A membrane-bound dissimilatory nitrate reductase from Rhodobacter sphaeroides f.sp. denitrificans

Biochimica et Biophysica Acta 915 (1987) 120-124

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Elsevier BBA 32936

A membrane.bound dissimilatory nitrate reductase from Rkodobacter sphaeroides f.sp. denitrificans Michael D. Byrne and D.J.D. Nicholas Department of Agricultural Biochemistry, Waite Agricultural Research Institute, Unioersity of Adelaide, Glen Osmond (Australia) (Received 16 March 1987)

Key words: Dissimilatory nitrate reductase; Nitrate reduetase; Denitrification; (Rh. sphaeroides)

(1) A dissimilatory nitrate reductase (nitrite:(acceptor) oxidoreductase, EC 1.7.99.4) which is associated with cell membranes has been purified from a photosynthetic bacterium, Rhodobaeter sphaeroides f.sp. denitrificans. (2) The heat-released enzyme, which had a molecular mass of 180 kDa was composed of a major catalytic subunit (120 kDa) and another subunit at 60 kDa, a membrane at*atclunent protein. (3) Molybdenum (1 atom per mol), but not iron, was found in rite purified enzyme; cytochrome was not detected. (4) This molecular structure for nitrate rMuctase is similar to that of many other dissimiintory nitrate reductases.

Introduction The photosynthetic bacterium Rhodobacter sphaeroides f.sp. denitrificans strain IL106 is very versatile in that it fixes dinitrogen gas and respires to oxygen as well as to nitrate [1]. Denitrification in this bacterium follows a pathway similar to that of other bacteria [2], i.e., NO~- --, NO~- --* N20 -'* N 2

The NO 2 reductase, [3,4] and N20-reductase, [5] from this bacterium have been purified to homogeneity and partially characterized. It has also been shown that some of the dinitrogen gas derived from dinitrification can be recycled via nitrogenase [6]. Abbreviations: PMSF, phenylmethylsulphonyl fluoride; LDS, lithium dodecyl sulphate. Correspondence: D.J.D. Nicholas, Department of Agricultural Biochemistry, Waite Agricultural Research Institute, University of Adelaide, Glen Osmond, 5064, South Australia, Australia.

Membrane-bound dissimilatory nitrate reductases have been purified from a variety of bacteria, e.g., Micrococcus denitrificans [7], Bacillus stearothermophilus [8], Paracoccus denitrificans [9], Pseudomonas denitrificans [10] and Escherichia coli [11,12]. Whilst there are many anomalous reports, it is generally considered that dissimilatory nitrate reductases (reviewed in Ref. 13) consist of three subunits, designated a, fl and "y, with approximate molecular masses of 150, 60 and 20 kDa, respectively. The a-subunit has a catalytic function, while the fl-subunit, which can exist in multiple forms (fl and fl'), has a structural function in membrane attachment. The ~,-subunit, which is not found in heat-released preparations, is a b-type cytochrome. Satoh [14,15] has isolated a soluble nitrate reductase containing a c-type cytochrome from Rh. sphaeroides.f.sp, denitrificans strain ILl06. McEwan et al. [16] have examined nitrate reductase activities of several strains of Rhodopseudomonas capsulata grown phototrophically in the presence of nitrate as the sole nitrogen source. They estab-

0167-4838/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

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lished that strains AD2 and BK5 resembled the spontaneous mutant N22DNAR +, in that nitrate reduction was inhibited by either illumination or oxygen but not by NH~-, and that electron flow to nitrate under dark anaerobic conditions generated a cytoplasmic membrane potential. Although they claim that nitrate reductase was located in the periplasmic space of strains AD2 and the mutant N22DNAR + the enzyme was more strongly associated with the cytoplasmic membrane in strain BK5. We report here the purification and partial characterization of a membrane-bound dissimilatory nitrate reductase (nitrite:(acceptor) oxidoreductase, EC 1.7.99.4) from Rh. sphaeroides f.sp. denitrificans strain ILl06, which has properties similar to most other dissimilatory nitrate reductases. Materials and Methods

Bacterium. Rh. sphaeroides f.sp. denitrificans strain ILl06 was kindly provided by Dr. T. Satoh, Department of Biology, Tokyo Metropolitan University, Tokyo 158, Japan. Cells grown photoheterotrophically under denitrifying conditions (20 mM KNO3) in 10 1 sealed bottles were harvested in the late exponential phase of growth and stored at - 15 o C [17]. Enzyme assay. Nitrate reductase activity was measured in open test-tubes (10 × 1.2 cm) at 30 o C. The assay mixture, in a final volume of 1 ml, consisted of 10 #1 enzyme/320 #mol sodium phosphate buffer (pH 7.5)/10 #mol potassium nitrate/0.4 #mol benzyl viologen. The reaction was initiated by the addition of 3 #mol of freshly prepared sodium dithionite in 1% (w/v) NaHCO 3. After 10 min, the reaction was terminated by oxidation of the reduced viologen with vigorous shaking in a vortex mixer. The nitrite produced was measured spectrophotometrically at 540 nm [18] (LKB Ultrospec, model 4050). Purification. All operations were carried out at 4°C. Washed cells were resuspended in 3 vol. 0.1 M Tris-HCl buffer (pH 7.5) with 0.5 mM EDTA, 0.5 mM phenylmethylsulphonyl fluoride (PMSF), 20 # g / m l RNAse, 200 # g / m l DNAse. Cells were broken at 7 MPa in a French pressure cell and the crude extract was centrifuged at 20 000 × g for 20

min. The supernatant ($20) was centrifuged at 200000 x g for 90 min. The pellet (P20o) w a s resuspended to its initial volume in 0.1 M Tris-HC1 buffer (pH 9.0), with 0.5 M NaC1, 0.5 mM EDTA, and 0.5 mM PMSF. This was incubated with shaking at 60 °C for 20 min, cooled on ice, and then centrifuged at 200000 × g for 60 min. The supernatant (Heat $200), dialyzed against 0.1 M Tris-HC1 buffer (pH 9.0) for 15 h, was then loaded onto a DEAE-Sephacel (Pharmacia) column (6 × 2.2 cm) previously equilibrated with the dialysis buffer at a flow rate of 25 ml/h. A linear 0-0.5 M NaC1 gradient (2 × 200 ml) was used to elute the enzyme. The pooled active fractions were concentrated 5-fold in an Amicon ultra.filtration cell (Model 52) fitted with an XM-100 membrane. The concentrated preparation was further purified on a Sephacryl S-200 (Pharmacia) column (70 × 2.2 cm) by eluting with 0.1 M Tris-HC1 buffer (pH 8.0) with 0.1 M NaC1 and deionized M urea. Fractions (2 ml) were collected at a flow rate of 14 ml/h. Calibration of the Sephacryl S-200 column was carried out using blue dextran, aldolase 158 kDa, bovine serum albumin (67 kDa) and ovalbumin (43 kDa) (Pharmacia gel-filtration calibration kits, Pharmacia Fine chemicals). Enzyme kinetics. K m values for NO 3 and reduced benzyl viologen were calculated using an iterative weighted least-squares regression method [19]. Electrophoresis. Native, sodium dodecyl sulphate (SDS) and lithium dodecyl sulphate (LDS) electrophoresis were carried out in vertical slabs according to the system of Laemmli [20]. Gels were fixed in 50% (w/v) trichloroacetic acid and then stained with Coomassie brilliant blue R-250 (0.25%, w/v) in ethanol/acetic acid/water (2.5 : 1 :6.5, v/v) a n d / o r silver [21]. Molecular masses were determined using the following markers: phosphorylase b, 94 kDa; bovine serum albumin, 67 kDa and ovalbumin 43 kDa (Pharmacia electrophoresis molecular weight marker kit, Pharmacia). LDS-gels were stained for haem proteins with 3,3',5,5'-tetramethylbenzidine [22]. Antibody production. Purified nitrate reductase (fraction 6, Table I) was used as antigen for the production of antibodies. A rabbit was injected subcutaneously with 0.4 mg of protein (1 ml) emulsified with an equal volume of Freund's corn-

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plete adjuvant. A second sample was injected intramuscularly a fortnight later. Subsequent intramuscular injections, which did not contain adjuvant, were administered at weekly intervals. The animal was bled 1 week after the fourth injection. The IgG fraction was purified from serum by ammonium sulphate fractionation and DEAE-cellulose chromatography [23]. Immunoelectrophoresis. Rocket and crossed-immunoelectrophoresis was carried out in 1 mm agarose gels, (type C, Pharmacia) cast on polyester sheets (Gel-Bound, FMC Corporation), according to the method of Weeke [24]. Analytical methods. Difference spectra were measured in a Perkin Elmer Lambda 5 dual-beam spectrophotometer. Inductively coupled plasmaoptical emission spectrometry (ICP-OES) was used to measure metal contents of nitric acid digests of purified nitrate reductase [25]. Nitrate reductase activity was determined in native polyacrylamide gels after electrophoresis by sectioning the gels into 1 mm slices and preincubating these in nitrate reductase assay mixture for 30 min at 30 o C. Activity was then determined as described above. Protein content was determined by the method of Bradford [26], using bovine serum albumin (Sigma, A-4503) as a standard. Results and Discussion

The dissimilatory nitrate reductase was purified 120-fold with a yield of 30~ (Table I). The presence of a nitrite reductase interfered with the estimation of nitrate reductase activity in crude extracts. It was found that 0.1 mM KCN inhibited

TABLE I PURIFICATION OF NITRATE REDUCTASE Fraction

Total activity =

Specific activity b

Purity (fold)

Yield (%)

1 Crude extract 2 $2oo 3 P20o 4 heat-S2oo 5 DEAE-Sepha¢~I 6 Scphacryl S-200

184471 4916 186918 136 706 100376 55670

117 12 322 2 389 8431 14038

1.0 0.1 2.8 20.4 72.1 120.0

100 5 101 74 54 30

a nmol N O 2 producexi/min. b nmol N O r p r o d u c e d / r a i n per m g protein.

nitrite reductase activity by 80% whereas that for nitrate reductase was unaffected. Thus, the nitrate reductase activity of the crude extracts in Table I was measured in the presence of 0.1 mM KCN. In our hands, nitrate reductase activity was inhibited by about 8096 with 1 mM KCN, whereas, in his studies, Satoh used this concentration to inhibit nitrite reductase activity [15]. The data in Table I show that nitrate reductase activity was associated with the membrane fraction (fraction 3, Table I), and this is supported by the rocket immunoelectrophoresis data in Fig. 1A. No rocket was formed in lane c (soluble proteins, fraction 3, Table I), confirming the absence of the nitrate reductase antigen from this fraction, whereas strong cross-reactions are shown when washed cells (lane a) and membrane-c, ontaining fractions were used (lanes b and d). These findings are at variance with those reported previously, where the enzyme was located in the soluble fraction [14,15]. Nitrate reductase was eluted from the DEAESephacel column at 0.35 M NaC1. In addition, a completely resolved cytochrome component was eluted at 0.2 M NaCl which was identified as a c-552 cytochrome with a molecular mass of 13.5 kDa and a redox potential of 225 mV [27]. This cytochrome, which is readily dissociated from the membrane-bound enzyme, has been claimed to be an electron donor for nitrate reductase [28]. The K m value for N O r was 0.6 mM, which is lower than 1.6 mM measured in the presence of 1 mM KCN by Satoh [15]. The K m for reduced benzyl viologen was 0.2 #M. Gel-filtration chromatography resulted in two incompletely resolved peaks of nitrate reductase activity, as has also been found for the enzyme from other bacteria [9-12,15]. The inclusion of M urea in the elution buffer, however, resulted in a single reproducible protein peak without loss in enzyme activity. The molecular mass of this ureatreated nitrate reductase was 180 kDa compared with values varying from 60 to 112 kDa for the soluble nitrate reductase reported by Satoh [15]. Crossed-immunoelectrophoresis of this antigen (fraction 6, Table I) into anti-nitrate reductase, resulted in a single precipitin peak (Fig. 1B), indicating that the preparation was immunologicaUy homogeneous. Molybdenum (1 atom per mol cata-

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a

b

c

d

Fig. 2. Two-dimensional electrophoresis of nitrate reductase. First-dimension native electrophoresis (7.5% acrylamide, stained with Coomassie brilliant blue, 75 gg fraction 6, Table I), followed by second-dimension SDS electrophoresis (10% acrylamide, stained with silver). Subunit a is derived from a single parent band (120 kDa) which has not dissociated into subunits b and c which correspond with the fl and fl' subunits from E. coli.

~

!~i!!ii¸!~¸

Fig. 1. (A) Subcellular localization of nitrate reductase by rocket immunoelectrophoresis. Aliquots were electrophoresed (3 h at 10 V/cm) against anti-nitrate reductase (1% (v/v) IgG fraction). Lane 1, washed cells (10 gg protein, suspended in 0.1 M Tris-HC1 buffer (pH 7.5) with 2% (v/v) Nonidet P-40); lane b, crude extract (30 gg); lane c, soluble fraction ($200, 30 gg); lane d, membrane fraction (P2oo, 10 gg). (B) Crossed-immunoelectrophoresis (16 h at 2 V/cm) of membrane fraction (10 #g of P200 fraction, with 2% (v/v) Nonidet P-40) against anti-nitrate reductase (1% (v/v) IgG fraction).

lytic subunit), but no iron, was detected in the purified nitrate reductase by inductive coupled plasma analysis. This is in agreement with the

absence of cytochromes from this preparation confirmed by spectral studies and 3,Y,5,5'-tetramethylbenzidine stained LDS gels. Although this purified enzyme appeared homogeneous by gel filtration chromatography and antigenic reactivity, electrophoresis showed that it was heterogeneous (Fig. 2), as has also been found in dissimilatory nitrate reductases in other bacteria [8-12]. Native electrophoresis of the purified enzyme resulted in one major protein band (120 kDa) plus two weak bands (approx. 60 kDa) of which only the major band had reduced benzyl viologen linked nitrate reductase activity. SDS electrophoresis revealed a similar banding pattern for the purified enzyme. The large-molecular-mass protein (120 kDa) could not be dissociated into the the smaller subunits, even in the presence of 8 M urea. We conclude from these results and the gel-filtration data that the nitrate reductase consists of one a- and one fl-subunit. Two-dimensional electrophoresis of the purified enzyme (Fig. 2) showed that the smaller proteins (b and c) were not derived from the larger one (a). These proteins (a, b and c, Fig. 2) correspond with the a, t - and fl'-subunits from other dissimilatory nitrate reductases, and confirm that the fl-subunit is not a

124 p r e c u r s o r for the a - s u b u n i t [28]. O u r results also show that when the dissimilatory nitrate reductase is p u r i f i e d a f t e r a l k a l i n e h e a t t r e a t m e n t , the y - s u b u n i t is n o t present, in a g r e e m e n t with the results for the e n z y m e f r o m o t h e r b a c t e r i a [8-12]. Thus, the d i s s i m i l a t o r y n i t r a t e r e d u c t a s e f r o m Rh. sphaeroides f.sp. denitrifications is a s s o c i a t e d w i t h m e m b r a n e s a n d h a s similar m o l e c u l a r p r o p erties to those f r o m o t h e r d e n i t r i f y i n g bacteria. Acknowledgements This w o r k was s u p p o r t e d b y a g r a n t to D . J . D . N . f r o m the A u s t r a l i a n R e s e a r c h G r a n t s Scheme. W e a r e grateful to Dr. W.P. M i c h a l s l d for the i m m u n o e l e c t r o p h o r e t i c d a t a as well as for h e l p f u l discussion.

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