Biochem. J. (1983) 209,43-50Printed in Great Britain

43

Purification and characterization of the inactive MoFe protein (NifB-Kpj)of the nitrogenase from nifB mutants ofKlebsiellapneumoniae

Timothy R. HAWKES and Barry E. SMITHSchool ofChemical and Molecular Sciences andARC Unit ofNitrogen Fixation, University ofSussex,

Brighton, BNI 9RQ, U.K.

(Received 22 March 1982/Accepted 10 September 1982)

The inactive MoFe protein of nitrogenase, NifB-Kp 1, from two distinct niB mutants ofKlebsiella pneumoniae, Kp5058 (a nifB point mutant) and UNF1718 (a nijB, nifJdouble mutant) has been purified and characterized. NifB-Kpl can be activated byreaction with the iron-molybdenum cofactor, FeMoco, extracted from active MoFeprotein. NifB-Kpl purified from either source had similar properties and was

contaminated with an approximately equimolar amount of protein of mol.wt. 21000.Like active wild-type Kp 1, it was an a2/J2 tetramer, but it was far less stable than Kpl,deteriorating rapidly at temperatures above 8°C or on mild oxidation. NifB-Kplpreparations contained 0.4-0.9 Mo and 9.0 + 0.9 Fe atoms mol-h and, when activatedby FeMoco, had a specific activity of approx. 500 units mg-'. The Mo in our

preparations was not associated with the e.p.r. signal normally observed from FeMoco.All preparations exhibited a weak gav = 1.95 e.p.r. signal which was probably notassociated with activatable protein.

The enzyme nitrogenase consists of two proteins:the MoFe protein, and the Fe protein. The mostactive preparations of the MoFe protein contain 2Mo and about 30 Fe atoms . mol-1 (Eady & Smith,1979; Mortenson & Thorneley, 1979) and itsbiosynthesis involves the products of the niWK, D, V,B, N and E genes (Dixon et al., 1981). Mutants innifB, N or E synthesize inactive MoFe protein,which can be activated by adding the FeMo cofactor(FeMoco) extracted from active MoFe protein(Shah & Brill, 1977). FeMoco probably includes thesite(s) for binding reducible substrates (Smith, 1977)and has a characteristic e.p.r. signal, with g-valuesnear 4.3, 3.7 and 2.01 in the Na2S204-reducedprotein (Eady & Smith, 1979). This e.p.r. signalintegrates to 1-electron spin/Mo atom and 6 Featoms are associated with each electron spin(Rawlings et al., 1978). However, analyses ofFeMoco after extraction into N-methylformamideindicated 7-8 Fe atoms per Mo atom (Shah & Brill,1977; Smith, 1980; Burgess et al., 1980). The extra1-2 Fe atoms may be those designated 'S' in theMossbauer spectrum (Miinck et al., 1975; Shah &Brill, 1981). The other Fe atoms in the MoFe proteinare probably arranged as four unusual [4Fe-4S]

Abbreviations used: Me, methyl; But, t-butyl; Hepes,4-(2-hydroxyethyl)- 1-piperazine-ethanesulphonic acid;SDS, sodium dodecyl sulphate; IgG, immunoglobulin G.

Vol. 209

clusters, designated 'P' clusters, per molecule (Zim-mermann et al., 1978).

Isolation of the two metal groupings would assistdefinition of their structural properties and inter-actions. FeMoco can be extracted from the MoFeprotein into N-methylformamide but this extractioneither destroys the 'P' clusters, or yields an insolubleresidual protein. The 'P' clusters should be present inthe inactive MoFe protein from mutants unable tosynthesize FeMoco. Here we present details of thepurification and characterization of this protein fromtwo distinct nifB mutants of K. pneumoniae. Wehave designated this protein 'NifB-Kp 1 in order tospecify its phenotype and to distinguish it fromKpl, the active MoFe protein from wild-type K.pneumoniae. A preliminary account has been pub-lished elsewhere (Smith & Hawkes, 1981).

Materials and methodsMaterials

Most laboratory reagents were obtained fromBDH Chemicals, Poole, Dorset, U.K., and bio-chemicals from Sigma (London) Chemical Co.,Poole, Dorset, U.K. Other chemicals were of thehighest grade available. The Fe-S clusters,Fe4S4(SBut)4(Me4N)2 and Fe4S4C14(Me4N)2 were agift from Dr. R. Burt of this Unit.

0306-3275/83/010043-08$02.00 © 1983 The Biochemical Society

T. R. Hawkes and B. E. Smith

Sources ofgrowth oforganismsKlebsiella pneumoniae M5al was grown and

harvested as described previously (Eady et al.,1972). Two nifB mutants strains of K. pneumoniaewere used. Kp5058, a hisD nifB point mutant(Cannon et al., 1977) and UNF1718, a nifBdeletion, nifJ insertion double mutant [A(his-nifB)nif2485, nifJ2009:: TnJO, a gift from Dr. M.Merrick of this Unit]. In growth experiments thebasic medium included glucose (20g.1-1), MgSO4(0.1 g * 1-1), NaCl (0.01 g * 1-1), FeSO4 (0.025 g * 1-1),Na2MoO4,2H20 (0.025 g * 1-1), L-histidine(0.025 g 1-1), K2HPO4 (12.05 g * 1-1) and KH2PO4(3.4g 1-) and was sparged with N2 at 29°C; thepH was maintained above 6.5. The two mostsuccessful protocols were: (a) medium containing(NH4)2SO4 (0.021g of N-1-1) was inoculated withcells grown anaerobically on NH4C1 (0.26 g ofN *1-1).After 6h, L-aspartate (0.01g -l1) was added. Thecells were harvested after a further 16 h; (b) mediumcontaining 1 g of L-arginine -11 and 1% or 2.5%'L-broth' was inoculated with cells grown anaerobi-cally on L-broth. Cells were harvested, generallyafter 16-20h, when growth had stopped or was veryslow. [L-broth contained Oxoid yeast extract(5 g * I-1), Difco Bactotryptone (lOg * 1-') and NaCl(10g .1-1)]. French-pressing of cells from 5-10 litrecultures yielded crude extracts with NifB-Kp 1specific activities of 20-30 units . mg-' (a unit isdefined under 'Analytical techniques' below).However, cells from 100, 400 or 1000 litres growthgenerally yielded crude extracts with specific ac-tivities of only 10-15 units * mg-'.

Purification ofnitrogenaseThe nitrogenase proteins from K. pneumoniae

M5al were purified by the method of Eady et al.(1972) as modified by Smith et al. (1976).

Preparation ofFeMocoFeMoco was prepared as described by Smith

(1980).

Analytical techniquesProtein concentrations, C2H2 reduction activities

(1 unit of activity = 1 nmol of C2H4 produced/min),and metal contents were measured by the tech-niques described by Eady et al. (1972).To assay NifB-Kpl it had first to be saturated

with FeMoco and then the Kp 1 thus formed assayedfor C2H2 reduction with excess Kp2. Solutions ofNifB-Kp 1, FeMoco and buffer were mixed to give afinal volume of 0.6 ml in N2-flushed 7.8 ml serumvials capped with Suba-Seal rubber closures. Thefinal buffer concentration was made 40mM-Hepes,pH 7.5, containing 2 mM-Na2S204. A positive press-ure of N2 gas was established in each vial.Activation, at 30°C, was monitored by removing

50u1 samples for C2H2 reduction assay with 3-5#uM-Kp2, and was complete in 30 min. Activitieswere independent of the activation temperaturebetween 300 and 48°C (but at 2.50C were only 50%of the maximum even after 3.5h), and the specificactivity of the FeMoco preparations used (specificactivities 177-230 units/ng-atom of Mo). Loweractivities were observed if the concentration of N-methylformamide exceeded 10% in the activationmixture or 3% in the assay mixture.

SDS/polyacrylamide-gel electrophoresisThe gel system of Laemmli (1970) was used with

fixing, staining and destaining as described by Weber& Osborn (1969). A Unicam SP. 1800 spectro-photometer was used for scanning densitometry ofstained gels at 600nm.

Double immunodifusionAntigen-antibody interaction between the pro-

teins and purified IgG fraction of antibodies raisedagainst Kpl in rabbits ('anti-Kpl', a gift from Dr.R. R. Eady of this Unit) was studied by using thetechnique of Ouchterlony (1948).

ImmunoelectrophoresisThe technique of Laurell (1972) was modified by

use of Tris/HCl in place of barbital buffer. Theconcentration of MoFe protein in crude extracts wasmeasured by comparing the heights of rockets with astandard of purified Kpl at loadings between 0 and2.5,pg. The gels contained 70,ug of purified anti-Kpl * ml-'.

Analytical centrifugationSedimentation coefficients were measured in

anaerobically filled cells at 40C and 50000rev./minon an MSE Centriscan 74 ultracentrifuge withabsorption optics at 400 nm.

E.p.r. spectroscopyE.p.r. spectra were measured on anaerobically

prepared frozen samples with a Varian E9 spectro-meter (Lowe et al., 1972) as described in the legendsto the Figures.

U.v. and visible absorption spectroscopySpectra were measured with a Unicam SP.1800

spectrophotometer, and fixed-wavelength measure-ments were recorded with a Perkin-Elmer Coleman550 double-beam spectrophotometer.

Redox measurementsPotentiometric titrations were carried out essen-

tially by the method of Dutton (1971) as describedby O'Donnell & Smith (1978).

1983

44

The MoFe protein of nitrogenase from nipB mutants

Results and discussion

Activation ofNifB-KplTitrations of crude or purified pre

NifB-Kp1 with FeMoco showed linearactivity followed by plateau regions, in(stoichiometric binding of FeMoco. Fig.effect of varying the purified protein conthe activation reaction under cosaturation with FeMoco. A dilutionobserved, the specific activity at 0.Abeing only 50% of the maximum v;centrations above 1.5 um. The dilutiolargely relieved, but the maximum spewas unaffected, when Kp2 (5 pM) was inactivation mixture.

Purification ofNifB-KplPurification (Fig. 2) was at 3 4

NifB-Kp 1 was thermolabile. In 721preparation lost 55% of its activity at 2only 14% at 6+20C. NifB-Kpl alsoof its activity at 40C during gel filtraSephadex G-200, pH 9.1, passage thro100, pH 6.8, and DEAE-cellulose chr(in 50 mM-P,, pH 7.4. The protein wxfrozen beads in liquid nitrogen. Table Idata from a typical purification. As wit]et al., 1972; Smith et al., 1976), goodunits (at least 65%) was recorded ir

100;z

C0E

(UXv cd 50(A E- t_

0__4._11.0 2.0

[NifB-Kpll (uM)

Fig. 1. Dilution effect on NifB-Kpl specThe concentration of NifB-Kp 1 in themixture was varied in the presence (E) t(0, A) of 5.uM-Kp2 and samples removedmeasurements as described in the Memethods section. At high NifB-Kpl conits specific activity was 430 units * mg-1activity a purity of 75% was estimated tcalculate the NifB-Kp I concentratioabscissa. The FeMoco concentrations wing at all times. Two FeMoco preparatioactivity 180 units/ng-atom of Mo) were(O, 0) with 2.2 and the other (A) with 4ofMo. ml-,.

fication step, indicating that at no stage wasNifB-Kpl sustaining gross damage. If the specificactivity of the starting crude extract was <10

parations of units-mg-', hydroxyapatite chromatography (Stepr increases in 7) was required to obtained NifB-Kpl of highdicating tight activity.1 shows the Quantitative rocket immunoelectrophoresis and

icentration in scanning densitometry of stained SDS/polyacryl-)nditions of amide gels of purified NifB-Kpl (Fig. 3) indicated a

effect was purity of -90%. One contaminant was identified as35,uM-protein the nifJ product (mol.wt. 120000), since it wasalue at con- absent from nifJ mutant crude extracts and frombn effect was NifB-Kpl purified from UNF1718, the nifB, niJYXcific activity double mutant (Fig. 3). This observation demon-cluded in the strated that the nifJ product was not necessary for

activation of NifB-Kpl by FeMoco. The mostabundant contaminant of NifB-Kp1 from both

:20C, since Kp5058 and UNF1718 had a subunit mol.wt. ofh

2°Cpurfined 21 000 (Fig. 3) and was present at a molar ratio ofh a purified about 0.75. It was not found in wild-type Kpl>1+ 10C, but preparations nor in preparations of Kpl purifiedlost 40-85% from UNF1718 crude extracts after saturation withtion through FeMoco. We cannot rule out the possibility that thisough Chelex protein assists in the activation of NifB-Kp1 by)matography FeMoco.as stored as Before activation with FeMoco, purifiedsummarizes NifB-Kp 1 had a specific activity of 7 units- mg-'

h Kpl (Eady from Kp5058 and < 1.5 units * mg-' from UNF 1718.l recovery of After activation with FeMoco (all NifB-Kpln each puri- specific activities reported below are after activation

with FeMoco) the maximum NifB-Kp1 specificactivity obtained (Table 1) from either organism wasabout 500 units mg-' and was low in comparisonwith Kpl, the bulk of which generally has a specificactivity of 1000-1500 units-mg-', with some atactivities around 2000 units-mg-'. However, thefinal specific activity agrees well with that of420 + 50 units * mg-' calculated from the NifB-Kplconcentration (estimated by immunoelectro-phoresis) in Kp5058 and UNF1718 crude extracts.Furthermore FeMoco was equally active whetherassayed with crude extracts or purified NifB-Kpl.Extrapolating from results with the most active

3.0 4.0 FeMoco preparation used here (230 units/ng-atomof Mo), a specific activity of 2100 units * mg-' can be

7ific activity calculated for Kpl of mol.wt. 218000 with 2- activation FeMoco . mol-1. These data indicated that, withinand absence the population of NifB-Kpl molecules in both crudefor activity extracts and purified preparations, there was a

aterials and species capable of activating FeMoco to its fullesticentrations extent.. From thisand used to)n on theere saturat-ins (specifice used, one1.0 ng-atom

Properties ofNifB-KplImmunological cross-reaction with anti-Kpl.

Figs. 4(a) and 4(b) are photographs of Ouchterlonyplates monitoring the cross-reactions of anti-Kpland Kpl, two preparations of NifB-Kpl and anextract of de-repressed UNF 107 (pMF260) cells (a

Vol. 209

45

T. R. Hawkes and B. E. Smith

500g of cells (Kp5058 or UNF1718)Add 500ml of lOOmM-Tris, pH 8.7

Step (1) French press (10 Pa)Centrifuge (30000g, 3 h)

Pellet Supernatant(discard) Step (2) 52%4NHJ2SO4 precipitadon

Centrifuge (30000g. 1 h)

Pellet Supernatant(discard)

Resuspend in buffer to (NH4)2S04 1 <0.02 MStep (3) Bind to DEAE-ceilulos l500ml in 5cm (i.d.) columnI

Wash with 0.08 M-NaCI (500ml)4 Elute with 0.4 M-NaCI

Eluate

Step (4) Fracdonal (NHJ)2SO, precipiadonStep (4) Centrifuge at 30000g for l h. Discard supernatant.

32-45%-(NH4)2SO4 pelletResuspend in buffer to 30mg ml-'.Centrifuge at 49 OOOg for I h. Discard pellet.

Step (5)Sephadex G-200 gel filtration of 40 ml on 5 cm x 45 cm column in buffer + 50 mm-MgCI2

Main fraction (60-75 ml)Dilute to 15 mM-MgCI2

Step (6) T Bind to DEAE-celWlose (2.6 cm x 14 cm column).Elute with a linear gradient from 0.1 to 0.25 M-NaCI (225 ml of each)

Main fractionIf specific activity <400 units-mg -'. dilute fivefold with 0.01 M-potassium phosphate buffer. pH 6.8

Step _7) Bind to hydroxyapadte (2.6 cm x 12 cm column).Wash with 3 bed-volumes of 0.01 M-potassium phosphate, pH6.8.Elute with a linear gradient from 0.01 to 0.2 M-potassium phosphate buffer at pH 6.8(240 ml of each)

Final fraction

Fig. 2. Purification schemefor NifB-KpJAll steps were carried out anaerobically at 40C. The buffer was 5OmM-Tris/HCl, pH7.8, except where otherwisestated, and contained 0.1mg of dithiothreitol* ml-l and 2 mM-Na2S204.

nifD insertion mutant of K. pneumoniae which lacksKp 1). The nif'D mutant control showed a singleprecipitin line, demonstrating that our anti-Kp 1 wasnot wholly specific to Kp 1. This band was present inpurified Kp 1 and in NifB-Kp 1 at Step (6) (Fig. 2) ofpurification, but was removed by hydroxyapatitechromatography (Step 7). The other precipitin lineshowed that NifB-Kpl had the antigenic determi-nants of Kpl.

Molecular weight and subunit composition.Densitometry of the gels in Fig. 3 indicated that, likeKpl, NifB-Kpl consists of equivalent amounts of a-and 4-subunits. NifB-Kpl behaved similarly to Kplduring gel filtration through Sephadex G-200 and insedimentation-velocity experiments and thus we

conclude that it is an a2fl2 tetramer of mol.wt.-218000.

Susceptibility to damage by oxidation. NifB-Kp 1was irreversibly inactivated by oxygen and, duringpotentiometric redox titrations, was inactivated atpotentials above -350mV, whereas Kpl was stableat potentials up to OmV (O'Donnell & Smith, 1978).However NifB-Kp 1 could be oxidized for short(<10min) periods with K3Fe(CN)6 or dyes withoutan appreciable loss of activity.Mo and Fe content. Mo and Fe were found in all

our preparations of NifB-Kp1. The presence of Mowas surprising, since NifB-Kpl was FeMoco-deficient. The Mo content was variable and did notseem to bear any relationship to the specific activity

1983

46

The MoFe protein of nitrogenase from nifB mutants

b!

fnifjl proudct-

MoFe pdiot-iI :2,

MoFcr protein

Fe protein-

1 2 3 4

-2 5000

21 000-Mil %v-

1 uteif

-1 2 bOO

5 6 7 8 9 10 11 12 13 14 15

Fig. 3. SDS/polyacrylamide gels of (a) crude extracts and (b) NifWB-KpJ from KpS058 and UNF1718 purified to step (7)in Fig. 2

Channel 1 contained purified Kpl; 2, Klebsiella pneumoniae MSal crude extract; 3, Kp5058 crude extract; 4,UNF1718 crude extract; 5-8, 5-40ug of NifB-Kpl from Kp5058 (specific activity 500 units * mg-'); 9-13, 5-40,ugof NifB-Kp from UNF1 718 (specific activity 450 units * mg-'); 14-15, standard molecular-weight markers.

Step no.(see Fig. 2)

(1)(2)(3)(4)(5)(6)

(7)t

Table 1. Typical purification datafrom 5OOg ofcells (KpS058)Total protein 10-3 x Total activity NifB-Kpl specific activity

(g) (units) [units (mg of protein)-']*15 223 14.97.9 200 25.31.71 140.5 82.20.45 82.0 1810.152 53.3 350

Fraction 1 0.014 5.77 4102 0.021 10.75 500 83 0.018 8.55 480 8.6%

4 0.030 9.0 300Fraction 1 0.07 7 10

2 0.123 28.25 2303 0.094 35.70 3855 0.063 15.91 255

Yield(%)100906336.7523.8

15.4%

}85

* C2H2 reduction measured after saturation with FeMoco and assayed with excess Kp2.t Optional; e.g. starting material from Step 6, 0.39 g of protein, specific activity 240 units * mg-1; total units = 93 x 103.

.. ... ...

..

Fig. 4. Double immunodifusionThe Figure shows double immunodiffusion of anti-Kp 1, in the centre well, against: (1) purified Kpl; (2) BifB-Kplpurified to Step (6) (Fig. 2); (3) NifB-Kpl purified to Step (7) (Fig. 2); (4) UNF107 (pMF260) crude extract. (a)Shows removal of an impurity from NifB-Kpl; (b) shows that the impurity is probably not specific to nif

Vol. 209

[}l

nIf.J pro&.ci

Mol.wt

A-1 000-59 600N51 300

-33 500

47

.: 1

T. R. Hawkes and B. E. Smith

of NifB-Kpl preparations. Since the NifB-Kplpreparations were not 100% pure, the possibilitythat some of the metal was associated with proteinsother than NifB-Kpl could not be eliminated. Forthe most pure preparations the only contaminant ata molar concentration comparable with NifB-Kplwas the 21 000-mol.wt. protein. The metal content ofNifB-Kp I estimated from fractions with specificactivities >380 units . mg-' was 0.4-0.9 Mo and9.0 + 0.9 Feg-atom* (mol of NifB-Kp 1)-'. [Tablesof data supporting these conclusions are availablefrom B.E.S.]

Attempted insertion of extra Fe-S clusters intoNifB-Kpl. A possible reason for the low NifB-Kplspecific activities was that some molecules lacked 'P'clusters. At about 9 Fe atom * mol-h the Fe contentof NifB-Kp 1 was lower than expected. Attemptswere therefore made to insert additional Fe-Sclusters. Reconstitution under conditions similar tothose described by Hong & Rabinowitz (1970) forclostridial ferredoxin, did not affect the specificactivity of NifB-Kpl. The specific activity was alsounaffected when synthetic 4Fe-4S clusters [70UM-Fe4S4C14(Me4N)2 or Fe4S4(SBut4(Me4N)21 wereincluded in the activation reaction with FeMoco.

Optical absorption spectrum. The optical ab-sorption spectrum of Na2S204-reduced NifB-Kplbetween 400 and 850nm (Fig. 5) was almostfeatureless and similar to that from Kpl, except thatthe change in slope observed at about 430nm forKpl (Eady et al., 1972) was at about 470nm. Onthe assumption of 100% purity the preparationcontained 0.48 Mo and 8.6 Fe atom * mol-1.

I

co

400 500 600 700 800

Wavelength (nm)Fig. 5. Optical absorbance spectrum ofNa2S204-reduced

NifB-KpJ from KpSO58The protein (specific activity 430 units * mg-';concentration 3.5 mg * ml-') was dissolved in5OmM-Tris/HCl (pH 7.4)/0.2M-NaCl/dithiothreitol(0.1mg * ml-')/2 mM-Na2S204. The ordinatescale was calculated on the assumption that theprotein was 100% pure (estimated purity= 87+8%).

The absorption spectrum of isolated FeMoco isalso featureless (Shah & Brill, 1977). Subtractingthis spectrum (1 Mo: 6-8 Fe) from the publishedspectrum of Kp 1 [1 atom of Mo and 17.5 atoms ofFe * mol-h (Eady et al., 1972)], the absorbance dueto the 'P' centres alone (approx. 10 Fe atom * mol-')can be estimated as e6"27 litre * mmol-h . cm-' and1. 19 litre . mmolh cm-1. These numbers are inreasonable agreement with those calculated fromFig. 5 of c" = 23.5 litre * mmol-[ cm-' and16" = 15.5 litre * mmol-h e cm-'. The absorptionspectrum (Fig. 5) appears similar to those offerredoxins containing [4Fe-451 + clusters but not[2Fe-2S]l+ or [2Fe-2S]2+ clusters (Stephens et al.,1978; Thomson et al., 1977).

E.p.r. spectroscopy. No e.p.r. signal with g-valuesof 4.3, 3.7 and 2.01 was observed from ourNa2S204-reduced preparations of NifB-Kpl, con-firming the absence of normal FeMoco. However,these preparations invariably gave an almost axiale.p.r. signal (Figs. 6a and 6b) with g, = 2.034,92= 1.928 and g3= 1.896, similar to those fromreduced ferredoxins. The integrated intensity of thee.p.r. signal varied from 0.013 to 0.081 electrons/mol, did not correlate with the specific activity of theNifB-Kpl, and often decreased during purification.It is therefore unlikely that the signal arises from aform of NifB-Kp 1 that can be activated byFeMoco. (Tables of data supporting these con-clusions are available from B.E.S.) This signaldisappeared at potentials above -360mV. Consist-ent with experiments on Kpl (O'Donnell & Smith,1978), no other e.p.r. signals were observed fromNifB-Kp 1 at potentials up to 0mV.

The gay. = 1.95 e.p.r. signal is similar to thatobserved by Zumft & Mortenson (1973) from theinactive 'demolybdo' form (0.5 Mo, 8 Featom * mol-1) of Cpl (the MoFe protein fromClostridium pasteurianum) and to that observedfrom low-specific-activity Kpl, in early fractionsfrom DEAE-ceflulose during gradient elution withMgCl2. Such a preparation of Kpl (>90% pure onSDS/polyacrylamide gels) gave the e.p.r. signalshown in Fig. 6(c), with g-values at g, = 2.038,92= 1.933 and g3 = 1.881, with an integrated inten-sity corresponding to -0.08 electron spin. (mol ofKpl)-'. This preparation was not further activatedby FeMoco. It probably contained an inactive formof Kp 1, analogous to that from Cpl.

Conclusions

The Mo content and relatively low specificactivities of our preparations are puzzling, but wefound these properties with two distinct nijBmutants and therefore they seem to be a function ofthe phenotype. It is possible that the Mo in ourpreparations was inserted by the nifN or E gene

1983

48

The MoFe protein of nitrogenase from nifB mutants

g, = 2.034 g0= 1.928

T l93= 1.896

(a)

(b)

9g = 2.038 92 = 1.933

93 = 1.881

(c)

Fig. 6. E.p.r. spectra ofpreparations ofNa2S24-reduced NiWB-Kpl and low-specific-activity Kpl(a) NifB-Kp 1 from UNF1 718; specific activity 360 units . mg-', concentration 11.4mg ml-'; (b) NifB-Kpl fromKp5058; specific activity 450 units* mg-', concentration 3.7mg ml-'; (c) Kpl, specific activity 550 units mg-',concentration 15.2mg ml-'. E.p.r. spectra were measured at 23 K, with a microwave frequency of 9.22 GHz andpower of lOmW using 1.OmT field modulation at 3.3kHz. The g-values shown are relatively comparable within+0.001 but are only accurate to +0.004.

products in place of FeMoco, thus preventing fullactivation of the protein. Wild-type Kp 1 prep-arations are a mixture of ill-defined species ofdiffering specific activities which are only partiallyseparated by DEAE-cellulose chromatography. It isprobably that our NifB-Kp1 preparations alsocontained a mixture of species, with only some ofthem being activatable by FeMoco, since the latterwas activated by our preparations to a levelconsistent with a Kpl specific activity of 2100units* mg-'. Furthermore, the gay. = 1.95 e.p.r. sig-nal in our NifB-Kp 1 preparations was probablyassociated with an inactivated species.

Burgess et al. (1981) published a preliminaryreport on the purification of the MoFe protein from

Vol. 209

a FeMoco-deficient mutant, UW45, of Azotobactervinelandii. This protein, although only 70% pure,had a specific activity (after activation by FeMoco)of 1900 units * mg-' and contained no Mo. Un-fortunately the nature of the mutation in UW45 hasnot been established, but comparison with our datawould suggest that it is not in the nifB gene.

Despite its low activity, the purified NifB-Kpldescribed here should prove useful in investigationsof the role(s) of FeMoco and the P clusters in Kpl,particularly in experiments in which they areselectivity enriched with "7Fe. Such experimentsshould allow unequivocal assignment of e.p.r.signals, seen on oxidation or during turnover (Smithet aL, 1972; Lowe et al., 1978).

49

50 T. R. Hawkes and B. E. Smith

We gratefully acknowledge the construction of strainUNF1718 by Dr. M. Merrick, the preliminary room-temperature purification work of Miss Denise Elsdon, andthe growth experiments of Mr. J. F. Bowden. We alsothank Professor J. R. Postgate and Dr. R. R. Eady forcomments on the manuscript and Dr. D. J. Lowe forrunning e.p.r. spectra. Mr. K. Baker and Miss KarinaWhite provided cells from large-scale cultures. T. R. H.was supported by a Science Research Councilstudentship.

ReferencesBurgess, B. K., Jacobs, D. B. & Stiefel, E. I. (1980)

Biochim. Biophys. Acta 614, 196-209Burgess, B. K., Yang, S.-S., You, C.-B., Li, J.-G., Friesen,

G. D., Pan, W.-H., Stiefel, E. I. & Newton, W. E.(1981) in Current Perspectives in Nitrogen Fixation(Gibson, A. H. & Newton, W. E., eds.), pp. 71-74,Australian Academy of Science, Canberra

Cannon, F. C., Reidel, G. E. & Ausubel, F. M. (1977)Proc. Natl. A cad. Sci. U.SA. 74, 2693-2697

Dixon, R., Kennedy, C. & Merrick, M. (1981) in Geneticsas a Tool in Microbiology (Glover, S. W. & Hopwood,D. A., eds.), pp. 161-185, Cambridge University Press,Cambridge

Dutton, P. L. (1971) Biochim. Biophys. Acta 226, 63-80Eady, R. R. & Smith, B. E. (1979) in A Treatise on

Dinitrogen Fixation (Hardy, R. W. F., Bottemely, F.& Bums, R. C., eds.), sect. I and II, pp. 399-490,Wiley, New York

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