Vol. 173, No. 8

Menaquinol-Nitrate Oxidoreductase of Bacillus halodenitrificansPAUL A. KETCHUM,' GERARD DENARIAZ,2 JEAN LEGALL,2 AND WILLIAM J. PAYNE3*

Department of Biological Sciences, Oakland University, Rochester, Michigan 483094401,1 and Departments ofBiochemistry2 and Microbiology,3 The University of Georgia, Athens, Georgia 30602

Received 26 October 1990/Accepted 4 February 1991

When grown anaerobically on nitrate-containing medium, Bacillus halodenitrificans exhibited a membrane-bound nitrate reductase (NR) that was solubilized by 2% Triton X-100 but not by 1% cholate or deoxycholate.Purification on columns of DE-52, hydroxylapatite, and Sephacryl S-300 yielded reduced methyl viologen NR(MVH-NR) with specific activities of 20 to 35 U/mg of protein that was stable when stored in 40% sucrose at-20°C for 6 weeks. 3-[(3-cholamidopr6pyl)dimethylammonio]-2-hydroxypropone-l-sulfonate (CHAPSO) anddodecyl-o-D-maltoside stimulated enzyme activity three- to fourfold. Membrane extractions yielded purifiedNR that separated after electrophoresis into a 145-kDa a subunit, a 58-kDa I8 subunit, and a 23-kDa y subunit.The electronic spectrum of dithionite-reduced, purified NR displayed peaks at 424.6, 527, and 557 nm,indicative of the presence of a cytochrome b, an interpretation consistent with the pyridine hemochromespectrum formed. Analyses revealed a molybdenum-heme-non-heme iron ratio of 1:1:8 for the NR and thepresence of molybdopterin. Electron paramagnetic resonance (EPR) signals characteristic of iron-sulfurcenters were detected at low temperature. EPR also revealed a minor signal centered in the g = 2 region of thespectra. Upon reduction with dithionite, the enzyme displayed signals at g = 2.064, 2.026, 1.906, and 1.888,indicative of the presence of low-potential iron-sulfur centers, which resolve most probably as two [4Fe-4S]+1clusters. With menadiol as the substrate for nitrate reduction, the Km for nitrate was 50-fold less than that seen

when MVH was the electron donor. The cytochrome b557-containing enzyme from B. halodenitrificans ischaracterized as a menaquinol-nitrate:oxidoreductase.

The moderate halophile Bacillus halodenitrificans was

selectively isolated from a solar saltern as a denitrifier able togrow on the high nitrate concentrations encountered inuranium processing wastewater. It tolerates 1.65 M (14%)nitrate and 0.29 M (2%) nitrite and grows optimally at pHsbetween 7.6 and 8.0. Cells readily reduce both nitrate andnitrite under anaerobic conditions, producing nitrous oxideas the end product of denitrification (17, 18). The electrontransfer components of the denitrification pathway includecytochrome b559, a cytochrome c550, and a copper nitritereductase (16).

Bacterial nitrate reductase (NR) from denitrifying bacteriais typically a membrane bound, heteromultimeric proteinthat may or may not purify with a cytochrome b moiety,depending on the method of solubilization (28, 42). Quinonesor menaquinones donate electrons directly to the nitratereductase complex in many of the gram-negative bacteriastudied to date (13, 20, 37). The quinone is postulated to beinvolved in the proton pump mechanism for energy genera-tion (36), while the cytochrome b transfers electrons to theiron-molybdenum centers of the nitrate reductase andthereby to nitrate. The active quinone can be reduced bydehydrogenases acting on NADH, formate, succinate, hy-drogen, and others and perhaps themselves possess a b-typecytochrome (29).

Studies of NR in gram-negative denitrifiers are moreextensive, but some years ago, Downey reported membranelocation and involvement of a cytochrome b in the NR ofBacillus stearothermophilus (19). Van't Riet et al. (44) laterpurified the deoxycholate-extracted respiratory NR frommembranes of Bacillus licheniformis and found that it wascomposed of a 150-kDa ot subunit and a 57-kDa , subunit.The a. subunit broke down into smaller peptides during

* Corresponding author.

storage at 4°C, explaining the instability of this NR. The use

of nonionic detergents to solubilize NR from B. stearother-mophilus provided greater stability (11). Unlike the respira-tory NR solubilized from the membranes of gram-negativebacteria with nonionic detergents (10, 12, 20), enzyme ex-tracted from bacilli with deoxycholate lacked cytochrome(44).The present study was designed to isolate a stable, purified

form of NR from B. halodenitrificans, to determine itspolypeptide structure, metal centers, and cytochrome binvolvement, and to relate these aspects to its membranelocation and physiological function in denitrification.

MATERIALS AND METHODS

Growth conditions. B. halodenitrificans (ATCC 49067) wasgrown at room temperature under anaerobic conditions in a

complex medium containing (per liter of distilled water): 8 gof nutrient broth (Difco), 5 g of yeast extract, 1 g ofMgSO4 7H20, 30 g of NaCl, and 5 g of NaNO3, andadjusted to pH 7.4. In addition, a 10-ml/liter solution of themetal solution contained (per liter) CaCl2 2H20, 2 g;FeSO4- 7H20, 1 g; Na2MoO4- 2H20, 0.1 g; CuSO4 5H20,0.1 g; and concentrated HCl, 8.5 ml (13).

Preparation of washed membranes. Cells harvested with a

Sharples centrifuge were suspended in an equal volume of 20mM Tris-HCl buffer (pH 7.6) supplemented with 10 mMphenylmethylsulfonyl fluoride (PMSF) to inhibit proteolysis.All subsequent procedures were carried out at 4°C. The cellsuspension was passed twice through a Manton-Gaulin ho-mogenizer at a pressure of 633 kg/cm2. A few milligrams ofDNase I and DNase II were added to decrease viscositybefore treating with neutralized streptomycin sulfate (0.5mg/mg of protein). After stirring for 20 min, the mixture wascentrifuged at 13,200 x g for 30 min. The pellet was

discarded, and the supernatant solution was centrifuged at

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NITRATE REDUCTASE OF B. HALODENITRIFICANS

144,000 x g for 90 min. The membranes thus sedimentedwere extracted once with 50 mM Tris-HCI (pH 7.6)-600 mMNaCl (final protein concentration, 10 mg/ml) at room tem-perature for 1 h before being pelleted by centrifugation at144,000 x g for 90 min. The dark red pellet, suspended in asmall volume of 50 mM Tris-HCl (pH 7.6) and called themembrane fraction, was frozen at -80°C until used.

Purification of NR. Frozen membranes were suspended in10 mM phosphate buffer (pH 7.6) containing 10 mM PMSFbefore extraction for 4 h at 4°C in 2% Triton X-100. Beforeuse, Triton X-100 was prepared as a 10% (vol/vol) solutionand filtered to remove bacteria. Debris was removed bycentrifugation at 144,000 x g for 90 min, and the supernatantsolution was dialyzed against 10 mM Tris-HCl, pH 7.6,before loading on a DE-52 column (4.5 by 28 cm) equili-brated in 10 mM Tris-HCl buffer (pH 7.6) containing 1%Triton X-100. After washing with 2 column volumes of thisbuffer, the column was eluted with a 0 to 400 mM NaClgradient (1,600 ml total) in the equilibration buffer. A cy-tochrome b emerged at 120 to 140 mM NaCl, NR emergedbetween 173 and 200 mM NaCl, and cytochrome c550emerged at 200 mM NaCl. The pooled NR fractions wereloaded on a hydroxylapatite column equilibrated with 3 mMphosphate (KH2PO4-K2HPO4) buffer (pH 7.6) containing0.1% Triton X-100. The column was washed with 2 columnvolumes of the equilibration buffer and then subjected to a 5to 200 mM gradient of KH2PO4-K2HPO4 (pH 7.6) containing0.1% Triton X-100. A major peak of cytochrome c eluted at75 mM phosphate, whereas NR eluted at 79 mM phosphate.Fractions with high NR activity were pooled and concen-trated to a small volume with an Amicon YP-10 membrane.Treating the pooled fraction with ethylene glycol during theconcentration process (24) helped remove Triton X-100 todecrease viscosity.The concentrated NR sample (2 to 3 ml) was loaded on a

Sephacryl S-300 column (3 by 90 cm) equilibrated in 50 mMKH2PO4-K2HPO4 buffer containing 0.1% Triton X-100 andeluted at a flow rate between S and 7 ml/h. NR elutedbetween 280 and 294 ml and was separated from contami-nating cytochrome c, which eluted in two peaks centered at315 and 416 ml. Samples containing peak NR activity werepooled and concentrated.

Subunit structure and molecular weight analysis. Molecularweights were estimated (46) by sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis (PAGE) (33) andthe following protein standards obtained from Bio-Rad:myosin (200 kDa), P-galactosidase (116.25 kDa), phosphory-lase A (97.4 kDa), bovine serum albumin (66.2 kDa), oval-bumin (42.7 kDa), carbonic anhydrase (31 kDa), soybeantrypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa). Gra-dient gels of 7.5 to 20% acrylamide were used for subunitseparation, whereas 7.5% acrylamide gels were used todetermine the mass of the a subunit. Samples were sus-pended in an equal volume of sample buffer containing 4%SDS, 0.05 mg% bromophenol blue tracking dye, 1% mercap-toethanol, and 2% glycerol, heated to 100°C for 2 min, andthen cooled on ice prior to loading. Separate unheatedsamples of NR were subjected to electrophoresis for b-typecytochrome analysis (10). All SDS-PAGE analyses werecarried out in a Protean II cell (Bio-Rad) at 100 V for 5 h, andthe gels were stained with Coomassie blue dye.

Optical spectra and metal analysis. Spectra were obtainedfor samples assayed at room temperature with a ShimadzuUV-265 double-beam spectrophotometer calibrated with aholmium filter. Heme content was determined from spectraof the alkaline pyridine hemochrome prepared in 0.075 M

NaOH and 25% pyridine (23). The OD550_570 after reductionwith a few crystals of dithionite was used to calculate hemeb content with the millimolar extinction coefficient of 34.4(41). Flavin was assayed spectrophotometrically after ex-tracting the sample with 8% trichloroacetic acid (40).

Electron paramagnetic resonance (EPR) spectra wereobtained for native enzyme and samples reduced by additionby gas-tight Hamilton syringe of aliquots of anaerobicallyprepared dithionite solutions (0.1 M in Tris-HCl buffer [pH9.0]). The effects of reduction times of 1 and 15 min and pHsof 8.5 and 6.2 were tested. Data were recorded with a BrukerER 200D SRC spectrometer equipped with an Oxford Instru-ments ESR 910 continuous-flow cryostat. After acquisitionwith a Bruker Aspect 2000 computer, data were transferredto an IBM AT computer for analysis.

Metal content was determined by plasma emission spec-troscopy with a Mark II Jarrel-Ash model 965 ICAP. Sepa-rate calibration curves were run for Fe and Mo in TritonX-100.Enzyme and protein assays. Reduced methyl viologen NR

(MVH-NR) was assayed at room temperature in 50 mMKH2PO4-K2HPO4 buffer pH 7.2, by the diazo procedure formeasuring nitrite (25). One unit of MVH-NR equals 1 ,umolof nitrite formed per min. Alternatively, the oxidation ofMVH under anaerobic conditions was followed by measur-ing the OD600 (2, 31).Menadiol was prepared daily by reducing a solution of

menadione in acidified methanol with crystals of sodiumborohydride (37). Menadiol oxidation by nitrate was fol-lowed at 266 nm (molar extinction coefficient of oxidizedminus reduced of 9,290 M-1 cm-') under anaerobic condi-tions in 50 mM Tris-HCl, pH 8.2, containing menadiol (333riM), sodium nitrate, and enzyme. Molybdopterin (MPT)was assayed by the 4°C-24-h procedure (27) for the recon-stitution of NADPH-NR in extracts of a Neurospora crassanit-i mutant (32). The units of NADPH-NR formed duringcomplementation were converted to MPT by using the valueof 26 ,umol of N02 per min per ng-atom of Mo in reconsti-tuted NR (27). MPT in purified NR and buttermilk xanthineoxidase was released by the dimethyl sulfoxide procedure ofHawkes and Bray (27). Protein was determined by a modi-fication of the Bradford method as described by Read andNorthcote (39) with bovine serum albumin as the standard.

Materials. Hydroxylapatite Bio-Gel-HTP and acrylamidewere obtained from Bio-Rad Laboratories, Richmond, Calif.;Sephacryl S-300 was from Pharmacia, Uppsala, Sweden; 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate(CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hy-droxypropane-1-sulfonate (CHAPSO), octyl-p-D-glucopyra-noside, and dodecyl-p-D-maltoside were from Calbiochem,San Diego, Calif.; DE-52 was from Whatman Ltd., Kent,England; and buttermilk xanthine oxidase, PMSF, duroqui-none, menadione, and hexyl agarose were from SigmaChemical Co., St. Louis, Mo.

RESULTS

The NR of anaerobically grown B. halodenitrificans ap-peared as a membrane-bound enzyme functionally associ-ated with a b-type cytochrome. Malate, succinate (notshown), and NADH reduced the b- and c-type cy-tochrome(s) present in membrane vesicles prepared fromdenitrifying cells (Fig. 1). Nitrate, nitrite, and nitric oxideoxidized both the reduced b- and reduced c-type cy-tochromes. The oxidation occurred in two steps: (i) a steady-state level of reduced cytochromes was reached immediately

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A B c

10.02 10.02 d iox

C)

z

.4

co

reduced membranes. Samples were made anaerobic by repeatedevacuation and argon flushing and then reduced by 1 mM NADH( .Protein concentration, 1.25 mg/ml in 0.1 M Tris-HCI (pH

7.6). (A) 10 mM nitrate added: after 2 and 8 min; after 15min. (B) SmM nitrite added: ,after 2 and 20 min; ........ after 25min. (C) 100p5 ofNO added: after 2 and 30 min; 550....after 50min.

after addition of the oxidant and lasted until depletion ofNADH (fo1Eowed at 360 nm), and (ii) a rapid oxidation ofboth cytochromes occuSmed when NADH was depleted. Thepattern of oxidation changed in the presence of diethyldithio-carbamate (DDC), which inhibits the copper nitrite reduc-tase of B. halodenitrificans. Nitrate oxidized only the re-duced b-typecytochr)me, whereas nitrite had no effect onthe reduced spectrum (Fig. 2). The di2ference spectrum (Fig.2B) resolved the oxidation of the cytochrome b59afrom thelarge amount of reduced c-type cytochrome(s) present. Ab-type cytochrome thus appears to be involved in thereduction of nitrate to nitrite, whereas both cytochrome b5reand cytochromeC550seem to be involved in nitnte and nitricoxide reduction.

Detergentsolubet idation of MVH-NR. Membranes sus-pended in 5omMKH2Prc4-K2HPo4 bucfer, pH 7.2, at 22.3mg of protein per ml were stiared for 1 h at4iC in thepresence of 2%Trtton X-100, 1% deoxycholate, or 1%cholate. The supernatant solutions of the detergent-treatedmembranes contained >50% of the total protein after cen-trifDgation (144,000 x g for 90 min), whereas <10% of theprotein in the untreated control remained suspended. Aftercentrifugation, 84, 8.6, and 8.9% of the MVH-NR present inthe control membranes was in the supernatant solutions of

e

0

.04

Wavelength (nm) Wavelength (nm)

FIG. 2. Effect of DDC on reduction of membrane cytochromesby NADH and their subsequent oxidation by nitrate. DDC concen-tration was 10 mM in all experiments; protein concentration was

1.25 mg/ml. (A) , 1 mM NADH added; ---, 2.5 mM nitrateadded after 15 min. (B) Difference spectrum of NADH plus nitrateminus NADH-reduced.

TABLE 1. Purification of NR

(Vo TotalVotal Sp act RecoveryProcedure ~ activity' protein (U/mg) (%)M(m) (U) (mg)

Triton X-100 high- 545 1,789 6,610 0.27 100speed supematant

DE-52 pool 124 1,218 372 3.27 68Hydroxylapatite pool 115 951 57 16.68 53Sephacryl S-300 13.5 518 17 30.124 28

a MVH-NR. Representative of six repetitions of the purification proce-dures.

the Triton X-100, cholate, and deoxycholate preparations,respectively. Cholate and deoxycholate appear to inactivateMVH-NR, since <16% of the MVH-NR originally presentwas recovered by subsequent extraction with 2% TritonX-100. Triton X-100 at a final concentration of 2% thusserved for extraction of MVH-NR from membranes for thepurification of NR in all other experiments described.

Purification of MVH-NR. Care was taken to inhibit prote-ases with PMSF during the cell breakage and again duringTriton X-100 extraction of the membranes. Passage of NRthrough DE-52, Bio-Gel-HT, and Sephacryl S-300 columnsresulted in >100-fold purification and 28% recovery (Table1). Cytochrome c550 was solubilized during Triton X-100extraction and remained a contaminant of NR up to Sepha-cryl gel filtration. Likewise, cytochrome b559 was extractedwith Triton X-100, but eluted from the DE-52 column at 120mM NaCl. The Triton X-100 content of the sample used forgel filtration was diminished by adding ethylene glycolduring concentration by ultrafiltration. Our purified enzymepreparations appeared dark- brown and possessed no cy-tochrome c550.We investigated the effects of glycerol, sucrose, and

ethylene glycol and freezing temperatures on the stability ofNR. NR was stable in 30 or 40%o sucrose at -20°C for 49days, whereas preparations stored at -80°C lost 45% ormore of their activity regardless of the supportive mediumprovided.Enzyme purity. The presence of Triton X-100 and the

heteromultimeric structure of NR complicate normal assaysfor enzyme purity. When components were separated ondisk gels containing Triton X-100 (45), one or two high-molecular-weight bands of NR activity and one or morefaster-migrating peptide bands appeared, perhaps as a resultof association-dissociation reactions like those demonstratedfor the Escherichia coli NR (35). Analyzing NR preparationson 7.5 to 20% SDS-PAGE (Fig. 3) provided partial clarifica-tion, and the expected a, 5, and -y subunits of bacterial NRemerged. The three major bands in these gels account formore than 95% of the Coomassie blue-staining protein.Different ratios of subunits within the NR complex mayaccount for the multiple high-molecular-weight bands ob-served in disk gels. Use of other detergents in either theenzyme preparations or the gels or both gave no furtherbenefit.The specific activity of the purified B. halodenitrificans

NR (20 to 35 U/mg) is greater than those reported for theNRs from B. licheniformis (44) and Pseudomonas aerugi-nosa (5) and approaches that of the cytochrome-lackingrespiratory NR isolated from gram-negative bacteria (13, 21,37).

Subunit structure of NR. SDS-PAGE of purified prepara-tions of NR permitted visualization of the a, 53, and y

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NITRATE REDUCTASE OF B. HALODENITRIFICANS

of _- 145K

%g-. P 58K

D1c

y_..# 23K

FIG. 3. SDS-PAGE of purified nitrate reductase (40 ,ug of pro-

tein) run on a 10 to 20% acrylamide gel, showing the ao, ,3, and y

subunits. Sizes are shown in kilodaltons.

subunits characteristic of other NRs solubilized withnonionic detergents (Fig. 3). Molecular mass estimates were:

ot subunit, 145 kDa; subunit, 58 kDa; and -y subunit, 23kDa. After aging for 60 days at 4°C, a minor P, band with a

mass of 46 kDa appeared beneath the band. The P, appearsto be derived from P. Assuming a 1:1:1 ratio of the a, P, and-y subunits permits estimation of a molecular mass of 226 kDafor the NR complex.

Effect of detergents and NaCI on MVH-NR. Detergents canmarkedly affect MVH-NR activity. Treatment of purifiedNR with CHAPSO (0.5%) and dodecyl-p-D-maltoside (0.5%)stimulated the MVH-NR activity 3.6- and 4.0-fold, respec-

tively, whereas CHAPS (0.5%) and oCtyl-p-D-gluCopyrano-side (0.5%) increased activity by approximately 1.2-fold.The detergents may alter the orientation of the enzyme

complex in the micelles or the structure of the enzyme itself,a possibility that warrants further investigation.Because B. halodenitrificans is a halophile, we investi-

gated the effect of NaCl on MVH-NR activity and found thatNaCl (<0.8 M) had no effect on MVH-NR activity, butconcentrations of 1.2 and 2 M NaCl inhibited MVH-NRactivity by 37 and 78%, respectively, in striking contrast tothe twofold stimulation of the activity of nitrite reductasefrom the same organism in the presence of 1 to 3 M NaCl(16).

Spectral analysis of purified NR. The electronic spectra ofNR as prepared (native) exhibit a broad absorption between400 and 450 nm with a major peak at 411 nm (Fig. 4).Absorption maxima at 424, 527, and 557 nm observed uponreduction with dithionite are characteristic of b-type cy-tochromes. The absorption spectrum of the pyridine hemo-chrome extracted from purified NR was characteristic of aprotoporphyrin IX, identifying the heme of NR as b type.Flavin was not detected in trichloroacetic acid extracts ofthe purified enzyme. We therefore attribute the high absorp-tion between 400 and 450 nm to the presence of iron-sulfurclusters.

0.04

450 550 65o

Wavelength (nm)

FIG. 4. Spectrum of purified NR (0.5 mg of protein per ml) asprepared ( ) and reduced with a few crystals of dithionite (---).

EPR and metal analysis of purified NR. The native enzymedisplayed a minor signal at g = 2.03 in samples assayed attemperatures below 20 K, probably owing to the existence ofa minor fraction of the iron-sulfur centers as [3Fe-4S]clusters (Fig. 5). Samples reduced for 1 min at pH 8.5exhibited signals at g = 2.064, 2.026, 1.906, and 1.888,assigned to low-potential iron-sulfur centers. The overallshape of the EPR spectra and the microwave power depen-dence of the EPR signals (not shown) indicate resolution ofthe spectral features into two [4Fe-4S]+1 clusters. At tem-peratures of >25 K, only the central feature at g = 1.96 wasdetected, the iron-sulfur centers being too broad for detec-tion. The central feature is probably attributable to Mo(V),but the low intensity and lack of resolution preclude furtheranalysis. EPR spectral features for samples reduced for 1min at pH 6.2 were not altered, and prolonged reduction withdithionite (15 min) resulted in a decrease in the intensities ofthe EPR signal.Four separate determinations on two preparations of NR

were made for molybdenum, heme iron, and total iron (Table2). Both the molybdenum and the iron analyses took accountof standards tested with quantities of Triton X-100 equiva-lent to those present in the samples. These data indicate thatthe molybdenum-heme-non-heme iron ratio is 1:1:8, sug-gesting that for each molybdenum atom there is one hemeiron and two [4Fe-4S] clusters.Two preparations of purified B. halodenitrificans NR

exhibited MPT reconstitution of NADPH-NR in the assaywith extracts of N. crassa nit-i. Although restoration ofNADPH-NR was proportional to the amount of denaturedNR added to the bacterial extract, less than 1% of theexpected MPT in the bacterial NR was converted toNADPH-NR (0.138 pmol of MPT per 35 pmol of bacterialNR). The same procedure was more efficient in transferringMPT from xanthine oxidase to NADPH-NR (2.08 pmol ofMPT per 45 pmol of xanthine oxidase). Perhaps the presenceof Triton X-100 in the NR preparations interfered withdenaturation and release of MPT from the NR complex.Menadiol oxidation by NR. Menaquinone (MK-7) is the

major quinone in B. halodenitrificans (17). Cytochrome b inpurified NR was quickly reduced by menadiol and reoxi-dized by nitrate added anaerobically (Fig. 6). The oxidationof menadiol was proportional to NR concentration, although

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2.033

2.064 1.9

1 2.026

A

1.906

A

0

E

A.

-W

a

ut

Magnetic Field (T) -'

FIG. 5. Low-temperature, X-band EPR spectra of NR. Experimental conditions: temperature, 8 K; microwave power, 2 mW; gain, 3.2x 105; modulation amplitude, 1 mT. (A) Enzyme as prepared (native); (B) dithionite reduced (1 min) at pH 8.5; (C) dithionite reduced (1 min)at pH 6.2.

the reaction required 10 times more enzyme than the MVHassay, a finding consistent with reports of duroquinol oxida-tion by other nitrate reductases (13, 37). Menadiol oxidationby NR was reflected by increases in peak height at 266 nmunder strict anaerobiosis in the presence of nitrate, whichwas converted to nitrite. The pH optimum for this reactionwas 8.0 to 8.2. The oxidation kinetics for menadiol and MVHwere obtained under identical conditions (except for enzymeconcentration). The Km for nitrate with MVH as the reduc-tant (2,730 jiM) was 50 times higher than with menadiol asthe reductant (54 ,uM). With menadiol as the reductant, theVmax was 10.7 nmol/min. MVH sustains a much greater Vmax(670 nmol/min) than menadiol and is thus preferred forroutine assays. We were unsuccessful in demonstratingnitrate reduction with duroquinol or the oxidation of duro-quinol by the B. halodenitrificans NR.

DISCUSSION

The nitrate reductase of B. halodenitrificans is a mem-brane-bound, heme-containing, molybdo-iron protein thatoxidizes menadiol and reduces nitrate to nitrite. This en-zyme complex separates into three subunits on SDS-PAGE:a 145-kDa ot subunit, a 58-kDa 1 subunit, and a 23-kDa rysubunit. Cytochrome b557 copurifies with the enzyme (pre-sumably the y subunit) and appears to be an integral part ofthe complex. The relationship of cytochrome b559 present inthe membranes and separated during the DE-52 purificationstep to nitrate reduction has yet to be resolved; however,

TABLE 2. Metal analysis of purified NRa

Mol of metal/mol of enzymeMetal

Range Avg Proposed

Molybdenum 0.43-1.08 0.705 1Non-heme iron 5.03-9.19 6.57 8Heme iron 0.99-1.08 1.035 1

a Four determinations on two preparations of purified NR (assuming 226kDa for NR).

two distinct b-type cytochromes appear to be involved in thedenitrification pathway.

B. halodenitrificans NR differs in structure and stabilityfrom the 193-kDa NR of B. licheniformis extracted withdeoxycholate (44). The latter enzyme has the typical 150-kDa ot and 57-kDa 1 subunits, but no cytochrome. Inaddition, the at subunit of B. licheniformis NR was unstableat 4°C, perhaps a result of exposure to ionic detergents.When purified in Triton X-100, our enzyme possessed cy-tochrome b and was stable at 4°C; however, it was unstablewhen switched from Triton X-100 to cholate. Cholate anddeoxycholate altered or inactivated membrane-bound NR,suggesting that ionic detergents somehow modify this NR.The absence of the b-type cytochrome in extracted NRcomplexes is not unique to the Bacillus enzyme; NRssolubilized from E. coli by heat (2, 35) and from P. aerugi-nosa by deoxycholate (5) also lack cytochromes. Suchresults relate specifically to extraction technique, since theinvolvement of b-type cytochromes with the NRs extractedwith nonionic detergents from E. coli and Paracoccus den-itrificans is well established (10-13, 20, 21).The mass and subunit structure of our NR resemble those

of the enzyme purified from E. coli with nonionic deter-gents (10-13, 21). The NR of E. coli displays a 150-kDa atsubunit with MPT at the active site (9), a 60-kDa P subunit(8, 15), and a ry subunit possessing the spectral propertiesof cytochrome b556 (10, 12, 20). The function of the 13 sub-unit is unknown; however, it may bind the NR complexto the membrane (42). An alternative function was pro-posed by Blasco et al. (3), who sequenced the 1 gene andproposed from such data alone that the P subunit is theiron-sulfur protein involved in electron transfer in NR.This proposal offers no accounting for the non-heme ironin the isolated at subunit (9), so the function of the 13 sub-unit remains unclear (9). Nevertheless, the striking similar-ities in the subunit size and composition of the E. coliand B. halodenitrificans NRs may indicate analogous func-tions.The most variable component of the E. coli enzyme is the

1 subunit, variously reported as a 60-, 58-, or 43-kDasubunit. The 60-kDa 1 subunit is present in membrane-bound NR (8, 15), the 58-kDa 13 subunit in most solubilized

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NITRATE REDUCTASE OF B. HALODENITRIFICANS

.0 \%,~~./ ~ 525 550 01

.0

0l400 5O0 60

Wavelength (nm)FIG. 6. NR (400 ,ug) in 2.0 ml of 50 mM KH2PO4-K2HPO4 buffer (pH 7.2) as prepared ( ), 1 min after addition of 500 nmol of menadiol

( ), and 1 min after addition of 1 ,umol of NaNO3 ( ---). Inset presents expanded view of the part of the spectrum responsive to menadiolreduction and oxidation by nitrate.

NR preparations (15), and the 43-kDa , subunit results fromtrypsin treatment of heat-dissociated NR (14, 15, 35). Weobserved two forms of the P subunit, a 58-kDa ,B present inall preparations containing cytochrome b and a 46-kDa ,B thatappeared in addition to the 58-kDa P in aged preparations. Inone preparation we removed cytochrome b from CHAPSO-treated purified NR by chromatography on a hexylagarosecolumn. This enzyme contained the 46-kDa I8 subunit and a12.9-kDa peptide in place of the 58-kDa P. A similar disso-ciation of the E. coli P subunit into protein bands at 43 and10 kDa was observed by Lund and DeMoss (35), a resultattributed to proteolysis of 58-kDa P. Alternatively, thesmall peptide(s) may constitute tightly bound 8 subunit(s)strongly bound to P, similar to the anchor proteins ofsuccinate dehydrogenase (1) and fumarate reductase (34) ofE. coli, now known to be required for quinol oxidation byfumarate reductase (6, 7).Each 226-kDa NR molecule from B. halodenitrificans

possesses at least one molybdenum atom, presumably asso-ciated with the MPT detected in the purified NR prepara-tions by the N. crassa nit-i assay. Our average value of 0.7nmol of molybdenum per nmol of NR is consistent with thelow values reported by others, which ranged from 0.24 to 1.0(21, 43). In addition, eight non-heme irons, probably ar-ranged in [4Fe-4S] clusters, and one heme iron are present.The 1:8 ratio of molybdenum to non-heme iron is consistentwith the metal contents of the NR from Klebsiella aerogenes(43) and B. licheniformis (44), but lower than the 1:12 and1:16 ratios reported by Chaudhry and MacGregor (10) and byAdams and Mortenson (2), respectively, for the E. coli NR.Demonstration by EPR that the states of Mo(V) appeared tobe pH dependent indicates that proton coupling and bindingby anions occurs (26). The reductive profile is consistentwith Mo(VI) -- Mo(V) -* Mo(IV) steps, and the Mo(V) EPRsignals represent a small fraction of the chemically deter-mined Mo (9% for P. aeruginosa [26]). The detectableMo(V) species were also very low in the B. halodenitrificans

NR, even during short reduction times or in reductiveconditions in which nonstoichiometric quantities of dithio-nite were used.Burke and Lascelles (4) demonstrated involvement of qui-

nones in nitrate reduction by gram-positive bacteria by show-ing that 2-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) in-hibited lactate-dependent NR in membrane preparations ofStaphylococcus aureus. The soluble, reduced benzyl violo-gen-NR was not inhibited by HOQNO (4). The main iso-prenoid quinone in Bacillus species is menaquinone withseven isoprene units (MK-7), of which menadione is a solubleanalog. With reduced menadione (menadiol) as the substratefor B. halodenitrificans NR, the Km for nitrate was 50-foldlower than that with MVH, a result consistent with thesuggestion that menadiol serves as the reductant in vivo. Useof duroquinol and MVH yielded similar kinetics for nitrate inthe NR activity of the reductases from E. coli (37) and P.denitrificans (13). Neither duroquinol nor ubiquinol donateselectrons for NR activity in preparations that lack cy-tochrome b (13, 37), suggesting that quinols donate electronsto the heme iron of NR. The direct reduction of cytochromeb557 in NR by menadiol suggests that the B. halodenitrificansenzyme follows this pathway as well.Menaquinone appears to play a pivotal role in the electron

transfer chain of B. halodenitrificans during denitrification.The membrane studies with succinate, malate, and NADHdemonstrate the presence of dehydrogenases able to reducecytochrome b. The presence of cytochrome b557 in purifiedNR and its reduction by menadiol and oxidation by nitrateare consistent with the conclusion that B. halodenitrificansNR is a menaquinol:nitrate oxidoreductase.

ACKNOWLEDGMENTSThis work was supported by National Science Foundation grant

DMB-8718646 to J.L. and W.J.P. and Research Opportunity AwardDMB-8942839 to P.A.K.

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We thank J. J. G. Moura for the EPR analyses and Isabel Mouraand Ming-Yih Liu for helpful discussion and advice.

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