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EVIDENCE FOR nifU AND nifS PARTICIPATION IN THEBIOSYNTHESIS OF THE IRON-MOLYBDENUM COFACTOR OF

NITROGENASEDehua Zhao, Leonardo Curatti, and Luis M. Rubio*

From the Department of Plant and Microbial Biology, University of California-Berkeley,Berkeley, CA 94720

Running Title: Role of NifU and NifS in FeMo-co biosynthesis* Address correspondence to: Luis M. Rubio. From the Department of Plant and Microbial Biology,

University of California-Berkeley, Berkeley, CA 94720. Telephone: 1-510-643-3940. FAX: 1-510-642-4995. E-mail: lrubio@nature.berkeley.edu

The ni fU and n i f S genes encode thecomponents of a cellular machinery dedicatedto the assembly of [2Fe-2S] and [4Fe-4S]clusters required for growth under nitrogen-fixing conditions. The NifU and NifS proteinsare involved in the production of active formsof the nitrogenase component proteins, NifHand NifDK. While NifH contains a [4Fe-4S]cluster, the NifDK component carries twocomplex metalloclusters, the iron-molybdenumcofactor (FeMo-co) and the [8Fe-7S] P-cluster.FeMo-co, located at the active site of NifDK, iscomposed of 7 Fe, 9 S, 1 Mo, 1 unidentifiedlight atom, and homocitrate. To investigatewhether NifUS are required for FeMo-cobiosynthesis and to understand at what level(s)they might participate in this process, weanalyzed the effect of nifU and nifS mutationson the formation of active NifB protein and onthe accumulation of NifB-co, an isolatableintermediate of the FeMo-co biosyntheticpathway synthesized by the product of the nifBgene. The nifU and nifS genes were required toaccumulate NifB-co in a n i f N mutantbackground. This result clearly demonstratesthe participation of NifUS in NifB-co synthesisand suggests an specific role of NifUS as themajor provider of [Fe-S] clusters that serve asmetabolic substrates for the biosynthesis ofFeMo-co. Surprisingly, while nifB expressionwas attenuated in nifUS mutants, the assemblyof the [Fe-S] clusters of NifB was compensatedby other non-nif machinery for the assembly of[Fe-S] clusters, indicating that NifUS are notessential to synthesize active NifB.

The [Fe-S] clusters carried by the proteincomponents of the molybdenum nitrogenaseendow this enzyme with the ability to perform N2

fixation. The heterotetrameric NifDK proteincomponent (α2β2 dinitrogenase) contains the iron-molybdenum cofactor (FeMo-co) within the activesite in the α-subunit (NifD) and has the [8Fe-7S]P-cluster at the interface of the α- and β-subunits(1). The homodimeric NifH (dinitrogenasereductase) contains a [4Fe-4S] cubane and a sitefor Mg-ATP binding and hydrolysis (2). These Fe-S clusters of nitrogenase play a critical function inelectron transfer and in the reduction of substratesdriven by the free energy liberated from Mg-ATPhydrolysis (3). The [4Fe-4S] cluster carried byNifH is relatively ubiquitous in nature, but the P-cluster and FeMo-co are unique and regarded assome of the most complex metalloclusters knownin biology. FeMo-co is composed of 7 Fe, 9 S, 1Mo, 1 homocitrate, and 1 unidentified light atom(4-6).

A systematic genetic and biochemicalanalysis, mostly in Azotobacter vinelandii andKlebsiella pneumoniae, has revealed complex andspecialized cellular biosynthetic pathways for thematuration of the nitrogenase component proteins(see (7-9) for reviews). The products of thenitrogen fixation (nif) genes nifU and nifS arerequired to achieve full activity of bothnitrogenase component proteins. A. vinelandii nifUor nifS deletion mutants exhibited a 15-foldreduction in NifH activity and a 4-fold reductionin NifDK activity (10). Similar to A. vinelandii,n i fS mutants of K. pneumoniae exhibitednegligible NifH activity and a 25-fold reduction inNifDK activity (11). Since both nitrogenasecomponents are [Fe-S] proteins, it was promptlysuggested that NifU and NifS were involved in theformation of [Fe-S] clusters for NifH and NifDK(10). Later on, in vivo and in vitro experimentsdemonstrated that NifU and NifS were involved in

http://www.jbc.org/cgi/doi/10.1074/jbc.M708097200The latest version is at JBC Papers in Press. Published on October 24, 2007 as Manuscript M708097200

Copyright 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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the assembly of the [4Fe-4S] cluster of NifH (12-14).

A series of studies by Dean, Johnson, and co-workers showed that NifU and NifS work inconcert to synthesize [Fe-S] clusters undernitrogen fixing conditions and that their rolesrepresent a specialization of the roles performedby the homologous proteins IscU and IscS ingeneral [Fe-S] cluster assembly ((12) andreviewed in (15)). Each [Fe-S] cluster assemblymachinery minimally consists of a sulfur-providing cysteine desulfurase and a molecularscaffold where [2Fe-2S] or [4Fe-4S] clusters aretransiently assembled prior transfer to target apo-proteins. NifS is a pyridoxal phosphate (PLP)-containing enzyme that catalyzes thedesulfurization of L-cysteine to provide sulfur for[Fe-S] cluster formation (16,17), and NifU servesas the molecular scaffold for the NifS-directedassembly of [Fe-S] clusters (18,19). Indeed, invitro experiments showed that NifU could transfera [4Fe-4S] cluster to apo-NifH† and reconstitute anactive holo-NifH (14).

The involvement of NifU and NifS in theassembly of the P-cluster and the FeMo-coembedded within the NifDK protein is less clear.Strains lacking both nifU and nifS exhibited a 10-fold decrease in NifDK activity that could not berecovered by addition of FeMo-co (10).Complementation by FeMo-co addition is acharacteristic property of apo-NifDK containingthe P-clusters but lacking FeMo-co. Thephenotype of nifUS mutants thus suggests that theabsence of NifUS mostly impairs P-clustersynthesis, but does not clarify whether nifUSmutants are capable of synthesizing FeMo-co.

Other nif genes, nifB, nifE, nifH, nifN, nifQ,nifV, and nifX have been shown to be involved inthe biosynthesis of FeMo-co (7,8). The nifB geneencodes a SAM-radical protein required tosynthesize NifB-co, an [Fe-S] cluster of unknownstructure that serves as a biosynthetic intermediateduring the early steps of FeMo-co biosynthesis(20-22). Unlike the wild-type strain, nifN or nifEmutant strains accumulate a measurable amount ofNifB-co under nitrogen-fixing growing conditionsbecause the FeMo-co biosynthetic pathway isinterrupted at the level of NifB-co processing.NifB-co can be isolated from cytoplasmicmembranes of a K. pneumoniae nifN mutant strainby treatment with the detergent sarkosyl (20).

Radiolabelling experiments with 55Fe and 35Sisotopes have shown that Fe and S from NifB-coare transferred to FeMo-co during cofactorsynthesis in vitro (21). However, it is not knownwhether NifB-co is the only source of Fe and S toFeMo-co. NifB-co contains neither Mo nor anorganic acid, such as homocitrate (20).

A standing hypothesis has been that the simple[2Fe-2S] or [4Fe-4S] clusters assembled by NifUand NifS could serve as metabolic substratesduring the early steps of FeMo-co synthesis (i. e.the synthesis of NifB-co) (7-9). However, directexperimental evidence supporting this hypothesisis lacking. The impairment of nifU and nifS mutantstrains to synthesize active NifDK protein couldbe the result of cumulative effect over the activityof some of the [Fe-S] cluster-containing proteinsthat are involved in FeMo-co synthesis and NifDKmaturation, for example NifH, NifB or NifEN. Toaddress this question, we have investigated herethe effect of ni fU and nifS mutations on theaccumulation of NifB-co and on the activity of theNifB protein.

EXPERIMENTAL PROCEDURES

K. pneumoniae s trains and growthconditions—K. pneumoniae strains UN (wild-type) and UN1217 (nifN4536::mu) has beenpreviously described (23). Strains generatedduring the course of this study are described inSupplemental Table S1. Growth in minimalmedium, nif derepression, cell collection, and cellbreakage has been described (20). For growth onplates, LC medium (1% tryptone, 0.5% yeastextract, and 0.5% NaCl) was solidified withseparately autoclaved 1.5% agar solution.Kanamycin (50 µg/ml), spectinomycin (100µg/ml), chloramphenicol (17.5 µg/ml), andampicillin (25 µg/ml) were added as required. A.vinelandii strain UW45 (nifB mutant) (24) wascultivated in 20-l carboys and derepressed fornitrogenase expression as described before (25).Escherichia coli DH5α, BL21, and S17-1 strainswere grown in Luria-Bertani medium at 37˚C withshaking (200 rpm). For growth of E. coli on plates,medium solidified with 1.5% agar was used.Antibiotics were used at standard concentrations(26).

Plasmid cons truc t ions and DNAmanipulations—Plasmid constructions, PCR, and

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transformation of E. coli were carried out bystandard methods (26). Plasmids generated duringthe course of this work are listed in SupplementalTable S1. Isolation of genomic DNA from K.pneumoniae strains was carried out using theDNAeasyTM Tissue Kit (Qiagen). Procedures forK. pneumoniae transformation (27), conjugationand gene replacement (28) have been described.

Generation of K. pneumoniae nifU and nifSmutant strains—Mutations in ni fU or n i f Sconsisted of in-frame deletions spanning thecomplete amino acid coding sequences of nifU ornifS without altering any other DNA sequence inthe nif gene cluster (Fig. 1). These deletions wereconstructed on suicide plasmids to forcerecombination with the genomic DNA of K .pneumoniae UN1217 and to promote exchange ofgene alleles. Oligonucleotides were designed toamplify by PCR the nif DNA regions flankingnifU or nifS using K. pneumoniae genomic DNAas template. DNA fragments were amplified usingPfu DNA polymerase and ligated into thecorresponding restriction sites of plasmidpEP185.2 to construct pRHB288 (Δ ni fU ) ,pRHB289 (ΔnifS), and pRHB290 (Δ nifUS),respectively. In pRHB288 (ΔnifU), the 1.9-kbKpnI DNA fragment containing part of nifN andthe complete nifX was blunt-end ligated to the 2.5-kb PstI DNA fragment containing the nifSVWgenes. In pRHB289 (ΔnifS), the 2.73-kb KpnIDNA fragment containing part of nifN and thecomplete nifX and nifU genes was blunt-endligated to the 1.26-kb P s tI DNA fragmentcontaining the complete n i f V W genes. InpRHB290 (ΔnifUS), the 1.9-kb KpnI DNAfragment containing part of nifN and the completenifX gene was blunt-end ligated to the 1.8-kb NotIDNA fragment containing the complete nifVWZgenes. Plasmid pRHB291 was generated frompRHB290 by replacing the 630-bp DNA fragmentbetween two BamHI restriction sites within nifXand ni fV by the 1.2-kb kanamycin resistancecassette from pUC4K. Fidelity of all constructionswas confirmed by sequencing both DNA strands.

The nifU and nifS mutations were introducedinto the chromosome of strain UN1217 by allelicexchange events. First, UC0 was generated byconjugation of K. pneumoniae UN1217 with E.coli S17-1 (pRHB291) followed by selection of aKmr Cms phenotype. After isolating genomic

DNA from resulting Kmr Cms colonies,incorporation and segregation of mutant allele intothe chromosome was checked by PCR. Second,pRHB288 (ΔnifU), pRHB289 (Δ ni fS ), orpRHB290 (ΔnifUS) were transferred to strain UC0by conjugation to generate UC1, UC2, and UC3,respectively. Ampr Cmr clones with plasmidsintegrated into the chromosome by singlecrossover events were selected and confirmed byPCR analysis. Third, selected Ampr Cmr cloneswere continuously cultured in liquid LC mediumcontaining 25µg/ml ampicillin for more than 100generations to enrich for cells having the secondallelic exchange. Cultures were then diluted andplated onto solid LC medium containing 25 µg/mlampicillin, or 25 µg/ml ampicillin plus 50 µg/mlkanamycin. Kms Cms colonies (which had asecond allelic exchange event) were selected andthe deletions in nifU, nifS, or nifU and nifS wereconfirmed by PCR analysis (Fig. 1).

Genetic complementation of ΔnifU, ΔnifS, andΔnifUS mutant strains—To perform geneticcomplementation analysis of ΔnifU and ΔnifSmutants, strains UC1, UC2 and UC3 weretransformed with plasmid pRHB257 according to(27). Plasmid pRHB257 is a derivative of the lowcopy number plasmid pEXT21 that carries wild-type ni fUS genes. Plasmid pRHB257 wasgenerated by cloning a 2275-bp BamHI DNAfragment, which covers from the restrictionenzyme site at the 3’ end of nifX to the stop codonof nifS, into the BamHI and HindIII sites ofpEXT21 (Fig. 1).

Generation and expression of GST-NifB fusionproteins in K. pneumoniae— NifB fromK. pneumoniae was expressed as a glutathione-S-transferase (GST) fusion protein. The chimerawas constructed in the pRHB153 plasmid, aderivative of plasmid pGEX-4T-3 (GE Healthcare)(29). The nifB gene was PCR-amplified from thechromosome of K. pneumoniae UN1217 usingo l i g o n u c l e o t i d e s n i f B - N 1 5 ’ -CCCCATATGACTTCCTGCTCCTCTTTTTCTGG - 3 ’ a n d n i f B - C 1 5 ’ -GGGCTCGAGTCAGGCGACCCCCTTATGCG-3’ as primers. The nifB gene cartridge was thendigested with NdeI and BamHI and ligated into thecorresponding sites of plasmid pRHB153 togenerate plasmid pRHB233.

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Two strategies were used to express gst-nifB atdifferent cellular levels. First, to keep gst-nifBexpression at wild-type levels, the chromosomalcopy of nifB was replaced by a gst-nifB allele sothat expression was controlled by the natural nifBpromoter. A 1-kb DNA fragment containing thenifA gene, the nifB promoter (PnifB), and a XbaIrestriction site at the 5’ end, was PCR-amplifiedfrom the chromosome of K. pneumoniae UN1217using o l igonucleot ides n i fA-m1 5’ -CCCCTCTAGAATCGCCAACGCCATCCACCAT A A T - 3 ’ a n d n i f A - c 1 5’-GGTCGTACCTTCGTGGTTGGGC-3’ asprimers. In addition, a 2.1-kb DNA fragmentcontaining the gst-nifB gene and a SacI restrictionsite at the 3’end was PCR-amplified frompRHB233 using oligonucleotides RNF17 5’-ATGTCCCCTATACTAGGTTATTGGAAATTAA G - 3 ’ a n d n i f B - c 3 5 ’ -CCGAGCTCTCAGGCGACCCCCTTATGCGGCAA-3’ as primers. Both DNA fragments wereligated into the XbaI and SacI sites of the suicideplasmid pDS132, which carries a sacB gene (30),to generate plasmid pRHB292. Plasmid pRHB292was transferred to strains UN (wild-type), UN1217(ni fN ::mu), and UC3 (Δ nifUS nifN::mu) togenerate strains UC4, UC5, and UC8, respectively.After selecting Cmr clones, the integration ofpRHB292 into the chromosome was confirmed byPCR analysis. Clones with integrated pRHB292were cultured in liquid LC medium andsubsequently plated onto solid LB mediumsupplemented with 5% sucrose to select forplasmid excision in sucrose-resistant colonies.Substitution of gst-nifB for nifB was confirmed byPCR analysis of chromosomal DNA isolated fromstrains UC4, UC5 and UC8. Cells from K.pneumoniae UC5 and UC8 strains were grown,derepressed for nitrogenase, and collected bystandard procedures (20).

The second strategy aimed at boostingexpression of gst-nifB up to levels that facilitatedpurification of NifB. To achieve this, the wild-typecopy of nifB was removed from the chromosomeof UN1217 and the gst-nifB gene was expressedfrom plasmid pRHB233 so that expression wascontrolled by an IPTG-inducible Ptac promoter. A1-kb XbaI-EcoRI DNA fragment containing nifA,and a 1-kb EcoRI-SphI DNA fragment containingnifQ, were amplified from UN1217 genomic DNA

by PCR and ligated into the XbaI and SphI sites ofpDS132 to generate plasmid pRHB235 (ΔnifB).Plasmid pRHB235 was transferred to strains UN(wild-type), UN1217 (ni fN ::mu), and UC3(ΔnifUS nifN::mu) to generate strains UC9, UC10,and UC11, respectively, by a procedure analogousto that described above for gst-nifB replacement.Finally, plasmid pRHB233 was transferred toUC9, UC10, and UC11 mutant strains for GST-NifB expression under different geneticbackground generating strains UC16, UC17, andUC18, respectively. Cells from K. pneumoniaeUC16, UC17 and UC18 strains were subjected toITPG induction (5 µM IPTG) and nif-derepressionat the same time.

Generation of a K. pneumoniae nifENXmutant strain—A K. pneumoniae ΔnifENX mutantstrain (UC15) was generated. A nifTY-nifU DNAfragment having a complete deletion of the nifENXoperon deleted was first cloned in plasmidpDS132 and then introduced into the chromosomeof K. pneumoniae UN by allelic exchange togenerate UC15. A 986-bp nifTY DNA fragmentcarrying XbaI restriction site at the 5’ end andEcoRI restriction site at the 3’ end was amplifiedby PCR using primers n i f T -N2 (5’-CCCTCTAGATGCCCCGCGTCATGCGGCGGCA G - 3 ’ ) a n d n i f Y- C 2 ( 5 ’ -CCCGAATTCGAGCGTAACGTGGGGAAGAGCGTCC-3’). A 1053-bp nifU DNA fragmentcarrying EcoRI restriction site at the 5’ end and anSphI restriction site at the 3’ end was amplified byPCR us ing p r imers n i f U - p (5’-CCCGAATTCGATCCGGACCCGCGCCGCTAG C C - 3 ’ ) a n d n i f U - C 3 ( 5 ’ -CCCGCATGCTCAGGCCGCCACCACTTCCATATAA-3’). Both DNA fragments were digested bythe corresponding restriction enzymes and co-ligated into the XbaI and SphI sites of pDS132 togenerate plasmid pRHB294. Transfer of pRHB294into K. pneumoniae UN, clone selection, andsegregation of ΔnifENX mutation was performedusing plasmid pDS132 as described above.Deletion of nifENX genes from the chromosome ofUC15 was confirmed by PCR analysis. StrainUC15 did not exhibit nitrogenase activity in vivo,as expected.

Purification of GST-NifB from K. pneumoniaecells— GST-NifB proteins were purified fromcells of strains UC5, UC8, UC17, and UC18 by

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affinity chromatography using GSH-Sepharoseresin (GE Healthcare). For preparation of GST-NifB, twenty-five g of collected cell paste wereresuspended in 80 ml of 2x buffer A (10 mMsodium phosphate, 1.8 mM potassium phosphatebuffer, pH 8.5, 140 mM NaCl, 2.7 mM KCl, 10%glycerol, 5 mM β-mercaptoethanol, 0.02% DDM,0.2 mM PMSF, 0.5 µg/ml leupeptin, 5 µg/mlDNaseI, and 1 mM DTH). Cells were disrupted by10 cycles of sonication (1 min per cycle) using aFisher Sonic Dismembrator 550 equipped with 12mm tip at 25% power output inside an anaerobicglove box. After adjusting pH of lysate to pH 7.4,cell debris was removed by centrifugation at27,000 x g for 30 min in a Beckman Ti 50.2ultracentrifuge rotor. The clarified cell-free extract(supernatant) was applied onto a 5-ml GSH-Sepharose column. The column was then washedwith three column volumes of buffer Asupplemented with 1% Triton X-100 and 10column volumes of buffer A to removecontaminants. The GST-NifB was eluted from thecolumn applying three column volumes of bufferB (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10%glycerol, 5 mM β-mercaptoethanol, 0.02% DDM,0.2 mM phenylmethylsulfonyl fluoride, 0.5 µg/mlleupeptin, 10 mM reduced glutathione, and 1 mMDTH). Eluted GST-NifB protein was concentratedto 3-5 mg/ml inside an anaerobic glove box usingan Amicon cell equipped with YM100 Milliporeultrafiltration membranes. Purification of the GST-NifB fusion protein was accomplished within sixhours at 16°C. Purified NifB preparations weredrop-frozen and stored in liquid nitrogen.

In vivo and in vitro nitrogenase activities—Invivo nitrogenase activity was determined byethylene production at 30 °C for 30 min in 1-mlculture samples as previously described (31).NifDK activity in cell-free extracts was obtainedafter titration with an excess of the complementarycomponent, NifH, as described (32). Specificactivity is defined as nmol of ethylene formed permin/mg of protein in the extract.

In vitro FeMo-co-dependent or NifB-co-d e p e n d e n t a p o - N i f D K a c t i v a t i o nassays—Protocols for the isolation of FeMo-co(33), NifB-co (20) have been described.Preparation of crude NifB-co extracts was carriedout according to (20) with a modified cellbreakage procedure. K. pneumoniae cells in a 3 ml

suspension were broken inside an anaerobic glovebox by sonication for 2 minutes and 15% poweroutput using a Fisher Sonic Dismembrator 550equipped with a 3 mm tip. Crude NifB-co extractrefers to NifB-co solubilized from lysed cells of K.pneumoniae strains with Sarkosyl detergent (n-lauroyl sarcosine) but not purified through furtherchromatographic steps.

Assays for NifB-co dependent in vitroactivation of apo-NifDK present in extracts of A.vinelandii strain UW45 (nifB) were performed asdescribed in (20) with modifications. Nine-mlserum vials sealed with stoppers were repeatedlyevacuated and flushed with argon gas and rinsedwith 0.3 ml of anaerobic buffer. The completereactions contained: 100 µl of 25 mM Tris-HClbuffer pH 7.5, 10 µl of 1 mM Na2MoO4, 20 µl of 5mM homocitrate, 200 µl of ATP-regeneratingmixture (containing 3.6 mM ATP, 6.3 mM MgCl2,51 mM phosphocreatine, 20 units/ml creatinephosphokinase, and 6.3 mM DTH), 200 µl ofUW45 cell-free extracts (≈3 mg of protein), and50 µl of crude NifB-co extract from the K .pneumoniae strain being analyzed. The reactionswere incubated at 30°C for 35 min to allow for theFeMo-co synthesis and insertion reactions. Theresulting activation of apo-NifDK present inUW45 extract was analyzed by the acetylenereduction assay after adding 0.8 ml of ATP-regenerating mixture and an excess of purifiedNifH (0.2 mg of protein) (32).

FeMo-co precursor activity present in purifiedGST-NifB preparations was analyzed in the UW45crude extract–based assay as described aboveexcept that purified GST-NifB substituted forNifB-co in the reaction mixture.

FeMo-co insertion assays into apo-NifDKwere performed as described in (34).

Apo-NifDK activation assay with purifiedcomponents—FeMo-co synthesis and apo-NifDK§

activation assays with purified components wereset in 406 µ l reaction mixtures inside 9 mlanaerobic vials. Apo-NifDK reconstitution wasdependent on the NifB-co activity associated withpurified NifB protein and therefore no extra sourceof Fe, S or SAM was added to the assay. Two-hundred µl of ATP-regenerating mixture, 100 µlof 25 mM Tris-HCl pH 7.4, 20 µ l of 5 mMhomocitrate, and 10 µl of 1 mM Na2MoO4 weremixed and incubated at room temperature for 10

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min. Reactions were then initiated by adding thepurified protein components, namely precursor-free NifEN, NifH, apo-NifDK, and GST-NifB tofinal concentrations of 1.1 µM, 14.3 µM, 1.1 µM,and 4.5 µM, respectively. Precursor-free NifENwas purified from A. vinelandii strain UW243(29), NifH was purified from A. vinelandii strainDJ884, and apo-NifDK was purified from A .vinelandii strain DJ1143 (35). Additional 25 mMTris-HCl pH 7.4 buffer was added to bring thereaction volume to 406 µL when needed. Reactionmixtures were incubated for 35 min at 30°C in arotary shaker. The resulting activation of apo-NifDK present in the reaction mixtures wasroutinely analyzed by the acetylene reductionassay after addition of excess NifH and ATP-regenerating mixture (32).

SDS-PAGE, anoxic native-gel electrophoresis,and immunoblot analysis—The procedure forSDS-PAGE has been described (36). Immunoblotanalysis was performed as described by Brandneret al. (37). Purified preparations of K. pneumoniaeNifB were used to raise anti-NifB antibodies atCapralogics Inc, (Hardwick, MA). For anoxicnative-gel electrophoresis, proteins were separatedon gels with superimposed 7-20% acrylamide and0-20% sucrose gradients as described (29). Nativegels were then stained for proteins with CoomassieR-250 by standard procedures, or stained for ironas described in (38).

UV-Visible spectroscopy— The UV-visiblespectra were determined using a Shimadzu UV-1601 spectrophotometer.

Miscellaneous assays—Protein concentrationswere determined by the bicinchoninic acid methodusing BSA as standard (39). Fe content in purifiedGST-NifB preparations was determined accordingto (40).

RESULTS

K. pneumoniae nifU and nifS mutants fail toaccumulate NifB-co—Strain UN1217 carries amutation in nifN that impairs FeMo-co synthesisand results in the accumulation of NifB-co activity(20). NifB-co activity levels in K. pneumoniaestrains that combine mutations in ni fN withdeletions of nifU, nifS, or both nifU and nifS weredetermined and are presented in Table 1. NifB-coactivity levels in extracts of K. pneumoniae strainswere determined by using the NifB-co-dependent

FeMo-co synthesis and apo-NifDK activationassay (20). This assay is based on the biochemicalcomplementation of a cell-free extract froma ΔnifB A. vinelandii strain (UW45) with a samplecontaining NifB-co activity. In this assay, FeMo-co is synthesized in vitro and inserted into apo-NifDK present in the UW45 extract to reconstitutenitrogenase activity. Crude NifB-co preparationsfrom cell-free extracts of strains UC1 (ΔnifUnifN ::mu), UC2 (ΔnifS nifN ::mu), and UC3(ΔnifUS n i f N::mu) were unable to reconstituteNifDK activity, indicating that they did notaccumulate enough NifB-co to support NifB-co-dependent FeMo-co synthesis (Table 1).

Genetic complementation experimentsconfirmed that the lack of NifB-co activity inextracts of strains UC1, UC2, and UC3 wasspecifically due to mutations in nifU and nifSgenes. The nifU and nifS genes were cloned intothe low-copy plasmid pEXT21 under the controlof the n i fU promoter to generate plasmidpRHB257, which in turn was used to transformstrains UC1, UC2, and UC3 (Fig. 1). Table 1shows that NifB-co preparations from extracts ofstrains UC1, UC2 and UC3, carrying pRHB257,have the ability of support NifB-co-dependentapo-NifDK activation to levels similar to thoseobserved in strain UN1217, indicating that theexpression of ni fU and nifS from pRHB257restores the intracellular accumulation of NifB-co.

These results demonstrate that NifU and NifSwere required for the accumulation of NifB-co in anifN mutant background.

NifB overexpression partially overcomes theeffect of nifUS deletion on NifB-coaccumulation—Two strategies were used toexpress NifB at different levels and examine therelationship between NifB protein levels andNifB-co activity (Fig. 2). The first was tosubstitute gst-nifB for nifB in the chromosome ofK. pneumoniae while keeping wild-typeexpression levels (strains UC4, UC5 and UC8).This strategy allowed examination of GST-NifBand NifB-co levels under normal nif regulatoryconditions. The second was to delete nifB from thechromosome and introduce a plasmid carrying agst-nifB gene under an IPTG-inducible P t a cpromoter (strains UC16, UC17 and UC18). Thisstrategy uncouples nifB expression from niftranscriptional regulation. In addition, NifB

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expression is enhanced allowing purification andanalysis of purified GST-NifB from differentgenetic backgrounds (see below).

Strains UC4 (gst-nifB), UC5 (nifN::mu, gst-nifB) and UC8 (ΔnifUS nifN::mu, gst-nifB) weregenerated from the parental strains UN, UN1217and UC3, respectively (Fig. 2). Incorporation ofthe GST tag into NifB did not impair in vivonitrogenase activity in K. pneumoniae UC4 cells.K. pneumoniae strain UC4, which expresses GST-NifB protein in a wild-type genetic background,exhibited in vivo nitrogenase activity (943 nmolC2H4 formed/hour/OD600) very similar to that ofthe wild-type UN strain (817 nmol C2H4formed/hour/OD600).

Fig. 2B shows that strain UC8 accumulates10-fold less GST-NifB than strain UC5,suggesting an effect of ΔnifUS mutation in eitherexpression or stability of GST-NifB. In addition,UC8 cells were severely affected in theaccumulation of NifB-co, the metabolic product ofNifB activity. While UC5 cells exhibited similarNifB-co activity levels to those of the parentalstrain UN1217, NifB-co activity extracted fromUC8 cells was remarkably low and consistentlyundistinguishable from the background of theUW45 extracts used in that particular assay (Table2).

Several differences were apparent in strainUC17 in which GST-NifB expression was drivenby the tac promoter instead of the endogenous nifBpromoter. First, GST-expression (Fig. 2B) andNifB-co activity (Table 2) increased 10-fold and2-fold, respectively. The fact that UC17 cells overexpressing NifB contain higher levels of NifB-cosuggests that the NifB levels present in a wild typestrain could be a limiting factor for NifB-cosynthesis. Second, the ΔnifUS mutation did nothave a major effect in the in vivo levels of GST-NifB protein (compare GST-NifB levels in UC17versus UC18 to those in UC5 versus UC8), whichsuggests that the effect of the ΔnifUS mutation onGST-NifB levels observed in UC8 was mainly dueto changes in expression rather than in NifBstability. Third, in a ΔnifUS genetic background,UC18 cells over expressing GST-NifB exhibitedmore NifB-co activity than UC8 cells (Table 2),uncovering a NifUS-independent pathway that canpartially substitute for the function of NifUS whenNifB is overexpressed.

The products of nifU and nifS are not requiredto synthesize a functional NifB protein— T h efailure of strains UC1, UC2, and UC3 toaccumulate NifB-co in a nifN background led toseveral hypothetical scenarios: (i) it is possiblethat NifU and NifS directly provide the [Fe-S]cluster substrates for NifB activity to transforminto NifB-co, (ii) it is possible that NifU and NifSare involved in the synthesis of a functional NifBprotein, which itself is an [Fe-S] protein or, (iii) acombination of both scenarios. To discriminatebetween these possibilities, we compared the ironcontent, UV-visible absorption spectrum, andspecific activity of NifB protein purified from anifN mutant strain to those of NifB purified from amutant strain lacking nifN and nifUS.

GST-NifB proteins were purified from cells ofstrains UC17 (ΔnifB nifN::mu, Ptac-gst-nifB) andUC18 (ΔnifB ΔnifUS nifN::mu, Ptac-gst-nifB) thathad been derepressed for nitrogenase activity asdescribed in Experimental Procedures (Fig. 3A). Atypical purification procedure yielded 15 mg ofGST-NifB protein from 45 g of K. pneumoniaecells. The NifB purification yields from UC17 andUC18 cells were similar, what was consistent withthe similar levels of in vivo NifB expression inUC17 and UC18 strains, as determined byimmunoblot analysis (Fig. 2B), Hereafter, we referto NifB preparations as GST-NifB and ΔnifUSGST-NifB when purified from UC17 or UC18cells, respectively. Purified preparations of GST-NifB and ΔnifUS GST-NifB contained similaramounts of Fe (10.3 ± 1.1 mol Fe/mol GST-NifBmonomer compared to 8.7 ± 0.1 mol Fe/molΔnifUS GST-NifB monomer) and showed similarUV-visible absorption spectra (Fig. 3B),suggesting that nifU and nifS were not essential forthe synthesis of the [Fe-S] clusters of NifB. As aSAM-radical enzyme, each NifB monomer isexpected to contain at least one permanent [4Fe-4S] cluster coordinated by three cysteine residuesand a molecule of SAM. Thus, the Fe content ofpurified GST-NifB preparations would be enoughto account for the presence of the permanent [4Fe-4S] cluster and additional [Fe-S] cluster(s).

Purified GST-NifB was active in the in vitroFeMo-co synthesis assay in which GST-NifBsubstitutes for NifB-co. Titration of a FeMo-cosynthesis reaction based on UW45 cell-free extractwith purified GST-NifB shows that non-saturating

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concentrations of ΔnifUS GST-NifB provided 63 ±4 % precursor activity compared to the GST-NifBprotein (Fig. 4). This result suggests that the loadof FeMo-co precursor in purified ΔnifUS GST-NifB was less than in purified GST-NifB and isconsistent with the slightly lower Fe contentexhibited by ΔnifUS GST-NifB preparations. Bothtitrations saturated at similar activity levels atwhich another component of the reaction mixturebecomes limiting. The FeMo-co synthesis assaybased on UW45 extracts contains 3.2 mM DTH. Ithas been shown that neither NifS (41) nor NifU(19) are functional in the presence of 2 mM DTH.Thus, it is unlikely that an in vitro activity ofNifUS could load GST-NifB with a FeMo-coprecursor under the conditions of this assay.

To further rule out in vitro complementationof NifU and NifS present in an UW45 extract andconfirm that purified GST-NifB proteins carriedFeMo-co precursors, GST-NifB and ΔnifUS GST-NifB were assayed for in vitro FeMo-co synthesisin a reaction with purified components (Table 3).Both forms of GST-NifB proteins producedsimilar apo-NifDK activation levels in the assaywith purified components. Unlike what has beenreported for A. vinelandii purified NifB protein(22), the GST-NifB protein, as purified from K.pneumoniae cells, did not require addition of Fe,S, and SAM for in vitro activity. Thus, GST-NifBappeared to be loaded with FeMo-co precursoractivity (presumably NifB-co). In fact, addition of0.42 mM (NH4)2Fe(SO4)2, 0.42 mM Na2S, and0.88 mM SAM to these reaction mixtures resultedin a slight decrease in the level of reconstitutedNifDK activity (data not shown).

Accumulation of FeMo-co precursor on K.p n e u m o n i a e NifB was not caused by theinterruption of FeMo-co synthesis pathway in anifN::mu strain because GST-NifB purified from awild-type genetic background (strain UC16) alsocarried FeMo-co precursor activity (Table 3).

The above results can be summarized asfollows: (i) NifU and NifS are not required for thesynthesis of a functional SAM-radical NifBprotein, (ii) GST-NifB preparations purified froma ΔnifUS strain carry substantial amounts ofFeMo-co precursor (presumably NifB-co), and(iii) n i f U and nifS mutants do not synthesizeenough NifB-co as to observe its accumulation ina strain (nifN::mu) unable to process it into FeMo-

co. These results strongly support the hypothesisin which NifU and NifS are the main providers of[Fe-S] cluster substrates that are converted intoNifB-co by the activity of NifB. They also indicatethat in the absence of NifU and NifS, anothercellular [Fe-S] cluster assembly machinery is ableto support low levels of NifB-co synthesis.

R o l e o f N i f X i n N i f B - c oaccumulation—Although the NifB protein co-purifies with a small fraction of NifB-co activitypresent in cellular extracts of K. pneumoniaeUN1217 (nifN::mu), most of the NifB-co activitypool accumulates elsewhere in the cell, probablyin a protein-bound form. The main candidatewould be NifX, which is known to be a NifB-cobinding protein (42). The transfer of NifB-co fromNifB to NifX was tested in vitro and the results areshown in Fig. 5. Incubation of purified GST-NifBwith NifX followed by separation of both proteinsby anoxic native gel electrophoresis results in thetransfer of an entity containing Fe from NifB toNifX (Fig. 5, lanes 1-2). This Fe-containingmoiety presumably represents NifB-co because aNifX/NifB-co complex generated in vitro byincubating together purified samples NifX andNifB-co migrates to the same position in the gel(Fig. 5, lane 6). Importantly, NifX has been shownnot to bind free Fe (29). Purified ΔnifUS GST-NifB was also able to transfer Fe to NifX (Fig. 5,lanes 3-4), consistent with the presence of NifB-coactivity in ΔnifUS GST-NifB preparations. Thisresult shows that NifB-co can be transferred fromNifB to NifX in vitro and suggest this could alsobe the case in vivo.

To test whether the pool of NifB-co activityaccumulated in nif-derepressed UN1217 cells wasdependent on the presence of NifX, we analyzedthe accumulation of NifB-co activity in a K .pneumoniae ΔnifENX mutant strain (UC15). Table4 shows that strain UC15 exhibited similar levelsof NifB-co activity than strain UN1217, indicatingthat NifX was not essential for NifB-coaccumulation in vivo. In addition, the phenotype ofUC15 completely rules out the possibility thatNifB-co activity could be associated to theremaining NifE polypeptide of UN1217(nifN::mu). Thus, the majority of the NifB-co poolin strain UC15 and, probably, in UN1217 might bebound to an unidentified carrier other than theNifB, NifX, or NifEN proteins.

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DISCUSSION

Dean and co-workers first suggested that NifUand NifS were involved in the maturation orstability of both nitrogenase component proteinsbecause A. vinelandii nifU or nifS deletion mutantsexhibited a 15-fold reduction in NifH activity anda 4-fold reduction in NifDK activity (10). Similarto A. vinelandii, nifS mutants of K. pneumoniaeexhibited negligible NifH activity and a 25-foldreduction in NifDK activity (11). In vivo and invitro evidence have conclusively supported aspecific role of NifU and NifS in the assembly andincorporation of the [4Fe-4S] cluster into NifH(12-14).

The exact roles of NifU and NifS in theassembly of the complex [Fe-S] clusters of NifDKare less conclusive. Although their involvement inthe assembly of a functional NifDK protein hasbeen established from genetic experiments, findingthe specific step(s) at which NifUS participate inthis process has been particularly difficult. Manyof the proteins that participate in FeMo-cobiosynthesis, such as NifB, NifEN and NifH, are[Fe-S] proteins and an impairement in FeMo-cosynthesis could initially be attributed to a defect inany of those proteins. The only proteinconclusively shown to be involved in thebiosyntheses of the P-cluster and FeMo-co isNifH. Two lines of evidence are inconsistent withthe possibility that the effect of nifUS mutations onFeMo-co biosynthesis could be mediated by theinability to assemble the [4Fe-4S] cluster of NifH.First, it was shown that a [4Fe-4S] cluster-deficient form of NifH (apo-NifH) was fullycompetent for FeMo-co synthesis and apo-NifDKmaturation in vitro (43). Second, deletion of thernf gene clusters of A. vinelandii, which results indeficient incorporation of Fe into NifH and a 100-fold decrease in NifH activity, had no effect on thelevels of NifDK activity (44).

To investigate whether [Fe-S] clustersassembled by NifU and NifS could serve assubstrates for FeMo-co synthesis, we tookadvantage of the fact that a K. pneumoniaenifN::mu strain (UN1217) accumulates substantialamount of an isolatable FeMo-co precursor that issynthesized by the activity of NifB (NifB-co).Analyzing the effect of nifU and nifS mutations onthe accumulation of NifB-co and on the activity ofthe NifB protein was necessary to clarify the

participation of NifU and NifS in the early steps ofFeMo-co biosynthesis, because it excludes theeffects mediated by the activities of other [Fe-S]proteins involved in the pathway (e.g. NifEN,NifH, and apo-NifDK).

Two domains have been identified in NifB.The N-terminal domain has similarity to SAM-radical proteins and was proposed to contain a[4Fe-4S] center coordinated by three cysteineresidues and a molecule of SAM (22). The C-terminal domain is similar to NifX, and is sharedby a family of proteins that show FeMo-coprecursor binding activity (34). NifB proteinspurified from K. pneumoniae cells exhibit similarFe contents and UV-visible spectra regardless ofthe presence or absence of ni fUS. Thus, thefunction of NifUS in the assembly of (at least)some of the [Fe-S]-clusters of NifB can becompensated by other non-Nif machineries for theassembly of simple [Fe-S] clusters. The genome ofK. pneumoniae contains genes encoding thecomponents of ISC, SUF, and CSD bacterialsystems, all of which are known to participate inthe biosynthesis of [Fe-S] clusters.

Other functions of NifUS appeared not to becompensated by the additional [Fe-S] clusterassembly machineries, because a K. pneumoniaeΔnifUS n i f N : : m u mutant strain does notaccumulate NifB-co. The 10-fold decrease in NifBpolypeptides exhibited by this mutant wouldcontribute to its failure to accumulate NifB-co. Ageneral attenuation of accumulation of nif geneproducts in K. pneumoniae nifUS mutants has beenpreviously observed (11). The mechanismunderlying nifUS requirement for maximalexpression of nif genes is not understood. Tocircumvent this effect, nifB expression wasuncoupled from Nif regulation of transcription byusing an IPTG-inducible Ptac promoter. When nifBexpression was driven by the Ptac promoter, theabsence of nifUS had no effect on the levels ofNifB polypeptides. However, accumulation ofNifB-co activity was still low, suggesting that invivo NifB-co synthesis was ocurring at lower ratesin the absence of nifUS.

The data presented in this study suggest thatalthough a strain lacking n i f U S does notaccumulate significant levels of NifB-co, it has thecapability to assemble a functional NifB protein.The GST-NifB proteins purified from wild type orΔnifUS genetic backgrounds not only exhibited

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similar Fe contents and UV-vis spectra, but alsosimilar levels of associated NifB-co activity asdetermined by in vitro FeMo-co synthesis assays.In addition, both the GST-NifB and the ΔnifUSGST-NifB proteins seem to be able to transferNifB-co to NifX, as suggested by the transfer ofFe label between the two proteins determined byanoxic native-gel electrophoresis. However, theamount of NifB-co activity associated with NifBrepresents only a small fraction of the cellularNifB-co pool, which size was shown to be NifUS-dependent. The main reservoir of NifB-co does notinvolve the NifB-co binding proteins NifEN orNifX because a K. pneumoniae strain lacking thenifENX genes accumulated similar NifB-coactivity than UN1217. Whether NifB-co binds toanother unidentified NifB-co binding proteinremains uncertain.

Purified preparations of K. pneumoniae GST-NifB supported in vitro FeMo-co synthesiswithout a requirement for Fe, S, or SAM. Thisresult indicates that, as-isolated, K. pneumoniaeNifB carries a FeMo-co precursor (probably NifB-co). Unlike the purified preparations of K .pneumoniae GST-NifB, the preparations of “as-isolated” A. vinelandii His-NifB protein do notcontain NifB-co activity in any of the geneticbackgrounds analyzed so far (22,45) and requireaddition of Fe, S and SAM to support in vitroFeMo-co synthesis. In addition, GST-NifBpurified from K. pneumoniae contains 10 Fe atomsper monomer compared to 6 Fe atoms permonomer present in His-NifB purified from A.vinelandii. These differences between the “as-isolated” NifB proteins from K. pneumoniae andA. vinelandii could be caused by the different

purification procedures used, or it could actuallyreflect different properties of both enzymes.Interestingly, when chemically reconstituted,purifed A. vinelandii His-NifB proteinincorporates three extra Fe atoms per monomerand becomes active in vitro. Understanding thesedifferences might provide some insights into themechanism of the reaction catalyzed by NifB.

Taken together, the results of this work clearlydemonstrate the participation of NifU and NifS inNifB-co biosynthesis and hence in FeMo-cobiosynthesis. Two different interpretations of thephenotype of a ΔnifUS mutant strain and thespecific function(s) of NifUS in NifB-cobiosynthesis arise from the present study. First, theNifUS system is the major contributor of theoverall metabolic flux through NifB by providing[Fe-S] precursors for NifB-co synthesis. The lackof NifUS activities in ΔnifUS mutants can bemarginally compensated by the activity of any ofthe other general machineries for the assembly ofsimple [Fe-S] clusters present in K. pneumoniae(Fig. 6). Second, NifU and NifS would not berequired to assemble the [Fe-S] clusters that arehypothesized to become NifB-co by the SAMradical-dependent activity of NifB. Rather, NifUand NifS would be required to accumulate NifB-co after it has been synthesized by an unknownmechanism that does not involve NifEN or NifX.While the experimental evidence presented hereinstrongly supports the first interpretation and is inline with previous genetic information (10,11),identification and characterization of the putativeNifB-co accumulating mechanism would benecessary to support the second interpretation.

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KEY WORDSnitrogenase/nif/FeMo-co/iron-sulfur/molybdenum

ABBREVIATIONSFeMo-co, iron-molybdenum cofactor; NifB-co, NifB-cofactor; NifDK, MoFe protein or dinitrogenase;NifH, Fe protein or dinitrogenase reductase; nif, genes encoding proteins involved in nitrogen fixation;DTH, sodium dithionite; DDM, n-dodecyl-β-D-maltopyranoside; PMSF, phenylmethylsulfonyl fluoride;SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SAM, S-adenosyl methionine;Sarkosyl, n-lauroyl sarcosine.

ACKNOWLEDGEMENTSWe thank Professor Paul Ludden for helpful discussions and support. This work was supported byNIGMS, National Institutes of Health Grant 35332 (to P. W. L.).

FOOTNOTES† Apo-NifH refers to a form of NifH which [4Fe-4S] cluster has been removed by chelation.§ A form of apo-NifDK that contains the P-clusters but lacks FeMo-co (ΔnifB his-tagged apo-NifDK) wasused for these studies (35). When purified by metal-affinity and ion-exchange chromatography, this formof apo-NifDK does not contain the NafY subunit.

FIGURE LEGENDSFIGURE 1. Scheme showing the genetic organization of K. pneumoniae strains UN1217, UC0, UC1,UC2, and UC3 in the chromosomal region around nifU and nifS. Plasmid pRHB257, used for geneticcomplementation analysis, contains the nifU and nifS genes cloned into pEXT21.

FIGURE 2. GST-NifB protein levels in nifN::mu and nifN::mu ΔnifUS genetic backgrounds. A,scheme showing the wild-type chromosomal region around nifB and the genetic constructions to expressgst-nifB from the PnifB promoter in the chromosome (strains UC4, UC5 and UC8) or from the Ptacpromoter present in plasmid pRHB233 (strains UC16, UC17, and UC18). B, GST-NifB levels innitrogenase derepressed cells of UC5 (nifN::mu, gst-nifB), UC8 (nifN::mu ΔnifUS, gst-nifB), UC17(nifN::mu ΔnifB, Ptac-gst-nifB), and UC18 (nifN::mu ΔnifB ΔnifUS, Ptac-gst-nifB). Cells from nif-derepressed cultures were collected and boiled in SDS loading buffer. UC17 and UC18 cultures were nif-derepressed and IPTG-induced. To help comparing GST-NifB levels between strains, the amount of UC5and UC8 cells loaded in the gel was seven-fold higher than UC17 and UC18. After SDS-PAGE, proteinsin the gel were transferred to nitrocellulose membranes and GST-NifB was detected by immunoblotdeveloped with antibodies to NifB and quantified by densitometry.

FIGURE 3. Purification of GST-NifB proteins from K. pneumoniae cells. A, SDS-PAGE analysis ofGST-NifB as purified from strains UC17 and UC18. Lane 1, molecular weight markers; lane 2, GST-

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NifB (3.2 µg) purified from UC17 (nifN::mu ΔnifB, Ptac-gst-nifB); lane 3, ΔnifUS GST-NifB (3.2 µg)purified from UC18 (nifN::mu ΔnifB ΔnifUS, Ptac-gst-nifB). The lower arrow points to endogenous K.pneumoniae GST protein present in GST-NifB preparations. B, UV-visible spectra of GST-NifB and ΔnifUS GST-NifB proteins as isolated in 1 mM DTH. Excess DTH was removed by gel filtrationchromatography before recording the spectra. The A400 to A280

ratios were 0.19 for both GST-NifB andΔnifUS GST-NifB.

FIGURE 4. Activation of apo-NifDK present in UW45 (nifB) extracts with purified GST-NifBproteins. Purified GST-NifB or ΔnifUS GST-NifB proteins were used as FeMo-co precursor sources forFeMo-co synthesis and the activation of apo-NifDK present in UW45 extracts. Reaction details aredescribed in Experimental Procedures. UW45 extracts were titrated for apo-NifDK activation with 0.28-4.4 µM GST-NifB () or with 0.24-3.8 µM ΔnifUS GST-NifB (), respectively. Activation of apo-NifDK present in UW45 extracts with an amount of isolated NifB-co equivalent to 20 µM Fe resulted in12.9 ± 0.5 nmol ethylene formed per min/mg protein in the extract.

FIGURE 5. Anoxic native gel showing the transfer of Fe from GST-NifB to NifX. Samples frompurified GST-NifB and NifX preparations were incubated anaerobically on ice for 30 min and then loadedonto an anoxic native gel to separate the proteins. Lane 1, 0.4 nmol GST-NifB; lane 2, 0.4 nmol GST-NifB plus 1.7 nmol NifX; lane 3, 0.4 nmol ΔnifUS GST-NifB; lane 4, 0.4 nmol ΔnifUS GST-NifB plus1.7 nmol NifX; lane 5, 1.7 nmol NifX; lane 6, 1.7 nmol NifX plus sub-saturating amounts of NifB-co (4.5nmol Fe as NifB-co). The positions of GST-NifB in the native gel were determined by immunoblotanalysis developed with antibodies to NifB (data not shown). The protein band corresponding toendogenous K. pneumoniae GST protein is marked. A, gel stained with Coomassie to detect proteins. B,gel stained to detect Fe.

FIGURE 6. Model for [Fe-S] cluster flow during the early steps of FeMo-co biosynthesis. A, synthesisand fate of NifB-co in a K. pneumoniae strain lacking NifEN. B, residual NifB-co synthesis in a K.pneumoniae strain lacking NifUS. C, proposed role of NifU and NifS as [Fe-S] cluster substrate providersfor NifB-co/FeMo-co synthesis in a K. pneumoniae wild-type strain under nitrogen fixing conditions.

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Role of NifU and NifS in FeMo-co biosynthesis

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TABLE 1Determination of NifB-co activity in K. pneumoniae ΔnifU, ΔnifS, and ΔnifUS mutant strains

Details for the complete NifB-co dependent FeMo-co synthesis and apo-NifDK activation assay can befound at the “Experimental Procedures” section. The amount of apo-NifDK from UW45 extractsreconstituted in vitro was determined by measuring acetylene reduction activity of matured NifDK.Activation of apo-NifDK present in UW45 extracts with an excess of isolated FeMo-co gives 13 nmolethylene formed per min/mg protein in the extract.

Strain used as sourceof NifB-co

Genotype Activity a

UN1217 nifN::mu 13.6 ± 0.2UC1 ΔnifU nifN::mu 0.1 ± 0.0UC2 ΔnifS nifN::mu 0.1 ± 0.0UC3 ΔnifUS nifN::mu 0.1 ± 0.0UC1 (pRHB257) ΔnifU nifN::mu, pEXT21-nifUS 14.7 ± 1.2UC2 (pRHB257) ΔnifS nifN::mu, pEXT21-nifUS 10.1 ± 0.4UC3 (pRHB257) ΔnifUS nifN::mu, pEXT21-nifUS 13.7 ± 0.1UW45 nifB 0.1 ± 0.1

a Values are the averages of at least four assays performed separately. Specific activities are expressed asnmol ethylene formed per min/mg protein in UW45 extract.

TABLE 2NifB-co activity in K. pneumoniae nifN::mu and ΔnifUS nifN::mu strains expressing GST-NifB

Crude NifB-co extracts were prepared as described in “Experimental Procedures”. Reaction conditionswere the same as those in Table 1. Values are the averages of two to seven assays performed separately.Specific activities are expressed as nmol ethylene formed per min/mg protein in UW45 extract.Activation of apo-NifDK present in UW45 extracts with an excess of isolated NifB-co gives 12.9 ± 1.0nmol ethylene formed per min/mg protein in the extract.

Strain used as sourceof NifB-co

Genotype Activity

UC5 nifN::mu, gst-nifB 9.5 ± 2.0 (n=7)UC8 ΔnifUS nifN::mu, gst-nifB 0.3 ± 0.2 (n=6)UC17 ΔnifB nifN::mu, Ptac-gst-nifB 18.7 ± 1.0 (n=2)UC18 ΔnifB ΔnifUS nifN::mu, Ptac-gst-nifB 3.4 ± 0.1 (n=2)UW45 nifB 0.3 ± 0.1 (n=6)

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Zhao et al.

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TABLE 3GST-NifB activities in the in vitro FeMo-co synthesis system with purified components

Details for in vitro FeMo-co synthesis and apo-NifDK activation with purified components are describedin “Experimental Procedures”. Reaction mixtures contain 1.1 µM precursor-free NifEN, 14.3 µM NifH,1.1 µM apo-NifDK, and 4.5 µM GST-NifB. The amount of in vitro activated apo-NifDK was determinedby measuring its acetylene reduction activity after adding excess NifH. Values are the averages of at leasttwo assays performed separately. Specific activities are expressed as nmol ethylene formed per min permg of apo-NifDK.

Source of FeMo-co precursor ActivityGST-NifB (nifN::mu) 76 ± 1GST-NifB (ΔnifUS nifN::mu) 89 ± 5GST-NifB (wild-type) 107 ± 6No NifB added 0.3 ± 0.1NifB-co a 56 ± 4

a An amount of purified NifB-co equivalent to 27 µM Fe was added into the reaction mixture.

TABLE 4Comparison of NifB-co activity in K. pneumoniae nifN::mu and ΔnifENX strains

Crude NifB-co extracts were prepared as described in “Experimental Procedures”. Reaction conditionswere the same as those in Table 1. Values are the averages of two to four assays performed separately.Specific activities are expressed as nmol ethylene formed per min/mg protein in UW45 extract.

Strain used as source ofNifB-co

Activity

UN1217 (nifN::mu) 12.6 ± 0.5UC15 (ΔnifENX) 15.0 ± 1.1UW45 (nifB) 0.5 ± 0.1NifB-co a 13.2 ± 0.4

a An amount of purified NifB-co equivalent to 11 µM Fe was added into the reaction mixture.

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Figure 1

PnifUUC3nifE nifXnifN::mu nifV

PnifUUC1nifE nifX nifVnifN::mu nifS

PnifUpRHB257nifU nifS

UC0nifE Km nifV’nifX’nifN::mu

UN1217nifE nifX nifS nifVnifN::mu nifU

UC2nifE nifXnifN::mu nifVnifU

PnifU

PnifU

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Figure 2

PnifBnifA nifQgst-nifB

UC4UC5UC8

gst-nifB pRHB233

UC16UC17UC18

PnifBnifQnifA

Ptac

nifA nifB nifQ WTPnifB

A

UC5

UC1

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UC8

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GST-NifB

1 1.10.1 1.5

1 0.1 7.710.5

Relative amountof NifB in gel:

Relative level ofNifB in UC strains:

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7550

37

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100150250

15

MW

M

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UC1

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ifB)

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8 (∆

nifU

S N

ifB)

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Figure 3

B

Wavelength (nm)250 350 450 550 650 750 850

0.0

0.5

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2.0NifB∆nifUS NifB

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Figure 4

0 1 2 3 4 50.0

2.5

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NifB (µM)

(nm

ol C

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∆nifUS NifB

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Figure 5

A B

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1 2 3 4 5 6 1 2 3 4 5 6

GST

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Carrier

Figure 6

[Fe-S] [Fe-S] [Fe-S]

NifB-coNifB-coNifUS

[Fe-S]

NifB-co pool

NifB

Isc/Suf/Csd

NifXNifB-co

?

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[Fe-S] [Fe-S] [Fe-S]

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VK-cluster

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[Fe-S] [Fe-S] [Fe-S]

NifB-coNifB-co

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Dehua Zhao, Leonardo Curatti and Luis M. Rubiocofactor of nitrogenase

Evidence for nifU and nifS participation in the biosynthesis of the iron-molybdenum

published online October 24, 2007J. Biol. Chem. 

  10.1074/jbc.M708097200Access the most updated version of this article at doi:

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Supplemental material:

  http://www.jbc.org/content/suppl/2007/10/26/M708097200.DC1

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