Molecular Microbiology (1992) 6(2), 221-230
Involvement of the narJ or narW gene product in the formation of active nitrate reductase in Escherichia coii F. Blasco,'^ J. Pommier, V. Augier, M. Chippaux and G. Giordano Laboratoire de Chimie Bacterienne, CNRS, 31 chemin J. Aiguier, BP 71, 13277 Marseille, France. Summary Two membrane-bound nitrate reductases, NRA and NRZ, exist in Escherichia COIL Both isoenzymes are composed of three structural subunits, a, p and y encoded by narG/narZ, narH/narY and narl/narV, respectively. The genes are in transcription units which also contain a fourtii gene encoding a polypeptide, S, which is not part of the final enzyme. A strain which is devoid of, or does not express, the r}ar genes, was used to investigate the role of the 6 and y polypeptides in the formation and/or processing of the nitrate reductase. When only the a and [i potypeptides are produced, an (u|)) complex exists which is inactive and soluble. When the rx, p and 5 polypeptides are produced, the (a[i) complex is active with artificial donors such as benzyl viologen but is soluble. When the a, {^ and y polypeptides are produced, the ((x[5) complex is inactive but partially binds the membrane. It was concluded that the y polypeptide is involved in the binding of the (ap) complex to the membrane while the 6 polypeptide is indispensable for the (a[i) nitrate reductase activity. The activation by the 5 polypeptide does not seem to involve the insertion of the redox centres of the enzyme since the purified inactive (, complexes? Under anaerobic growth conditions, lactate or glycerol can be used as carbon source provided that their utilization is coupled to the presence of an active respiratory chain (Giordano etai, 1981; Wallace and Young, 1977). Therefore strains devoid of physiologically functional nitrate reductase cannot grow anaerobically in the presence of nitrate at the expense of these compounds. As reported in Table 2, only those strains in which all four a. P, Yand 6 polypeptides were produced, irrespective of the nar operons from which they were derived, expressed formate-dependent nitrate reduction and were able to grow anaerobically with glycerol in fhe presence of nitrate. Although a benzyl viologen-dependent nitrate reductase activity is present in ceils lacking the y polypeptide (Table 1), the activity was mainly in the soluble fraction and therefore could no longer be coupled to quinol oxidation In the membrane. Consequently, these cells cannot grow anaerobically on glycerol in the presence of nitrate.
Subunit composition and molecular weight When subjected to non-denaturing polyacrylamide gel electrophoresis and visualized with Coomassie Brilliant Blue, the purified inactive (UP)A complex migrated as a single band with an R^ value of 0.19, which is close to that of the active nitrate reductase enzyme (0.23). Furthermore, molecular filtration on BioGel A (0.5 M) indicated a relative molecular mass close to 200 000 (similar to that of solubilized active nitrate reductase). Finally, analysis of the subunit composition of this protein revealed the same aA ( H 150 000) and PA {M, 60 000) subunits as found for the active enzyme (Fig. 2. lanes 1 and 3). It is obvious from these results that the a and p polypeptides can associate in the absence of either the 5 and/or Y polypeptides. Presence of the molybdenum cofactor The molybdenum cofactor is very labile and is oxidized into stable products which exhibit characteristic fluorescence spectra. We have previously demonstrated that these derivatives can be extracted from purified molybdoenzymes by a heat treatment (100°C for 5 min) (Giordano
Table 2. Formate-nitrate reductase acti\nty and aerobic growth on glycerol-nltrate medium of strains transformed with various piasmids.
Strain/Plasmid
Produced nar polypeptides
Formate-nitrErte reductase activity
Growth on giycerol -nitrate medium (doubling time in mm)
(aP78)A
98
195
Characterization of the fap^^ complex present in strains lacking the 5 polypeptide
LCB 320 (parental strain) LCB 2048 LCB 2048 with: pFB76: pFB74 pFB76: pFB891 pFB76: pFB78 pFB76: pFB890 pFB76: pFB79 pFB76: pFB892
As demonstrated above, the absence of the 5 polypeptide resulted in an inactive (ap)A complex that was soluble or membrane-bound according to the absence or presence
Formate nitrate reductase activity from ceils grown anaerobically in the presence o! glucose (2 g I'') was measured as described by Blasco etal. (1992, accompanying paper). Activity is given as nmol nitrite formed min"' mg"^ dry weight.
None («P)A.(&y)A
(ap)A.(SY)z («P1A.(S)A («P)A.(8)Z («(i)A,(Y)A («P)A.(Y)Z
1-3 79 37 1-3 1-3 1-3 1-3
No o'owth
210 280 No growth No growth No growth No growth
Formation of active nitrate reductase in Escherichia coli Table 3. Purification of inactive (nPI^ complex from soluble extract of strain LCB 2048 carrying pFB76 and producing n^ and PA subunits.
Nitrate Reductase Immunoprecipitated
225
Stage of purification
Protein (mg)
mg protein
yield (%)
% of nitrate reductase
Soluble fraction 35% ammonium sulphate precipitation DEAE-Sepharose CL-6B Mono-QHIO/IOFPLC Superose12FPLC
7560 1120
550 300
100 54
7,3 26.7
219 110 61
158 102 61
29 18 11
72.1 92.7 99.6
This strain was grown anaerobically with nitrate. Purification was performed as described in the Experimental procedures. The amount of immunoprecipitated nitrate reductase was estimated by rocket immunoelectrophoresis and expressed in mg of prolein as in Table 1.
et ai. 1990). When the inactive (aP)^ complex was subjected to the same treatment, fluorescent material was obtained having excitation spectra maxima at 288 and 378 nm and an emission spectra maximum at 460 nm (not shown). These values are those expected for the molydenum cofactor derivatives extracted from purified moiybdoenzymes and thus indicate that the cofactor is present in the (aP)^ inactive complex. Further evidence for the presence of molybdenum cofactor in the purified complex was obtained from experiments using the Neurospora crassa niti complementation test for molybdenum cofactor (Hawkes and Bray, 1984). Because it lacks molybdenum cofactor, this mutant is devoid ot NADPH:nitrate oxidoreductase activity. This activity can be restored upon addition of exogenous molybdenum cofactor to a N. crassa niti crude extract preparation. As expected from the above-mentioned data, the heat-treated purified inactive (aP)A complex gave a positive result in the complementation test. Presence of non-haem iron and labile sulphur As for the native nitrate reductase enzyme, the purified inactive (aP) complex was brown, consistent with the presence of iron-sulphur centres. Atomic absorption analysis revealed, on average, 10 atoms of iron and 11 atoms of acid-labile sulphur per molecule (assuming a molecular weight of 200 000). These values are in the range of those currently reported by various authors for the purified nitrate reductases (Enoch and Lester, 1975; lobby-Nivolefa/. 1990). Protein which possess iron-sulphur centres gives difference spectra with a peak af about 400 nm (Guerlesquin et ai. 1980). The inactive (ap)A complex also gave such a peak, consistent with the iron and sulphur atoms being present as Fe-S centres (Blasco et ai, 1989:1990). Fragmentation by trypsin It is well documented that tryptic proteolysis of the purified
active nitrate reductase A leaves the a^ subunit unchanged but cleaves the PA subunit to give a PA' polypeptide of molecular weight 43 000 (DeMoss. 1977). Upon exposure to trypsin, the inactive (ap)A complex behaved differently. As shown on Fig. 2 (lane 4). both subunits were cleaved by the protease and were each almost totally degraded. This result was not unexpected since during the purification of the inactive (ap)A compiex we had noted the presence of partially degraded forms which were discarded. Discussion During our study of heterologous nitrate reductases, some of our data concerning the role of the 6 polypeptide encoded by narJ, the third gene of the narGHJI operon, were not in agreement with those reported in the literature (Sodergren et ai, 1988). We failed to find any nitrate reductase activity in strains lacking the 5 polypeptide. Therefore we reinvestigated the biochemical characteristics of the (ap)A complex to assess the influence of the presence or absence of 6 and/or y, the other two polypeptides encoded by fhe narGHJI operon. In order to follow quantitatively the (aP)A complex in cells, we employed an immunological technique. This choice was based on the rationale that this approach would quantify the complex, irrespective of its activity, and would be less misleading than the determination of nitrate reductase activity (as was previously used). Our data clearly indicate that (i) in all strains, the (ap)A complex is almost as abundant as in the control strain expressing all four genes, (ii) when only the a and p polypeptides are produced, an (aP)^ complex is present which is soluble and not active, (iii) when fhe a, (J and 5 polypeptides are produced, the (ap)A complex is soluble and is active with benzyl viologen as electron donor but is not physiologically functional, and (iv) when the a. p and y polypeptides are produced, the (ap)A complex is bound to the membrane in an inactive form regardless of the electron donor. In this last case, our results are in agreement with those reported by Sodergren et al. (1988). who unambiguously identified the y subunit to the Narf gene
226
F. Blasco etal
product and demonstrated that it is responsible for the attachment of the (aP)A complex to the membrane as was previously suggested (MacGregor, 1976). Our data further indicate, however, that the attachment also occurs when the complex is not active. When the a and p polypeptides are produced in fhe absence of the y and 5 polypeptides, our results clearly show that an inactive (ap)A complex is produced. This is not in agreement with a previous report (Sodergren etai, 1988) which reported a relatively strong nitrate reductase activity. In that system, the activity was not significantly enhanced by the concomitant production of the 5 polypeptide. In our hands, the inactive (ap)A complex is apparently formed solely by the a and p polypeptides but is 'activated' by the 3 polypeptide in such a way that its final activity is similar to that of the native complex. The reason for this difference probably arises from the strain used by Sodergren et ai (1988) in their experiments. Based on the presence of a TnTO inserted in the narJ gene, this strain should lack the nari gene product (y polypeptide) and be devoid of the narJ gene product (S polypeptide). However, the genotype of this strain was poorly documented and the localization of the transposon was inferred from Southern blotting experiments which indicated that it was lying 3' from a restriction site {SstW) present in the first half of the narJ gene. Since this strain presents a nitrate reductase activity, we suggest that it produced an active 8 polypeptide and that the transposon either is in the nari and not in the narJ gene or may actually be in narJ but at its very 3' end. which could result in a partially active 6" polypeptide. This latter proposition has a precedent in the narZYWV operon encoding nitrate reductase Z. Here, the elimination of the 3' end (from the Sal\ site) of the narWgene (homologous to narJ) results in the production of an (ap)^ complex which is active, suggesting that it produces an active Sz polypeptide (Blasco etai. 1990). How does the 8 polypeptide act on the inactive (ap)A complex so that it becomes active as a benzyl viologennitrate reductase? The active enzyme contains at least two different types of redox centres: molybdenum cofactor, and iron-sulphur centres (Johnson etai 1984; 1985). We have to discard the possibility that the 5 polypeptide inserts the molybdenum cofactor into the complex since both the direct determination of the pterin moiety and the niti complementation test show that the inactive (afi)A complex contains the cofactor. We also have eliminated the possibility that the iron-sulphur centres are not inserted in the (ap)A complex since (i) the iron content of the purified inactive complex is normal, and (ii) electron paramagnetic resonance (EPR) spectroscopic analysis confirmed that all four iron-sulphur centres are present in the inactive complex (P. Bertrand and B. Guigliarelli, personal communication).
Since all the redox centres known to be associated with the a and/or [5 subunits are present in the inactive complex, we must surmise that the action of the 8 polypeptide concerns some modification of the structure of the complex. This is supported by the fact that the patterns of tryptic proteolysis are radically different. The inactive (ap)A complex suffers a total degradation while the activated one is cleaved in a manner similar to that of the native complex. As trypsin cleaves polypeptides at the level of accessible lysine or arginine residues, it can be concluded that the access of trypsin to these residues is changed by the action of the 8 polypeptide. which probably modifies the overall conformation of the (aP)A complex. The role proposed for the 8 polypeptide strongly resembles that of a chaperone protein (Ellis, 1991) which, without being part of the final structure, controls the assembly of heteropolymeric complexes by lowering the probability of incorrect associations leading to structures devoid of biological function. The 8 polypeptide seems to fulfill all these conditions since, as discussed above, it plays a role in the final arrangement of the a and p subunits of the nitrate reductase without itself being part of fhe complex. However, two considerations must be taken into account which render this role unlikely for the 5 polypeptide. First, the peptide sequence of this protein does not resemble those of known chaperones. Second, it is generally considered for a given chaperone that it Is produced before. and independently of, the proteins the incorrect folding of which it is supposed to prevent. Such is not the case for the S polypeptide, which is encoded by the third gene of the narGHJi operon and is therefore expressed after both the a and p polypeptides. We have already mentioned the existence of active heterologous nitrate reduciase complexes composed of the a polypeptide encoded by one nar operon and of the p and y polypepfides encoded by the other nar operon (Blasco et al., 1992. accompanying paper). The present dafa extend this result. Heterologous active (ap)A complexes can be formed upon activation by the 5^ polypeptides and these complexes can be bound to the membrane provided that the yz polypeptide is present. It is worth noting that, whatever the role of the 5 polypeptide. the result of its action on the (aP)A complex does not depends upon the operon it comes from: with 8A or 6z, the complex is fully activated. Experimental procedures Strains The strains used in this work are listed in Table 4. Details of the construction of the double mutant strain LCB 2048 (nar'A:. miniMu,nar'Z::si) are given by Blasco et al. (1992) in the accompanying paper.
Formation of active nitrate reductase in Escherichia coli
227
Table 4. List of strains and piasmids. Plasmid/Strain
Description/Genotype
Sources
pBR322 derivative carrying r^arGHJl (NRA promoter) Ap" pBFI322 derivative carrying narZYWV{nHZ promoter) Ap" pJFi 19EH derivative carrying narGHJI (NRA promoter) Ap" pUCI 9 derivative carrying narGHJI (NRA promoter) Ap" pUCI 9 derivative, cloning vector (NRA promoter) Ap" pUCi 9 derivative carrying narJI (NRA promoter) Ap" pACYC184 derivative, cloning vector {NRA promoter) Cm" pACYCi84 derivative carrying narGH (NRA promoter) Cm'' pUCI 9 derivative carrying narJ (NRA promoter) Ap" pUCI 9 derivative carrying nari {NRA promoter) Ap'' pACYC184 derivative carrying narZV (NRA promoter) Cm" pUCI 9 derivative carryirig narW (NRA promoter) Ap" pUC19 derivative carrying na/'lW(NRA promoter) Ap" pUCi 9 derivative carrying narV(NRA promoter) Ap"
S. Rondeau V. Bonnefoy This work This work This work This work This work This work This work This work This work This work This work This work
dii'1, thr-i. leu-6, lacYI. supE44. rpsL175 LCB 320 with Anar2S(narG-narH). Km" LCB 333 with i[narV-narZ-),n (Spc")
M. Chippaux M. Chippaux F. Blasco
Piasmid pSR95 pLCB14 pVA70 pVA71 pFB73 pFB74 pFB75 pFB76 pFB78 pFB79 pFB87 pFB890 pFB891 pFB892 F. coli strain LCB 320 LCB 333 LCB 2048
Expressed nar genes and produced nar polypeptides LCB 2048: with pVA70 withpLCB14 with pFB76 with pFB87 with pFB74 with pFB891 with pFB76 and with pFe76 and with pFB76 and with pFB76 and with pFB87 and with pFB87 and witn pFB87 and with pFB76 and with pFB76 and
pFB74 pFB891 pFB892 pFB890 pFB892 pFB890 pFB74 pFB78 pFB79
narGHJUa^)/, narZYWV {a?>&y)z narGH {a^U narZY (0^)2 narJI (SY)A narWV{&i}z narGH: narJI (aP)A, (SY)A narGH: narWV{a?>U, (frf)z narGH] narV{a^)f^. (Y)Z narGH: narW(a^)f,. (8)z narZY: narV{a^)z. (yJz narZY. narW {ap)z. (8)z narZY; narjl {a^)z ,(&r)A narGH: narJ {ap)^. (5)A narGH: nari {a^)^, (y)^
This work TTiis work This work This work This work This work This work This work This work This work This work This work This work This work This work
)". ampicillin-resistant; Cm", chtoramphenicol-resistant; Km", kanamyctn-resistant; Spc", spectinomycin-resistant. /cin-resistant.
Piasmid constructior) In the piasmids listed above and described in Fig. 3. the nar genes are under the control of narGHJI promoter and are expressed in anaerobiosis and in the presence of nitrate. pVA70 was constructed by iigating the 7.5 kb Ssp\-Ssp\ fragment from pSR95 (Rondeau etai. 1984), containing all the narGHJI operon. into the Sma I site of pJF119 EH (Furste ef ai, 1986). pVA71 results from the same construction made in pUC19 (not shown). pFB73 was obtained by deleting the narG, narH. narJ and part of the nart genes from pVA71 by EcoRV digestion. The resulting piasmid contains the narGHJI promoter and a convenient fcoRV cloning site nearby. This site is used to clone the nargenes of interest under the narGHJ/promoter. pFB74 was constructed by deleting the 5 kb Sa/l-C/al fragment from pVA71 and recircularizlng. This piasmid only contains the YA a i d 5A subunit structural genes under the narGHJI promoter. pFB75 was obtained by inserting the 0amHI-H/ndlll fragment issued from pFB73 into the eamHI-H/ndlll sites of
pACYC184 (New England Biolabs) compatible with pBR322 and pUCi9 piasmids. Piasmid pFB75 is similar to pFB73 in that it allows the cloning of different genes under the narGHJI promoter in the EcoRV site. pFB76 was obtained by Iigating the 5.7 kb Sspl-SamHI fragment of pSR95 containing the narG and narH genes into the ecoRV-SamHI sites of pACYC184. pFB78 was obtained by cloning the 1.2 kb H/ndlll-XmnI fragment of pVA74 containing the narGHJ/promoter and narJ into pUCI 9 digested by Mndlll and Sma\. pFB79 was constructed by inserting the 1 kb Fsp\-EcoR\ fragment of pFB74 containing nar/into the EcoRV-EcoRI sites of pFB73. pFB87, a pACYCi84 derivative, was obtained by cloning the 5.4 kb XhoW-XhoW fragment from pLCB14, encoding the narZ and narVgenes into the EcoRV site of pFB75. pFB88 was obtained by inserting the Stu\-Fsp\ fragment encoding narlVand narV'genes, issued from pLCBI 4, into the EcoRV site of pBluescript SK* (Stratagene). pFB890 is a pUC19 derivative constructed by inserting the Smal-Hpal fragment of pFB88 carrying narWmXo the EcoRV site of pFB73.
228 F. e/asco etal. BamH 1 BanH I
Stpl
Sip I
I
pSR95 H
Sma I
EeoRl Kpn 1 /
B
J
1
S,p\
Bail Ftp]
EtoRV Smo. 1
pPB73
I
Smo I
Bat !
m.
pVA70
J
Hi/id i n
Hind in
pFB74
pFB75 Sip I Clal
5«p I
Sip I
EcaK V A'
Sma I
pFB76
pFB79 Ftp I Ami
Stul .Bwil iHpal
ScoRl
Fspl
1kb EeoRV
Hpa\
Kpnl
pPB88 BcaftV BtoRV
ScoKV
Sma\
Sma I Ecalt V
pFB891
pFB890 Sma I
Hpn I
S>p I
EeaRV
pFBS92 I ff V I Sip I Sma 1 Kpn t
Sat i
Sail
Fig. 3. Piasmids used in this study; the procedures for constnjcting piasmids from pSR95 and pLCBi4 are described in the Experimental procedures. The arrows indicate the directions of transcription of the operons. The nitrate reductase A promoter Is depicted by the black box. Hatched boxes represent the vectors used in these constructions (for details see the Experimental procedures and Table 4), The restriction sites are only indicated it relevant for cloning procedures which are explained in the Experimental procedures. Detailed restriction maps of pSR95 and pLCBi4 can be obtained from Bonnefoy et ai (1987). The locations of narK, narG. narH, narJ, nar/and nartl. narZ, narY. narWar\6 narUgenes are indicated by the open boxes and the corresponding letters.
pFB891 was constructed by inserting the Smal-Kpnl fragment coding for narlV and narV (issued from pFB88) into the ecoRV and Kpn\ sites of pFB73. pFB892 was obtained by cloning the blunt-ended Sa/I-Sa/I fragment carrying the narV^ gene issued from pFB88 into the EcoRV site of pFB73. Culture conditions, preparation of subcelluiar fractions, enzyme assays, limited proteclysis by trypsin, polyacryiamide gels and immunological analysis were as described by Blasco etai (1992) in the accompanying paper.
Purification of the inactive (ocpj^ complex The stages of the purification are listed in Table 3. Inactive (ap)A complex was detected using immunoelectrophoresis rocket technique (Graham et al.. 1980). All fractions containing
a and p subunits were analysed by Western blotting (Towbin et ai. 1979). Only fractions containing non-degraded a and [i subunits were retained. Wet cells (200 g) were suspended in 1200 ml of 50 mM TrisHCI, pH 7.6, containing 1 mM benzamidine-HCI. The crude extract (8860 mg of protein) obtained alter breakage of the cells was centrifuged twice at 120 000 x g for 90 min. The soluble fraction (7460 mg protein) which contains the (a^)^ compiex, was precipitated with ammonium sulphate at 35%, stirred for 30 min under nitrogen, and then centrifuged for 20 min at 18000 X g. The dark brown precipitate obtained was suspended in 200 ml of 50 mM Tris-HCI, pH 7.6, containing 1 mM benzamidine-HCI and dialysed overnight against 10 I of the same buffer. The dialysed material was centrifuged for 20 min at 40000 X g and the supernatant fraction was applied to a DEAE Sepharose CL-^6B column (150 x 3.5 cm) equilibrated in
Formation of active nitrate reductase in Escherichia coli the dialysis buffer; 1200 ml of a 0-400 mM linear NaCI gradient in the same buffer was applied- The fractions containing inactive nitrate reductase were eluted in the range of 0.25-0.28 M NaCI. Only fractions containing nitrate reductase with nondegraded a and [3 subunits were saved, dialysed, concentrated and purified by fast-protein liquid chromatography (FPLC) on a Mono-Q H10/10 ion exchange column equilibrated with 100 mM Tris-HCI, pH 7.6, 1 mM ben2amidine. Elution of samples (55 mg x 4) was effected by applying a 100-400 mM NaCI gradient in the same buffer. Fractions containing inactive nitrate reductase (61 mg of protein) were pooled, concentrated and eluted (27.5 mg x 4) on a Superosei 2 column. Purified enzyme was frozen in liquid nitrogen and kept at -80°C.
Iron content Iron was determined by atomic absotption analysis using a Varian AA 175 spectrometer. Inorganic sulphide was estimated by the method of Fogo and Popowsky (1949), as modified by Lovenberg etal. (1963).
Molybdenum cofactor isolation and identification Molybdenum cofactor associated with the (ap) complex was identified by complementation of the cofactor-deficient NADPH-dependent nitrate reductase produced by the nit1 mutant of N. crassa (Hawkes and Bray, 1984). Crude extracts of N. crassa were prepared as described by Amy and Rajagopalan (1979). Molybdenum cofactor and its derivatives were extracted by denaturing the enzyme (1 mg ml"') at lOO^C for 5 min under a nitrogen atmosphere and then rapidly cooling i1 to 0°C. Denatured proteins were removed by centrifugation at 15 600 X g for 2 min, and the supernatant fraction used for the reconstitution of N. crassa nitrate reductase activity in the complementation mixture. Fluorescence spectra of material released from purified nitrate reductase were obtained with a Kontron SFM 25 spectrofluorimeter. The heat-treated supernatant fraction was used for fluorescence spectroscopy as described by Johnson et ai. (1984). The excitation spectrum was obtained with emission at 455 nm, and the emission spectrum was obtained with excitation at 380 nm.
f\/tanipulation of DNA Restriction enzyme digestion, polymerase polishing, ligation, transformation and electrophoresis were essentialy adopted from Maniatis etal. (1982). Enzymes and chemicals were purchased from BRL and used as recommended by the suppliers.
Acknowledgements We wish to thank D. H. Boxer for critical reading of the manuscript, and F. Nunzi and M. C. Pascal for helpful discussion. We are grateful to J. M. Soulier and M. Denis for spectroscopic studies. This research was supported by the Centre National de la Recherche Scientifique and the Fondation pour la Recherche Medicale.
229
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F. Blasco e\a\.
lobbi-Nivol, C , Santini, C.L., Blasco, F., and Giordano, G. (1990) Purification and further characterization of the second nitrate reductase of Escherichia coli M 2. Eur J Biochem 188:679-687. Johnson, J.L., Hainline B.E.. and Rajagopalan, K.V. (1984) The ptehn component of the molybdenum cofactor. Structural characterization of the two fluorescent derivatives. J Biol Chem 259: 5414-5422. Johnson, M.K.. Bennett. D.E., Morningstar, J.E.. Adams. M.W.W., and Mortenson, L.E. (1985) The iron-sulfur composition of Escherichia coli Nitrate reductase. J Biol Chem 260: 5456-5463. Lovenberg, W.. Buchanan. B.B., and Rabinowitz, J.C. (1963) Studies on the chemical nature of Clostridial ferredoxin. J Biol Chem 238: 3899--3913. MacGregor, C.H. (1976) Biosynthesis of membrane-bound nitrate reductase in Escherichia coli: evidence for a soluble precursor. J Bacterioi 126:122-131. Maniatis,T., Frisch, E.F., and Sambrook, J, (1982) Moiecuiar Cloning. A Laboratory Manuai. Cold Spring Harbor. New York: Cold Spring Harbor Laboratory Press. Rondeau, S.S.. Hsu, P.Y., and DeMoss, J.A. (1984) Construc-
tion /n vitro of a cloned naroperon from Escherichia coli. J Bacterioi ^59: 159-166. Ruiz-Herrera, J., and DeMoss, J.A. (1969) Nitrate reductase complex of Escherichia coli K12: participation of specific formate dehydrogenase and cytochrome 6, components in nitrate reduction. JBacrer/o/99: 720-729. Sodergren. E.J., and DeMoss, J.A. (1988) nar/region of nilrate reductase {nat) operon contains two genes in Escherichia coli J Bacterioi ^70: 1721-1729. Sodergren. E.J., Hsu, P.Y., and DeMoss, J.A. (1988) Roles of the narJ and nari gene products in the expression of nitrate reductase in Escherichia coii. J Biol Chem 263: 16156-16162. Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications, Proc Nati Acad Sci USA 76: 4350-4356. Wallace. B.J., and Young, I.G. (1977) Role of quinones in electron transport to oxygen and nitrate in Escherichia coii. Studies with a ubia, menA double quinone mutant. Biochim S;dphys Acta 46-\:&4-: 00.
Involvement of the narJ or narW gene product in the formation of active nitrate reductase in Escherichia coii F. Blasco,'^ J. Pommier, V. Augier, M. Chippaux and G. Giordano Laboratoire de Chimie Bacterienne, CNRS, 31 chemin J. Aiguier, BP 71, 13277 Marseille, France. Summary Two membrane-bound nitrate reductases, NRA and NRZ, exist in Escherichia COIL Both isoenzymes are composed of three structural subunits, a, p and y encoded by narG/narZ, narH/narY and narl/narV, respectively. The genes are in transcription units which also contain a fourtii gene encoding a polypeptide, S, which is not part of the final enzyme. A strain which is devoid of, or does not express, the r}ar genes, was used to investigate the role of the 6 and y polypeptides in the formation and/or processing of the nitrate reductase. When only the a and [i potypeptides are produced, an (u|)) complex exists which is inactive and soluble. When the rx, p and 5 polypeptides are produced, the (a[i) complex is active with artificial donors such as benzyl viologen but is soluble. When the a, {^ and y polypeptides are produced, the ((x[5) complex is inactive but partially binds the membrane. It was concluded that the y polypeptide is involved in the binding of the (ap) complex to the membrane while the 6 polypeptide is indispensable for the (a[i) nitrate reductase activity. The activation by the 5 polypeptide does not seem to involve the insertion of the redox centres of the enzyme since the purified inactive (, complexes? Under anaerobic growth conditions, lactate or glycerol can be used as carbon source provided that their utilization is coupled to the presence of an active respiratory chain (Giordano etai, 1981; Wallace and Young, 1977). Therefore strains devoid of physiologically functional nitrate reductase cannot grow anaerobically in the presence of nitrate at the expense of these compounds. As reported in Table 2, only those strains in which all four a. P, Yand 6 polypeptides were produced, irrespective of the nar operons from which they were derived, expressed formate-dependent nitrate reduction and were able to grow anaerobically with glycerol in fhe presence of nitrate. Although a benzyl viologen-dependent nitrate reductase activity is present in ceils lacking the y polypeptide (Table 1), the activity was mainly in the soluble fraction and therefore could no longer be coupled to quinol oxidation In the membrane. Consequently, these cells cannot grow anaerobically on glycerol in the presence of nitrate.
Subunit composition and molecular weight When subjected to non-denaturing polyacrylamide gel electrophoresis and visualized with Coomassie Brilliant Blue, the purified inactive (UP)A complex migrated as a single band with an R^ value of 0.19, which is close to that of the active nitrate reductase enzyme (0.23). Furthermore, molecular filtration on BioGel A (0.5 M) indicated a relative molecular mass close to 200 000 (similar to that of solubilized active nitrate reductase). Finally, analysis of the subunit composition of this protein revealed the same aA ( H 150 000) and PA {M, 60 000) subunits as found for the active enzyme (Fig. 2. lanes 1 and 3). It is obvious from these results that the a and p polypeptides can associate in the absence of either the 5 and/or Y polypeptides. Presence of the molybdenum cofactor The molybdenum cofactor is very labile and is oxidized into stable products which exhibit characteristic fluorescence spectra. We have previously demonstrated that these derivatives can be extracted from purified molybdoenzymes by a heat treatment (100°C for 5 min) (Giordano
Table 2. Formate-nitrate reductase acti\nty and aerobic growth on glycerol-nltrate medium of strains transformed with various piasmids.
Strain/Plasmid
Produced nar polypeptides
Formate-nitrErte reductase activity
Growth on giycerol -nitrate medium (doubling time in mm)
(aP78)A
98
195
Characterization of the fap^^ complex present in strains lacking the 5 polypeptide
LCB 320 (parental strain) LCB 2048 LCB 2048 with: pFB76: pFB74 pFB76: pFB891 pFB76: pFB78 pFB76: pFB890 pFB76: pFB79 pFB76: pFB892
As demonstrated above, the absence of the 5 polypeptide resulted in an inactive (ap)A complex that was soluble or membrane-bound according to the absence or presence
Formate nitrate reductase activity from ceils grown anaerobically in the presence o! glucose (2 g I'') was measured as described by Blasco etal. (1992, accompanying paper). Activity is given as nmol nitrite formed min"' mg"^ dry weight.
None («P)A.(&y)A
(ap)A.(SY)z («P1A.(S)A («P)A.(8)Z («(i)A,(Y)A («P)A.(Y)Z
1-3 79 37 1-3 1-3 1-3 1-3
No o'owth
210 280 No growth No growth No growth No growth
Formation of active nitrate reductase in Escherichia coli Table 3. Purification of inactive (nPI^ complex from soluble extract of strain LCB 2048 carrying pFB76 and producing n^ and PA subunits.
Nitrate Reductase Immunoprecipitated
225
Stage of purification
Protein (mg)
mg protein
yield (%)
% of nitrate reductase
Soluble fraction 35% ammonium sulphate precipitation DEAE-Sepharose CL-6B Mono-QHIO/IOFPLC Superose12FPLC
7560 1120
550 300
100 54
7,3 26.7
219 110 61
158 102 61
29 18 11
72.1 92.7 99.6
This strain was grown anaerobically with nitrate. Purification was performed as described in the Experimental procedures. The amount of immunoprecipitated nitrate reductase was estimated by rocket immunoelectrophoresis and expressed in mg of prolein as in Table 1.
et ai. 1990). When the inactive (aP)^ complex was subjected to the same treatment, fluorescent material was obtained having excitation spectra maxima at 288 and 378 nm and an emission spectra maximum at 460 nm (not shown). These values are those expected for the molydenum cofactor derivatives extracted from purified moiybdoenzymes and thus indicate that the cofactor is present in the (aP)^ inactive complex. Further evidence for the presence of molybdenum cofactor in the purified complex was obtained from experiments using the Neurospora crassa niti complementation test for molybdenum cofactor (Hawkes and Bray, 1984). Because it lacks molybdenum cofactor, this mutant is devoid ot NADPH:nitrate oxidoreductase activity. This activity can be restored upon addition of exogenous molybdenum cofactor to a N. crassa niti crude extract preparation. As expected from the above-mentioned data, the heat-treated purified inactive (aP)A complex gave a positive result in the complementation test. Presence of non-haem iron and labile sulphur As for the native nitrate reductase enzyme, the purified inactive (aP) complex was brown, consistent with the presence of iron-sulphur centres. Atomic absorption analysis revealed, on average, 10 atoms of iron and 11 atoms of acid-labile sulphur per molecule (assuming a molecular weight of 200 000). These values are in the range of those currently reported by various authors for the purified nitrate reductases (Enoch and Lester, 1975; lobby-Nivolefa/. 1990). Protein which possess iron-sulphur centres gives difference spectra with a peak af about 400 nm (Guerlesquin et ai. 1980). The inactive (ap)A complex also gave such a peak, consistent with the iron and sulphur atoms being present as Fe-S centres (Blasco et ai, 1989:1990). Fragmentation by trypsin It is well documented that tryptic proteolysis of the purified
active nitrate reductase A leaves the a^ subunit unchanged but cleaves the PA subunit to give a PA' polypeptide of molecular weight 43 000 (DeMoss. 1977). Upon exposure to trypsin, the inactive (ap)A complex behaved differently. As shown on Fig. 2 (lane 4). both subunits were cleaved by the protease and were each almost totally degraded. This result was not unexpected since during the purification of the inactive (ap)A compiex we had noted the presence of partially degraded forms which were discarded. Discussion During our study of heterologous nitrate reductases, some of our data concerning the role of the 6 polypeptide encoded by narJ, the third gene of the narGHJI operon, were not in agreement with those reported in the literature (Sodergren et ai, 1988). We failed to find any nitrate reductase activity in strains lacking the 5 polypeptide. Therefore we reinvestigated the biochemical characteristics of the (ap)A complex to assess the influence of the presence or absence of 6 and/or y, the other two polypeptides encoded by fhe narGHJI operon. In order to follow quantitatively the (aP)A complex in cells, we employed an immunological technique. This choice was based on the rationale that this approach would quantify the complex, irrespective of its activity, and would be less misleading than the determination of nitrate reductase activity (as was previously used). Our data clearly indicate that (i) in all strains, the (ap)A complex is almost as abundant as in the control strain expressing all four genes, (ii) when only the a and p polypeptides are produced, an (aP)^ complex is present which is soluble and not active, (iii) when fhe a, (J and 5 polypeptides are produced, the (ap)A complex is soluble and is active with benzyl viologen as electron donor but is not physiologically functional, and (iv) when the a. p and y polypeptides are produced, the (ap)A complex is bound to the membrane in an inactive form regardless of the electron donor. In this last case, our results are in agreement with those reported by Sodergren et al. (1988). who unambiguously identified the y subunit to the Narf gene
226
F. Blasco etal
product and demonstrated that it is responsible for the attachment of the (aP)A complex to the membrane as was previously suggested (MacGregor, 1976). Our data further indicate, however, that the attachment also occurs when the complex is not active. When the a and p polypeptides are produced in fhe absence of the y and 5 polypeptides, our results clearly show that an inactive (ap)A complex is produced. This is not in agreement with a previous report (Sodergren etai, 1988) which reported a relatively strong nitrate reductase activity. In that system, the activity was not significantly enhanced by the concomitant production of the 5 polypeptide. In our hands, the inactive (ap)A complex is apparently formed solely by the a and p polypeptides but is 'activated' by the 3 polypeptide in such a way that its final activity is similar to that of the native complex. The reason for this difference probably arises from the strain used by Sodergren et ai (1988) in their experiments. Based on the presence of a TnTO inserted in the narJ gene, this strain should lack the nari gene product (y polypeptide) and be devoid of the narJ gene product (S polypeptide). However, the genotype of this strain was poorly documented and the localization of the transposon was inferred from Southern blotting experiments which indicated that it was lying 3' from a restriction site {SstW) present in the first half of the narJ gene. Since this strain presents a nitrate reductase activity, we suggest that it produced an active 8 polypeptide and that the transposon either is in the nari and not in the narJ gene or may actually be in narJ but at its very 3' end. which could result in a partially active 6" polypeptide. This latter proposition has a precedent in the narZYWV operon encoding nitrate reductase Z. Here, the elimination of the 3' end (from the Sal\ site) of the narWgene (homologous to narJ) results in the production of an (ap)^ complex which is active, suggesting that it produces an active Sz polypeptide (Blasco etai. 1990). How does the 8 polypeptide act on the inactive (ap)A complex so that it becomes active as a benzyl viologennitrate reductase? The active enzyme contains at least two different types of redox centres: molybdenum cofactor, and iron-sulphur centres (Johnson etai 1984; 1985). We have to discard the possibility that the 5 polypeptide inserts the molybdenum cofactor into the complex since both the direct determination of the pterin moiety and the niti complementation test show that the inactive (afi)A complex contains the cofactor. We also have eliminated the possibility that the iron-sulphur centres are not inserted in the (ap)A complex since (i) the iron content of the purified inactive complex is normal, and (ii) electron paramagnetic resonance (EPR) spectroscopic analysis confirmed that all four iron-sulphur centres are present in the inactive complex (P. Bertrand and B. Guigliarelli, personal communication).
Since all the redox centres known to be associated with the a and/or [5 subunits are present in the inactive complex, we must surmise that the action of the 8 polypeptide concerns some modification of the structure of the complex. This is supported by the fact that the patterns of tryptic proteolysis are radically different. The inactive (ap)A complex suffers a total degradation while the activated one is cleaved in a manner similar to that of the native complex. As trypsin cleaves polypeptides at the level of accessible lysine or arginine residues, it can be concluded that the access of trypsin to these residues is changed by the action of the 8 polypeptide. which probably modifies the overall conformation of the (aP)A complex. The role proposed for the 8 polypeptide strongly resembles that of a chaperone protein (Ellis, 1991) which, without being part of the final structure, controls the assembly of heteropolymeric complexes by lowering the probability of incorrect associations leading to structures devoid of biological function. The 8 polypeptide seems to fulfill all these conditions since, as discussed above, it plays a role in the final arrangement of the a and p subunits of the nitrate reductase without itself being part of fhe complex. However, two considerations must be taken into account which render this role unlikely for the 5 polypeptide. First, the peptide sequence of this protein does not resemble those of known chaperones. Second, it is generally considered for a given chaperone that it Is produced before. and independently of, the proteins the incorrect folding of which it is supposed to prevent. Such is not the case for the S polypeptide, which is encoded by the third gene of the narGHJi operon and is therefore expressed after both the a and p polypeptides. We have already mentioned the existence of active heterologous nitrate reduciase complexes composed of the a polypeptide encoded by one nar operon and of the p and y polypepfides encoded by the other nar operon (Blasco et al., 1992. accompanying paper). The present dafa extend this result. Heterologous active (ap)A complexes can be formed upon activation by the 5^ polypeptides and these complexes can be bound to the membrane provided that the yz polypeptide is present. It is worth noting that, whatever the role of the 5 polypeptide. the result of its action on the (aP)A complex does not depends upon the operon it comes from: with 8A or 6z, the complex is fully activated. Experimental procedures Strains The strains used in this work are listed in Table 4. Details of the construction of the double mutant strain LCB 2048 (nar'A:. miniMu,nar'Z::si) are given by Blasco et al. (1992) in the accompanying paper.
Formation of active nitrate reductase in Escherichia coli
227
Table 4. List of strains and piasmids. Plasmid/Strain
Description/Genotype
Sources
pBR322 derivative carrying r^arGHJl (NRA promoter) Ap" pBFI322 derivative carrying narZYWV{nHZ promoter) Ap" pJFi 19EH derivative carrying narGHJI (NRA promoter) Ap" pUCI 9 derivative carrying narGHJI (NRA promoter) Ap" pUCI 9 derivative, cloning vector (NRA promoter) Ap" pUCi 9 derivative carrying narJI (NRA promoter) Ap" pACYC184 derivative, cloning vector {NRA promoter) Cm" pACYCi84 derivative carrying narGH (NRA promoter) Cm'' pUCI 9 derivative carrying narJ (NRA promoter) Ap" pUCI 9 derivative carrying nari {NRA promoter) Ap'' pACYC184 derivative carrying narZV (NRA promoter) Cm" pUCI 9 derivative carryirig narW (NRA promoter) Ap" pUC19 derivative carrying na/'lW(NRA promoter) Ap" pUCi 9 derivative carrying narV(NRA promoter) Ap"
S. Rondeau V. Bonnefoy This work This work This work This work This work This work This work This work This work This work This work This work
dii'1, thr-i. leu-6, lacYI. supE44. rpsL175 LCB 320 with Anar2S(narG-narH). Km" LCB 333 with i[narV-narZ-),n (Spc")
M. Chippaux M. Chippaux F. Blasco
Piasmid pSR95 pLCB14 pVA70 pVA71 pFB73 pFB74 pFB75 pFB76 pFB78 pFB79 pFB87 pFB890 pFB891 pFB892 F. coli strain LCB 320 LCB 333 LCB 2048
Expressed nar genes and produced nar polypeptides LCB 2048: with pVA70 withpLCB14 with pFB76 with pFB87 with pFB74 with pFB891 with pFB76 and with pFe76 and with pFB76 and with pFB76 and with pFB87 and with pFB87 and witn pFB87 and with pFB76 and with pFB76 and
pFB74 pFB891 pFB892 pFB890 pFB892 pFB890 pFB74 pFB78 pFB79
narGHJUa^)/, narZYWV {a?>&y)z narGH {a^U narZY (0^)2 narJI (SY)A narWV{&i}z narGH: narJI (aP)A, (SY)A narGH: narWV{a?>U, (frf)z narGH] narV{a^)f^. (Y)Z narGH: narW(a^)f,. (8)z narZY: narV{a^)z. (yJz narZY. narW {ap)z. (8)z narZY; narjl {a^)z ,(&r)A narGH: narJ {ap)^. (5)A narGH: nari {a^)^, (y)^
This work TTiis work This work This work This work This work This work This work This work This work This work This work This work This work This work
)". ampicillin-resistant; Cm", chtoramphenicol-resistant; Km", kanamyctn-resistant; Spc", spectinomycin-resistant. /cin-resistant.
Piasmid constructior) In the piasmids listed above and described in Fig. 3. the nar genes are under the control of narGHJI promoter and are expressed in anaerobiosis and in the presence of nitrate. pVA70 was constructed by iigating the 7.5 kb Ssp\-Ssp\ fragment from pSR95 (Rondeau etai. 1984), containing all the narGHJI operon. into the Sma I site of pJF119 EH (Furste ef ai, 1986). pVA71 results from the same construction made in pUC19 (not shown). pFB73 was obtained by deleting the narG, narH. narJ and part of the nart genes from pVA71 by EcoRV digestion. The resulting piasmid contains the narGHJI promoter and a convenient fcoRV cloning site nearby. This site is used to clone the nargenes of interest under the narGHJ/promoter. pFB74 was constructed by deleting the 5 kb Sa/l-C/al fragment from pVA71 and recircularizlng. This piasmid only contains the YA a i d 5A subunit structural genes under the narGHJI promoter. pFB75 was obtained by inserting the 0amHI-H/ndlll fragment issued from pFB73 into the eamHI-H/ndlll sites of
pACYC184 (New England Biolabs) compatible with pBR322 and pUCi9 piasmids. Piasmid pFB75 is similar to pFB73 in that it allows the cloning of different genes under the narGHJI promoter in the EcoRV site. pFB76 was obtained by Iigating the 5.7 kb Sspl-SamHI fragment of pSR95 containing the narG and narH genes into the ecoRV-SamHI sites of pACYC184. pFB78 was obtained by cloning the 1.2 kb H/ndlll-XmnI fragment of pVA74 containing the narGHJ/promoter and narJ into pUCI 9 digested by Mndlll and Sma\. pFB79 was constructed by inserting the 1 kb Fsp\-EcoR\ fragment of pFB74 containing nar/into the EcoRV-EcoRI sites of pFB73. pFB87, a pACYCi84 derivative, was obtained by cloning the 5.4 kb XhoW-XhoW fragment from pLCB14, encoding the narZ and narVgenes into the EcoRV site of pFB75. pFB88 was obtained by inserting the Stu\-Fsp\ fragment encoding narlVand narV'genes, issued from pLCBI 4, into the EcoRV site of pBluescript SK* (Stratagene). pFB890 is a pUC19 derivative constructed by inserting the Smal-Hpal fragment of pFB88 carrying narWmXo the EcoRV site of pFB73.
228 F. e/asco etal. BamH 1 BanH I
Stpl
Sip I
I
pSR95 H
Sma I
EeoRl Kpn 1 /
B
J
1
S,p\
Bail Ftp]
EtoRV Smo. 1
pPB73
I
Smo I
Bat !
m.
pVA70
J
Hi/id i n
Hind in
pFB74
pFB75 Sip I Clal
5«p I
Sip I
EcaK V A'
Sma I
pFB76
pFB79 Ftp I Ami
Stul .Bwil iHpal
ScoRl
Fspl
1kb EeoRV
Hpa\
Kpnl
pPB88 BcaftV BtoRV
ScoKV
Sma\
Sma I Ecalt V
pFB891
pFB890 Sma I
Hpn I
S>p I
EeaRV
pFBS92 I ff V I Sip I Sma 1 Kpn t
Sat i
Sail
Fig. 3. Piasmids used in this study; the procedures for constnjcting piasmids from pSR95 and pLCBi4 are described in the Experimental procedures. The arrows indicate the directions of transcription of the operons. The nitrate reductase A promoter Is depicted by the black box. Hatched boxes represent the vectors used in these constructions (for details see the Experimental procedures and Table 4), The restriction sites are only indicated it relevant for cloning procedures which are explained in the Experimental procedures. Detailed restriction maps of pSR95 and pLCBi4 can be obtained from Bonnefoy et ai (1987). The locations of narK, narG. narH, narJ, nar/and nartl. narZ, narY. narWar\6 narUgenes are indicated by the open boxes and the corresponding letters.
pFB891 was constructed by inserting the Smal-Kpnl fragment coding for narlV and narV (issued from pFB88) into the ecoRV and Kpn\ sites of pFB73. pFB892 was obtained by cloning the blunt-ended Sa/I-Sa/I fragment carrying the narV^ gene issued from pFB88 into the EcoRV site of pFB73. Culture conditions, preparation of subcelluiar fractions, enzyme assays, limited proteclysis by trypsin, polyacryiamide gels and immunological analysis were as described by Blasco etai (1992) in the accompanying paper.
Purification of the inactive (ocpj^ complex The stages of the purification are listed in Table 3. Inactive (ap)A complex was detected using immunoelectrophoresis rocket technique (Graham et al.. 1980). All fractions containing
a and p subunits were analysed by Western blotting (Towbin et ai. 1979). Only fractions containing non-degraded a and [i subunits were retained. Wet cells (200 g) were suspended in 1200 ml of 50 mM TrisHCI, pH 7.6, containing 1 mM benzamidine-HCI. The crude extract (8860 mg of protein) obtained alter breakage of the cells was centrifuged twice at 120 000 x g for 90 min. The soluble fraction (7460 mg protein) which contains the (a^)^ compiex, was precipitated with ammonium sulphate at 35%, stirred for 30 min under nitrogen, and then centrifuged for 20 min at 18000 X g. The dark brown precipitate obtained was suspended in 200 ml of 50 mM Tris-HCI, pH 7.6, containing 1 mM benzamidine-HCI and dialysed overnight against 10 I of the same buffer. The dialysed material was centrifuged for 20 min at 40000 X g and the supernatant fraction was applied to a DEAE Sepharose CL-^6B column (150 x 3.5 cm) equilibrated in
Formation of active nitrate reductase in Escherichia coli the dialysis buffer; 1200 ml of a 0-400 mM linear NaCI gradient in the same buffer was applied- The fractions containing inactive nitrate reductase were eluted in the range of 0.25-0.28 M NaCI. Only fractions containing nitrate reductase with nondegraded a and [3 subunits were saved, dialysed, concentrated and purified by fast-protein liquid chromatography (FPLC) on a Mono-Q H10/10 ion exchange column equilibrated with 100 mM Tris-HCI, pH 7.6, 1 mM ben2amidine. Elution of samples (55 mg x 4) was effected by applying a 100-400 mM NaCI gradient in the same buffer. Fractions containing inactive nitrate reductase (61 mg of protein) were pooled, concentrated and eluted (27.5 mg x 4) on a Superosei 2 column. Purified enzyme was frozen in liquid nitrogen and kept at -80°C.
Iron content Iron was determined by atomic absotption analysis using a Varian AA 175 spectrometer. Inorganic sulphide was estimated by the method of Fogo and Popowsky (1949), as modified by Lovenberg etal. (1963).
Molybdenum cofactor isolation and identification Molybdenum cofactor associated with the (ap) complex was identified by complementation of the cofactor-deficient NADPH-dependent nitrate reductase produced by the nit1 mutant of N. crassa (Hawkes and Bray, 1984). Crude extracts of N. crassa were prepared as described by Amy and Rajagopalan (1979). Molybdenum cofactor and its derivatives were extracted by denaturing the enzyme (1 mg ml"') at lOO^C for 5 min under a nitrogen atmosphere and then rapidly cooling i1 to 0°C. Denatured proteins were removed by centrifugation at 15 600 X g for 2 min, and the supernatant fraction used for the reconstitution of N. crassa nitrate reductase activity in the complementation mixture. Fluorescence spectra of material released from purified nitrate reductase were obtained with a Kontron SFM 25 spectrofluorimeter. The heat-treated supernatant fraction was used for fluorescence spectroscopy as described by Johnson et ai. (1984). The excitation spectrum was obtained with emission at 455 nm, and the emission spectrum was obtained with excitation at 380 nm.
f\/tanipulation of DNA Restriction enzyme digestion, polymerase polishing, ligation, transformation and electrophoresis were essentialy adopted from Maniatis etal. (1982). Enzymes and chemicals were purchased from BRL and used as recommended by the suppliers.
Acknowledgements We wish to thank D. H. Boxer for critical reading of the manuscript, and F. Nunzi and M. C. Pascal for helpful discussion. We are grateful to J. M. Soulier and M. Denis for spectroscopic studies. This research was supported by the Centre National de la Recherche Scientifique and the Fondation pour la Recherche Medicale.
229
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