum is also complicated by an overlapping Mo(V) signal at approximately g = 1.98. As the Eh is further reduced from +129 to +23 mV, the Mo(V) signal disappears, and the composite spectrum of the highest potential [4Fe-4S] cluster remains, revealing an additional peak atg = 2.01. Between −83 and −268 mV, there is a subtle increase in the intensity of the g = 2.05 peak and g = 1.89–1.87 peak-trough and a slight decrease in the intensity of the g = 1.95 peak-trough. Between −371 and −540 mV, the spectrum is extensively broadened by the interaction of the lowest potential [4Fe-4S] cluster with the other reduced clusters. This broadening is accompanied by the appearance of a deep trough at g = 1.92. Similar redox-poised spectra are obtained from TP1000/pVA700 (mobAB) membranes (Fig. 4), the notable exceptions being: (i) the absence of a Mo(V) signal atg = 1.98 in any of the spectra recorded; and (ii) at approximately +132 mV, there is an absence of the spectrum of the reduced highest potential [4Fe-4S] cluster, and a significant amount of oxidized [3Fe-4S] cluster remains. These results suggest that the absence of Mo-MGD in the mobAB mutant NarGHI affects the midpoint potentials of the two highest potential clusters of NarH. Redox-poised membranes from LCB79/pVA700 grown in the presence of 15 mm sodium tungstate to prevent Mo-MGD assembly yield essentially similar spectra to those reported for membranes from TP1000/pVA700 membranes (data not shown). Plots of the intensity of the g = 2.02 peak of the oxidized [3Fe-4S] cluster versus redox potential (Eh) (Fig. 5 A) indicate that in the wild-type and mobAB mutant enzymes, this center has two major subpopulations with" @default.
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• W2029198060 title "The Molybdenum Cofactor of Escherichia coli Nitrate Reductase A (NarGHI)" @default.
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