- Email: [email protected]
A spectrophotometric assay for dissimilatory nitrite reductases
ANALYTICAL
BIOCHEMISTRY
172,420-426
A Spectrophotometric
(1988)
Assay for Dissimilatory
Nitrite Reductases
C. L. HULSE,* J. M. TIEDJE,t AND B. A. AVERILL*” *Department qfChemistry. Universit.v of Virginia, Charlottesville, Virginia 22901, and?Department Soil Sciences, Michigan State University, East Lansing, Michigan 48824
of Crop and
Received January 20, 1988 A spectrophotometric assay for dissimilatory nitrite reductases has been developed utilizing mammalian cytochrome c (equine heart) as reductant and spectrophotometric agent. The copper-containing nitrite reductase from Achromobacter cvcloclastes has been shown to have apparent K,‘s for reduced cytochrome c and nitrite of 86 f 5 and 5.63 f 0.03 PM, respectively. The heme cd-containing enzyme from Pseudomonas stutzeri was shown to have apparent K,‘s for reduced cytochrome c and nitrite of 260 CL60 and I .8 + 0.8 pM, respectively. This assayrepresents a simple, general method for consistently evaluating the activity of the copper- and heme cd-containing nitrite reductases that are capable of utilizing readily available mammalian cytochrome c as electron donor and should be useful for mechanistic studies of these enzymes. 0 1988 Academic
Press. Inc.
denitrification; nitrite reductases; spectrophotometric assay;cytochrome c:Achromobacter cvcloclastes; Pseudomonas stutzeri KEY WORDS:
Denitrification is the respiratory or dissimilatory reduction of nitrate and nitrite to dinitrogen. The key step is the reduction of NO; to gaseous nitrogen oxides, catalyzed by two classes of nitrite reductases: the heme-containing (cytochrome cdl) enzymes isolated from Pseudomonas aeruginosa (l), Pseudo-
these systems, artificial redox mediators such as phenazine methosulfate (PMQ2 hydroquinone. methylviologen, benzylviologen, tetramethyl-p-phenylenediamine, and pphenylenediamine are used to shuttle electrons to the nitrite reductase from artificial electron donors such as ascorbate, dithionite, monas stutzeri (2), Paracoccus denitrificans FADH2, and NADH. (3-5). and Alcaligenes faecalis (6); and the The nitrite reductases isolated thus far have copper-containing enzymes isolated from also been shown to utilize endogenous proAchromobacter cycloclastes (7,8), Alcaligenes teins such as reduced c-type cytochromes or sp. (9), Rhodopseudomonas sphaeroides azurins as their natural electron donors (l( 10, 1 1), and Alcaligenes faecalis strain S-6 12). Kinetics parameters for these species have been determined ( 1- 12,15- 17), but ( 12). Mechanistic analysis of denitrification in these systems has been hindered by the their utility in spectrophotometric analyses lack of a simple and convenient spectrophohas been limited by their availability and/or tometric assay for nitrite reductase activity. spectral characteristics. Substitution of mammalian cytochromes for the endogenous cyTo date, most analyses are performed as time point assays utilizing manometric or gas tochrome species has also been reported chromatographic methods to follow gaseous (5,8,18). In this paper, we describe a spectroproduct formation, chemical methods to fol- photometric assay for the nitrite reductase activity for the copper-containing enzyme from low nitrite disappearance, or radioactive methods to follow both (13,14). In most of A. cycloclastes and the heme cd, enzyme ’ Abbreviations used: PMS, phenazine methosulfate; Mes, 2-(N-morpholino)ethanesulfonic acid.
I To whom all correspondence should be addressed. 0003-2697188
$3.00
Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
420
DISSIMILATORY
NITRITE
from P. stutzeri, utilizing the inexpensive, commercially available mammalian c-type cytochrome (equine heart) as the electron donor and spectrophotometric agent. The assay is linear in activity to 0.1 OD units (550 nm) or 15 nmol cytochrome c oxidized per minute, and the rate of cytochrome c oxidation is equal to that of the nitrite to nitric oxide conversion. The assay is used to determine the Michaelis constants for reduced cytochrome c and nitrite for both enzymes. The spectrophotometric assay represents a simple and convenient method for studying the nitrite reductases that are capable of substituting the more readily obtainable mammalian cytochrome c for the natural or more commonly used artificial electron donors. MATERIALS
AND
METHODS
A. cycloclastes (ATCC 2192 1) was obtained from the American Type Culture Collection, Rockville, Maryland. Difco-Bacto tryptic soy broth (0370-05-7) was purchased from Scientific Products, Columbia, Maryland. 2-(N-Morpholino)ethanesulfonic acid (Mes) and tris(hydroxymethyl)aminoethane (ultrapure) were obtained from Research Organics, Cleveland, Ohio. Cytochrome c (equine horse heart, type III), N-l-naphthylethylenediamine hydrochloride, and phenylSepharose CL-4B were purchased from Sigma Chemical Co., St. Louis, Missouri. Sephacryl S-200 and HA-Ultrogel were obtained from Pharmacia Fine Chemicals, Piscataway, New Jersey and LKB, Gaithersburg, Maryland, respectively. DE-52 anion exchange resin was purchased from Whatman, Clifton, New Jersey. All other reagents used were reagent grade. Preparation qfPurijied Nitrite Reductase from A. cyc,loclastes Growth of A. cycloclastes. Cultures were maintained at room temperature (20-25°C) in liquid cultures composed of 3.0% tryptic soy broth, 3.0 mM KN03, and 1.0 PM CuSO4. Cells were cultivated semi-anaerobi-
REDUCTASES
ASSAY
421
tally on 3.0% tryptic soy broth, 3.0 mM KN03, and 1.O PM CuS04. Twenty-liter cultures, prepared via a series of 10% inocula (20,200, and 2000 ml) grown at 30°C for 12 h, were grown at 30°C for 18 h, at which time production of gas and depletion of nitrite was apparent. Cells were harvested by centrifugation at 10,OOOg for 10 min at 0-4”C, yielding 80- 1OOg of cells (wet wt). Preparation of cell extract. Freshly harvested cells were suspended in 0.05 M potassium phosphate buffer, pH 7.0 (2 ml/g), and were disrupted in a stainless steel beaker with a Heat Systems sonicator (Model W-220F) operating at 60% power for 15 min at 0-4°C. The sonicated suspension was centrifuged at 10,OOOgat 0-4°C for 30 min to remove cellular debris. The resulting supernatant was dialyzed twice against 10 liters of 10 mM TrisHCl buffer, pH 7.5, at 0-4°C for 12 to 18 h. Precipitated material was removed by ultracentrifugation at 100,OOOg for 90 min at o-4°C. Purification of nitrite reductase. Coppercontaining nitrite reductase was isolated using modifications ofthe procedures described by Iwasaki and Matsubara (6) and Liu et al. (19). All column chromatography was carried out at 0-4°C. All chromatography buffers were made 1 PM in hydrogen peroxide3 and maintained at pH 7.5. The ultracentrifuged cell extract was applied to a DE-52 anion exchange column (5 X 30 cm) equilibrated with 10 mM Tris-HCl, pH 7.5 (buffer A). The column was washed with 150 ml of buffer A, followed by 150 ml of 100 mM Tris-HCl, pH 7.5 (buffer B), and protein was eluted with a 400-ml linear gradient (100 to 250 mM Tris-HCI, pH 7.5). Fractions (4 ml) were collected and assayed for the ability to reduce 1 mM NaN02/3 h/200 ~1 protein fraction using the PMS/ascorbate assay described below. ’ Hz02 is used to avoid autoreduction of the enzyme during chromatography, which results in a modified form of the enzyme that no longer reacts with reduced cytochrome c (C. L. Hulse and B. A. Averill, unpublished results).
422
HULSE.
TIEDJE.
The protein fraction was made 1 M in ammonium sulfate (132 g/liter) and centrifuged at 10,OOOgat 0-4°C for 30 min. The resulting supernatant was applied to a phenyl-Sepharose CL-4B column (5 X 20 cm) equilibrated with 1 M ammonium sulfate in buffer B (buffer C). The column was washed with 100 ml buffer C and protein eluted with a 400-ml linear gradient (1 to 0 M ammonium sulfate in buffer B). Green fractions (4 ml) were collected and concentrated to 4 ml with an Amicon ultrafiltration apparatus fitted with a PM 30 membrane. The concentrated sample was chromatographed on a Sephacryl S-200 column (5 X 60 cm) equilibrated and eluted with buffer B. Green fractions (2 ml) were pooled and applied to a HA-Ultrogel column (5 X 30 cm) equilibrated in buffer B. The column was washed with 100 ml of buffer B and the protein eluted with a 400-ml linear gradient (0 to 50 mM potassium phosphate in buffer B). Green fractions (2 ml) were combined and concentrated to 3 ml using an Amicon ultrafiltration device fitted with a PM 30 membrane. The concentrated sample was rechromatographed on a Sephacryl S-200 column as described above. Fractions having A180/A458 < 17 were collected, concentrated, and electrophoresed on 10% gels using the neutral pH, nondissociating, discontinuous system of Williams and Reisfeld as described by Hames and Rickwood (20), except that the reservoir buffer contained 75 mM glycine instead of diethylbarbituric acid. The protein recovered from the gels had an A&&s = 15. Concentration of protein was determined using t = 4.8 mrv-’ cm-’ at 458 nm (C. L. Hulse and B. A. Averill, unpublished results). Preparation qf denitr$jling extracts of P. stutzeri. Cultures of P. stutzeri (JM 300) maintained on agar slants, were grown on 3.0% tryptic soy broth and 3.0 mM KNOX at 30°C until nitrite was depleted. Cells were harvested by centrifugation at 10,OOOg for 30 min at 0-4°C. Cells were resuspended in 0.05 M potassium phosphate buffer, pH 7.0, and
AND
AVERILI
sonicated for 15 min at 0-4°C using a Heat Systems sonicator operating at 60% power. The resulting suspension was centrifuged at 10,OOOgfor 30 min at 0-4°C to remove cellular debris. The extract was assayed for its ability to reduce nitrite in the PMS/ascorbate assay and used without further purification. PMS/ascorbate assay. Nitrite reductase activity was monitored by nitrite disappearance utilizing ascorbate/PMS as reductant. Enzyme (200 ~1) was added to 2.8 ml of 1 mM nitrite, 100 pM PMS, and 50 mM ascorbate in 50 mM Mes buffer, pH 6.2. Reactions were carried out in 5-ml glass serum bottles sealed with rubber septa. After 3 h at room temperature, aliquots were analyzed for nitrite by the diazotization method described below. Enzyme fractions not producing a visible pink color (i.e., those which had completely consumed the nitrite) in the diazotization assay were collected. Determination of nitrite and nitrate. Nitrite and nitrate were determined calorimetrically by the diazotization method described by Nicholas and Nason (2 1). To 1.O ml of sample was added 0.5 ml of 58 mM sulfanilic acid in 1.5 N HCI and 0.5 ml of 775 PM N- 1-naphthylethylenediamine. HCI in 1.5 N HCI. The color of the resulting mixture was allowed to develop for 20-30 min. Nitrate was determined similarly after reduction to nitrite with powdered zinc. Cytochrome c assa!). Cytochrome c (125 mg) was dissolved in 25 ml of 50 mM potassium Mes buffer, pH 6.2, and stored at 0-4°C. An aliquot (6-8 ml) of the cytochrome stock solution was placed in the minichamber ofan Amicon 8MC ultrafiltration apparatus fitted with a YM5 membrane and reduced with excess solid sodium dithionite. The reduced solution was diafiltered at room temperature against at least 10 vol of 50 mM potassium Mes buffer, pH 6.2. Cytochrome c solutions were diluted to the indicated concentrations using t550 (reduced) = 29.5 mrv-’ cm-‘. Sodium nitrite solutions were prepared in 50 mM potassium Mes buffer, pH 6.2. All solutions were placed in serum bottles
DISSIMILATORY
NITRITE
(5- 10 ml), sealed with rubber septa, and deoxygenated by flushing the solutions with argon for at least 30 min prior to use. All solutions were maintained under a positive argon pressure throughout the procedure. Transfers of solutions were made utilizing Hamilton gas-tight syringes and standard anaerobic techniques. Assays were performed at room temperature in anaerobic cuvettes sealed with rubber septa or 5-ml serum bottles fitted with butyl rubber stoppers. Reactions were initiated by addition of enzyme or nitrite. Absorbance changes were followed using a Cary 2 19 or a Perkin-Elmer 320 spectrophotometer (At.&reduced-oxidized) = 2 1.2 mM-’ cm-’ for cytochrome c). Nitric oxide was the only gaseous product formed under these conditions; it was detected and quantitated using a Perkin-Elmer 9 10 gas chromatograph, fitted with a Porapak column and electron capture detector, operating under standard running conditions, which included an oven temperature of 55°C and argon-methane (95%-5%) carrier gas. Data were analyzed with standard least-squares programs and are reported as the value + 1 standard deviation. Concentrations of reduced cytochrome c and nitrite are indicated in the figure legends. RESUL.TS
AND
DISCUSSION
Studies on the Copper-Containing from ‘4. cycloclastes
Enzyme
Gas chromatographic versus cytochrome c assay. A comparison of the nitrite reductase activity obtained utilizing the cytochrome c spectrophotometric assay and the gas chromatographic assay for the appearance of nitric oxide is shown in Fig. 1. The rate of oxidation of cytochrome c is the same as the rate of nitric oxide formation, within experimental error. The major differences between the two assays sh’own in Fig. 1 are the time required to obtain the data, the number of mechanical manipulations involved, the number of calculations required to obtain useful data, and the reproducibility of the data. The
REDUCTASES
423
ASSAY
EOC q Reduced Cyt c oxldat,on 8 Nitric oxide producilon
150
v ; IOOE c
5oL
k
50
100
150
TIME (mans)
FIG. I. Gas chromatographic assay versus the cytochrome c assay.The rate of reduced cytochrome c’oxidation as measured in the spectrophotometricassay (Q was compared to the rate of nitric oxide production as measured by gas chromatography (0). The concentrations ot cytochrome (’ and nitrite were 335 PM and 1.7 mM. respectively. Achronzohactrr cycloclastc~.s nitrite reductase (0. I Kg) was assayed in a total assay volume of 625 ~1 in either a 2-mm path length anaerobic cuvette or a 5-ml anaerobic serum bottle at 20-2X. Error bars are shown for the gas chromatographic assay.Errors in the spectrophotometric assay were consistently and significantly smaller (<: lo%) than those for the CC assayand therefore the error bars are not shown for clarity. The rate ofoxidation of cytochrome c is the same as the rate of nitric oxide formation. within experimental error.
cytochrome c assay utilizes three solutions and three anaerobic transfers with immediate procurement of data from the spectrophotometer. The gas chromatographic analysis requires the manipulation of gaseous samples, the preparation of standard curves, and extensive data manipulations. The error in the spectrophotometric assay is consistently and significantly less than that for the GC assay. The cytochrome c assay has all the advantages of a continuous spectrophotometric assay, whereas the gas chromatographic analysis suffers from the disadvantages typical of a discontinuous timepoint assay. The utiliza-
HULSE. TIEDJE. AND AVERILL
/
/
1
30 TIME
(mlns)
FIG. 2. Linearity of the cytochrome c assay. Typical spectrophotometric tracings show the oxidation of reduced cytochrome c with varying amounts of enzyme. Conditions of the assay were similar to those in Fig. I. Inset: a replot ofthe absorbance change occurring during the first minute as a function of the amount of enzyme. The linearity of the reduced cytochrome c spectrophotometric assay was confirmed by demonstrating that the initial rate of cytochrome c oxidation was proportional to the amount of enzyme present. The assay is linear in the amount of enzyme to ASsoof 0.1 units ( 15 nmol cytochrome c oxidized) per minute.
that the assay is, to a very good approximation, linear in amount of enzyme to aA of 0.1 units (15 nmol cytochrome c oxidized) per minute. The spectrophotometric assay thus represents a convenient method for quantifying enzyme concentrations during enzyme purification. Km for c.vtochrome c and nitrite. In order for the assay to be applicable for determination of total enzyme activity over a wide range of enzyme concentrations, it is essential that cytochrome c and nitrite be present at saturating concentrations. The Michaelis constants for reduced cytochrome c and nitrite were therefore determined; the data are presented in Figs. 3 and 4. The K,,, for reduced cytochrome c in the presence of a saturating levels of nitrite is 86 + 5 PM, while the apparent Km for nitrite at saturating levels of reduced cytochrome c is 5.63 + 0.03 PM. (The deviation from linearity at high nitrite concentration in Fig. 4 is real and may be due to reaction of two nitrites to produce small amounts of N20 (C. L. Hulse and B. A. Averill, unpublished results).) Since typical concentrations of reduced cytochrome c and nitrite in the assays were at least 300 and 50 PM, respectively, saturating levels of substrates are present in the assay.
I
tion of reduced cytochrome c as both the electron source and spectrophotometric agent in the analysis of nitrite reductase activity is therefore of great technical importance, resulting in significantly greater precision with a major decrease in the time and effort required. Linearity of the cytochrome c assay. The linearity of the reduced cytochrome c spectrophotometric assay was confirmed by demonstrating that the initial rate of cytochrome oxidation was proportional to the amount of enzyme present. As shown in Fig. 2, a replot of the absorbance change per unit time as a function of enzyme concentration from a set of typical spectrophotometric traces reveals
I
I
I/vCrMe’
I
300.~ mln) zoo.-
25
0
25 l/[cyt
50
C] CmM-‘1
FIG. 3. Determination of the K, for reduced cytochrome c for the nitrite reductase from Achromobacfer cycloclastes. The K,,, for cytochrome c was determined from a Lineweaver-Burk double reciprocal plot. A. cycloclustes nitrite reductase (0.54 rg) was assayed in a total assayvolume of625 ~1 in a 2-mm path length anaerobic cuvette. The concentration of nitrite was I .7 mM. The Km for reduced cytochrome c in the presence of saturating levels of nitrite is 86 * 5 f.tM.
DISSIMILATORY
-020
I 0
-0 IO IdNO;]
1 010
NITRITE
I 0 20
CPM-‘1
FIG. 4. Determination of the K, for nitrite for the nitrite reductase from Achromobacter cycloclastes. The K,,, for nitrite was determined from a Lineweaver-Burk double reciprocal plot. A. cvc(oc(aste~ nitrite reductase (0.22 pg) was assayed in a total volume of 625 r.d in a 2-mm path length anaerobic cuvette. The concentration of cytochrome c was 280 ELM.The K, for nitrite in the presence of saturating levels of cytochrome c is 5.63 + 0.03 PM.
REDUCTASES
ASSAY
425
ited. As a result, only minimal data are available on the steady-state kinetics properties of the various nitrite reductases. Some of these data have been obtained using artificial electron donors and rather cumbersome and tedious techniques. The data presented here for the copper enzyme from A. cycloclastes and the heme cd enzyme from P. stutzeri demonstrate that precise and convenient kinetics measurements are possible with this system and that the assay can be utilized to study any nitrite reductase that is capable of oxidizing mammalian cytochrome c. Detailed mechanistic studies of the A. cycloclastes copper enzyme and the P. stutzeri heme cd enzyme are in progress. ACKNOWLEDGEMENTS
Studies on the cd Enzymefrom
P. stutzeri
To demonstrate the generality of the assay, it was used to determine the Michaelis constants for extracts containing the heme cdcontaining nitrite reductase from P. sfutzeri. Assays performed at pH 7.0 yielded values of 260 + 60 and 1.8 + 0.8 PM for the apparent K,,,‘s of reduced cytochrome c and nitrite, respectively (data not shown). Michaelis constants for nitrite ranging from 5.3 to 53 prvt have been reported for the cd enzyme from P. aeruginosa (22). Values for the K,,, of reduced cytochrome I~ have not been reported previously for the cd enzyme system. That the mammalian cytochrome is relatively inefficient as an electron donor, however, is suggested by the lower rate of electron transfer between equine heart cytochrome c and the cd system relative to the endogenous reductants (5). CONCLUSIONS
Although various cytochromes have been reported to act as electron donors and their kinetics parameters have been determined for the nitrite reductases isolated thus far, their utilization in spectrophotometric assays and steady-state kinetics studies has been lim-
We thank E. Weeg-Aerssens for helpful discussions and D. L. Fitzgerald for providing partially purified extracts of the P. stufzeri nitrite reductase. This research was supported by the USDA Competitive Research Grants Office (83-CRCR- 1292) and the NSF Chemistry of Life Processes Program (CHE-8607681). B.A.A. was an Alfred P. Sloan Foundation Fellow, I98 I - 1985.
REFERENCES I. Yamanaka, T., and Okunuki, K. (1983) B&him. Blophys. Acta 67,379-393. 2. Komama, T. (1970) Plant CellPhyssiol. 11,231-239. 3. Pichinoty. F. (1969) Arch. Microbial. 69,3 14-329. 4. Newton, N. (1969) Biochim. Biophys. .4cta 185, 316-331. 5. Timkovich, R.. Dhesi. R., Martinkus. K. J., Robinson. M. K., and Rea, T. M. ( 1982) Arch. Biochem. Blophys. 215,47-58. 6. Iwasaki, H., and Matsubara. T. (197 1) J. Biochem (Tokyo) 69,847-857. 7. Iwasaki, H., and Matsubara, T. (1972) J. Biochem (Tokyo) 71,645-652. 8. Iwasaki, H., Noji, S., and Shidara, S. (1975) J. Biothem (Tokyo) 78,355-36 I. 9. Masuko. M.. Iwasaki, H., Sakurai, T., Suzuki, S., and Nakahara, A. (1984) J. Biochem. (Tokyo) 96, 447-454. 10. Sawada, E., Satoh, T.. and Kitamura. H. (1978) Plant Ceil Physiol. 19, 1339- I35 1. I I. Michalski, W., and Nicholas, D. J. D. (1985) Biochim. Biophys. Acta 828,130- 137. 12. Katutani, T., Watanabe, H., Arima, K.. and Beppu, T. (1981) J. Biochem. (Tokyo) 89,453-461.
426
HULSE,
TIEDJE,
13. Payne, W. J. (198 1) Denitrification. pp. 54-67. Wiley. New York. 14. Tiedje, J. M. (1982) in Methods ofSoil Analysis. Part 2: Chemical and Microbiological Properties (Page, A. L., Miller, R. H., and Keeney. D. R., Eds.), Agronomy Monogr. No. 9, pp. 10 II- 1026. Amer. Sot. Agron., Madison, WI. 15. Bessieres, P., and Henry, Y. (1984) Biocltirnic 66, 313-318. 16. Tordi, M. G.. Silvestrini. M. C.. Colosimo. A., Tuttobello. L., and Brunori. M. (1985) Biochetn. J. 230.797-805. 17. Ranaweera, S. S., and Nicholas, D. J. D. (I 985) Biochew. IN. lo,41 5-423. 18. Robinson. M. K., Martinkus, K.. Kennelly, P. J..
AND
AVERILL and Timkovich, 392 l-3926.
R.
(1979)
Bioclrc~mhlq~
18,
19. Liu. M.-Y., Liu, M.-C., Payne, W. J.. and LeGall, ( 1986) J. Bac,leriol. 166.604-608.
J.
20. Hames. B. D. ( 198 1) in Gel Electrophoresis of Proteins: A Practical Approach (Hames, B. D.. and Rickwood, D., Eds.), pp. 30-31, IRL Press, London. 2 1. Nicholas, D. J. D.. and Nason. A. (1957) in Methods of Enzymology (Colowick. S. P., and Kaplan, N. 0.. Eds.). Vol. 3. pp. 981-984, Academic Press, New York. 22. Henry. Y.. and Bessieres, 259-289.
P. (1984)
Biochitnic66,
BIOCHEMISTRY
172,420-426
A Spectrophotometric
(1988)
Assay for Dissimilatory
Nitrite Reductases
C. L. HULSE,* J. M. TIEDJE,t AND B. A. AVERILL*” *Department qfChemistry. Universit.v of Virginia, Charlottesville, Virginia 22901, and?Department Soil Sciences, Michigan State University, East Lansing, Michigan 48824
of Crop and
Received January 20, 1988 A spectrophotometric assay for dissimilatory nitrite reductases has been developed utilizing mammalian cytochrome c (equine heart) as reductant and spectrophotometric agent. The copper-containing nitrite reductase from Achromobacter cvcloclastes has been shown to have apparent K,‘s for reduced cytochrome c and nitrite of 86 f 5 and 5.63 f 0.03 PM, respectively. The heme cd-containing enzyme from Pseudomonas stutzeri was shown to have apparent K,‘s for reduced cytochrome c and nitrite of 260 CL60 and I .8 + 0.8 pM, respectively. This assayrepresents a simple, general method for consistently evaluating the activity of the copper- and heme cd-containing nitrite reductases that are capable of utilizing readily available mammalian cytochrome c as electron donor and should be useful for mechanistic studies of these enzymes. 0 1988 Academic
Press. Inc.
denitrification; nitrite reductases; spectrophotometric assay;cytochrome c:Achromobacter cvcloclastes; Pseudomonas stutzeri KEY WORDS:
Denitrification is the respiratory or dissimilatory reduction of nitrate and nitrite to dinitrogen. The key step is the reduction of NO; to gaseous nitrogen oxides, catalyzed by two classes of nitrite reductases: the heme-containing (cytochrome cdl) enzymes isolated from Pseudomonas aeruginosa (l), Pseudo-
these systems, artificial redox mediators such as phenazine methosulfate (PMQ2 hydroquinone. methylviologen, benzylviologen, tetramethyl-p-phenylenediamine, and pphenylenediamine are used to shuttle electrons to the nitrite reductase from artificial electron donors such as ascorbate, dithionite, monas stutzeri (2), Paracoccus denitrificans FADH2, and NADH. (3-5). and Alcaligenes faecalis (6); and the The nitrite reductases isolated thus far have copper-containing enzymes isolated from also been shown to utilize endogenous proAchromobacter cycloclastes (7,8), Alcaligenes teins such as reduced c-type cytochromes or sp. (9), Rhodopseudomonas sphaeroides azurins as their natural electron donors (l( 10, 1 1), and Alcaligenes faecalis strain S-6 12). Kinetics parameters for these species have been determined ( 1- 12,15- 17), but ( 12). Mechanistic analysis of denitrification in these systems has been hindered by the their utility in spectrophotometric analyses lack of a simple and convenient spectrophohas been limited by their availability and/or tometric assay for nitrite reductase activity. spectral characteristics. Substitution of mammalian cytochromes for the endogenous cyTo date, most analyses are performed as time point assays utilizing manometric or gas tochrome species has also been reported chromatographic methods to follow gaseous (5,8,18). In this paper, we describe a spectroproduct formation, chemical methods to fol- photometric assay for the nitrite reductase activity for the copper-containing enzyme from low nitrite disappearance, or radioactive methods to follow both (13,14). In most of A. cycloclastes and the heme cd, enzyme ’ Abbreviations used: PMS, phenazine methosulfate; Mes, 2-(N-morpholino)ethanesulfonic acid.
I To whom all correspondence should be addressed. 0003-2697188
$3.00
Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
420
DISSIMILATORY
NITRITE
from P. stutzeri, utilizing the inexpensive, commercially available mammalian c-type cytochrome (equine heart) as the electron donor and spectrophotometric agent. The assay is linear in activity to 0.1 OD units (550 nm) or 15 nmol cytochrome c oxidized per minute, and the rate of cytochrome c oxidation is equal to that of the nitrite to nitric oxide conversion. The assay is used to determine the Michaelis constants for reduced cytochrome c and nitrite for both enzymes. The spectrophotometric assay represents a simple and convenient method for studying the nitrite reductases that are capable of substituting the more readily obtainable mammalian cytochrome c for the natural or more commonly used artificial electron donors. MATERIALS
AND
METHODS
A. cycloclastes (ATCC 2192 1) was obtained from the American Type Culture Collection, Rockville, Maryland. Difco-Bacto tryptic soy broth (0370-05-7) was purchased from Scientific Products, Columbia, Maryland. 2-(N-Morpholino)ethanesulfonic acid (Mes) and tris(hydroxymethyl)aminoethane (ultrapure) were obtained from Research Organics, Cleveland, Ohio. Cytochrome c (equine horse heart, type III), N-l-naphthylethylenediamine hydrochloride, and phenylSepharose CL-4B were purchased from Sigma Chemical Co., St. Louis, Missouri. Sephacryl S-200 and HA-Ultrogel were obtained from Pharmacia Fine Chemicals, Piscataway, New Jersey and LKB, Gaithersburg, Maryland, respectively. DE-52 anion exchange resin was purchased from Whatman, Clifton, New Jersey. All other reagents used were reagent grade. Preparation qfPurijied Nitrite Reductase from A. cyc,loclastes Growth of A. cycloclastes. Cultures were maintained at room temperature (20-25°C) in liquid cultures composed of 3.0% tryptic soy broth, 3.0 mM KN03, and 1.0 PM CuSO4. Cells were cultivated semi-anaerobi-
REDUCTASES
ASSAY
421
tally on 3.0% tryptic soy broth, 3.0 mM KN03, and 1.O PM CuS04. Twenty-liter cultures, prepared via a series of 10% inocula (20,200, and 2000 ml) grown at 30°C for 12 h, were grown at 30°C for 18 h, at which time production of gas and depletion of nitrite was apparent. Cells were harvested by centrifugation at 10,OOOg for 10 min at 0-4”C, yielding 80- 1OOg of cells (wet wt). Preparation of cell extract. Freshly harvested cells were suspended in 0.05 M potassium phosphate buffer, pH 7.0 (2 ml/g), and were disrupted in a stainless steel beaker with a Heat Systems sonicator (Model W-220F) operating at 60% power for 15 min at 0-4°C. The sonicated suspension was centrifuged at 10,OOOgat 0-4°C for 30 min to remove cellular debris. The resulting supernatant was dialyzed twice against 10 liters of 10 mM TrisHCl buffer, pH 7.5, at 0-4°C for 12 to 18 h. Precipitated material was removed by ultracentrifugation at 100,OOOg for 90 min at o-4°C. Purification of nitrite reductase. Coppercontaining nitrite reductase was isolated using modifications ofthe procedures described by Iwasaki and Matsubara (6) and Liu et al. (19). All column chromatography was carried out at 0-4°C. All chromatography buffers were made 1 PM in hydrogen peroxide3 and maintained at pH 7.5. The ultracentrifuged cell extract was applied to a DE-52 anion exchange column (5 X 30 cm) equilibrated with 10 mM Tris-HCl, pH 7.5 (buffer A). The column was washed with 150 ml of buffer A, followed by 150 ml of 100 mM Tris-HCl, pH 7.5 (buffer B), and protein was eluted with a 400-ml linear gradient (100 to 250 mM Tris-HCI, pH 7.5). Fractions (4 ml) were collected and assayed for the ability to reduce 1 mM NaN02/3 h/200 ~1 protein fraction using the PMS/ascorbate assay described below. ’ Hz02 is used to avoid autoreduction of the enzyme during chromatography, which results in a modified form of the enzyme that no longer reacts with reduced cytochrome c (C. L. Hulse and B. A. Averill, unpublished results).
422
HULSE.
TIEDJE.
The protein fraction was made 1 M in ammonium sulfate (132 g/liter) and centrifuged at 10,OOOgat 0-4°C for 30 min. The resulting supernatant was applied to a phenyl-Sepharose CL-4B column (5 X 20 cm) equilibrated with 1 M ammonium sulfate in buffer B (buffer C). The column was washed with 100 ml buffer C and protein eluted with a 400-ml linear gradient (1 to 0 M ammonium sulfate in buffer B). Green fractions (4 ml) were collected and concentrated to 4 ml with an Amicon ultrafiltration apparatus fitted with a PM 30 membrane. The concentrated sample was chromatographed on a Sephacryl S-200 column (5 X 60 cm) equilibrated and eluted with buffer B. Green fractions (2 ml) were pooled and applied to a HA-Ultrogel column (5 X 30 cm) equilibrated in buffer B. The column was washed with 100 ml of buffer B and the protein eluted with a 400-ml linear gradient (0 to 50 mM potassium phosphate in buffer B). Green fractions (2 ml) were combined and concentrated to 3 ml using an Amicon ultrafiltration device fitted with a PM 30 membrane. The concentrated sample was rechromatographed on a Sephacryl S-200 column as described above. Fractions having A180/A458 < 17 were collected, concentrated, and electrophoresed on 10% gels using the neutral pH, nondissociating, discontinuous system of Williams and Reisfeld as described by Hames and Rickwood (20), except that the reservoir buffer contained 75 mM glycine instead of diethylbarbituric acid. The protein recovered from the gels had an A&&s = 15. Concentration of protein was determined using t = 4.8 mrv-’ cm-’ at 458 nm (C. L. Hulse and B. A. Averill, unpublished results). Preparation qf denitr$jling extracts of P. stutzeri. Cultures of P. stutzeri (JM 300) maintained on agar slants, were grown on 3.0% tryptic soy broth and 3.0 mM KNOX at 30°C until nitrite was depleted. Cells were harvested by centrifugation at 10,OOOg for 30 min at 0-4°C. Cells were resuspended in 0.05 M potassium phosphate buffer, pH 7.0, and
AND
AVERILI
sonicated for 15 min at 0-4°C using a Heat Systems sonicator operating at 60% power. The resulting suspension was centrifuged at 10,OOOgfor 30 min at 0-4°C to remove cellular debris. The extract was assayed for its ability to reduce nitrite in the PMS/ascorbate assay and used without further purification. PMS/ascorbate assay. Nitrite reductase activity was monitored by nitrite disappearance utilizing ascorbate/PMS as reductant. Enzyme (200 ~1) was added to 2.8 ml of 1 mM nitrite, 100 pM PMS, and 50 mM ascorbate in 50 mM Mes buffer, pH 6.2. Reactions were carried out in 5-ml glass serum bottles sealed with rubber septa. After 3 h at room temperature, aliquots were analyzed for nitrite by the diazotization method described below. Enzyme fractions not producing a visible pink color (i.e., those which had completely consumed the nitrite) in the diazotization assay were collected. Determination of nitrite and nitrate. Nitrite and nitrate were determined calorimetrically by the diazotization method described by Nicholas and Nason (2 1). To 1.O ml of sample was added 0.5 ml of 58 mM sulfanilic acid in 1.5 N HCI and 0.5 ml of 775 PM N- 1-naphthylethylenediamine. HCI in 1.5 N HCI. The color of the resulting mixture was allowed to develop for 20-30 min. Nitrate was determined similarly after reduction to nitrite with powdered zinc. Cytochrome c assa!). Cytochrome c (125 mg) was dissolved in 25 ml of 50 mM potassium Mes buffer, pH 6.2, and stored at 0-4°C. An aliquot (6-8 ml) of the cytochrome stock solution was placed in the minichamber ofan Amicon 8MC ultrafiltration apparatus fitted with a YM5 membrane and reduced with excess solid sodium dithionite. The reduced solution was diafiltered at room temperature against at least 10 vol of 50 mM potassium Mes buffer, pH 6.2. Cytochrome c solutions were diluted to the indicated concentrations using t550 (reduced) = 29.5 mrv-’ cm-‘. Sodium nitrite solutions were prepared in 50 mM potassium Mes buffer, pH 6.2. All solutions were placed in serum bottles
DISSIMILATORY
NITRITE
(5- 10 ml), sealed with rubber septa, and deoxygenated by flushing the solutions with argon for at least 30 min prior to use. All solutions were maintained under a positive argon pressure throughout the procedure. Transfers of solutions were made utilizing Hamilton gas-tight syringes and standard anaerobic techniques. Assays were performed at room temperature in anaerobic cuvettes sealed with rubber septa or 5-ml serum bottles fitted with butyl rubber stoppers. Reactions were initiated by addition of enzyme or nitrite. Absorbance changes were followed using a Cary 2 19 or a Perkin-Elmer 320 spectrophotometer (At.&reduced-oxidized) = 2 1.2 mM-’ cm-’ for cytochrome c). Nitric oxide was the only gaseous product formed under these conditions; it was detected and quantitated using a Perkin-Elmer 9 10 gas chromatograph, fitted with a Porapak column and electron capture detector, operating under standard running conditions, which included an oven temperature of 55°C and argon-methane (95%-5%) carrier gas. Data were analyzed with standard least-squares programs and are reported as the value + 1 standard deviation. Concentrations of reduced cytochrome c and nitrite are indicated in the figure legends. RESUL.TS
AND
DISCUSSION
Studies on the Copper-Containing from ‘4. cycloclastes
Enzyme
Gas chromatographic versus cytochrome c assay. A comparison of the nitrite reductase activity obtained utilizing the cytochrome c spectrophotometric assay and the gas chromatographic assay for the appearance of nitric oxide is shown in Fig. 1. The rate of oxidation of cytochrome c is the same as the rate of nitric oxide formation, within experimental error. The major differences between the two assays sh’own in Fig. 1 are the time required to obtain the data, the number of mechanical manipulations involved, the number of calculations required to obtain useful data, and the reproducibility of the data. The
REDUCTASES
423
ASSAY
EOC q Reduced Cyt c oxldat,on 8 Nitric oxide producilon
150
v ; IOOE c
5oL
k
50
100
150
TIME (mans)
FIG. I. Gas chromatographic assay versus the cytochrome c assay.The rate of reduced cytochrome c’oxidation as measured in the spectrophotometricassay (Q was compared to the rate of nitric oxide production as measured by gas chromatography (0). The concentrations ot cytochrome (’ and nitrite were 335 PM and 1.7 mM. respectively. Achronzohactrr cycloclastc~.s nitrite reductase (0. I Kg) was assayed in a total assay volume of 625 ~1 in either a 2-mm path length anaerobic cuvette or a 5-ml anaerobic serum bottle at 20-2X. Error bars are shown for the gas chromatographic assay.Errors in the spectrophotometric assay were consistently and significantly smaller (<: lo%) than those for the CC assayand therefore the error bars are not shown for clarity. The rate ofoxidation of cytochrome c is the same as the rate of nitric oxide formation. within experimental error.
cytochrome c assay utilizes three solutions and three anaerobic transfers with immediate procurement of data from the spectrophotometer. The gas chromatographic analysis requires the manipulation of gaseous samples, the preparation of standard curves, and extensive data manipulations. The error in the spectrophotometric assay is consistently and significantly less than that for the GC assay. The cytochrome c assay has all the advantages of a continuous spectrophotometric assay, whereas the gas chromatographic analysis suffers from the disadvantages typical of a discontinuous timepoint assay. The utiliza-
HULSE. TIEDJE. AND AVERILL
/
/
1
30 TIME
(mlns)
FIG. 2. Linearity of the cytochrome c assay. Typical spectrophotometric tracings show the oxidation of reduced cytochrome c with varying amounts of enzyme. Conditions of the assay were similar to those in Fig. I. Inset: a replot ofthe absorbance change occurring during the first minute as a function of the amount of enzyme. The linearity of the reduced cytochrome c spectrophotometric assay was confirmed by demonstrating that the initial rate of cytochrome c oxidation was proportional to the amount of enzyme present. The assay is linear in the amount of enzyme to ASsoof 0.1 units ( 15 nmol cytochrome c oxidized) per minute.
that the assay is, to a very good approximation, linear in amount of enzyme to aA of 0.1 units (15 nmol cytochrome c oxidized) per minute. The spectrophotometric assay thus represents a convenient method for quantifying enzyme concentrations during enzyme purification. Km for c.vtochrome c and nitrite. In order for the assay to be applicable for determination of total enzyme activity over a wide range of enzyme concentrations, it is essential that cytochrome c and nitrite be present at saturating concentrations. The Michaelis constants for reduced cytochrome c and nitrite were therefore determined; the data are presented in Figs. 3 and 4. The K,,, for reduced cytochrome c in the presence of a saturating levels of nitrite is 86 + 5 PM, while the apparent Km for nitrite at saturating levels of reduced cytochrome c is 5.63 + 0.03 PM. (The deviation from linearity at high nitrite concentration in Fig. 4 is real and may be due to reaction of two nitrites to produce small amounts of N20 (C. L. Hulse and B. A. Averill, unpublished results).) Since typical concentrations of reduced cytochrome c and nitrite in the assays were at least 300 and 50 PM, respectively, saturating levels of substrates are present in the assay.
I
tion of reduced cytochrome c as both the electron source and spectrophotometric agent in the analysis of nitrite reductase activity is therefore of great technical importance, resulting in significantly greater precision with a major decrease in the time and effort required. Linearity of the cytochrome c assay. The linearity of the reduced cytochrome c spectrophotometric assay was confirmed by demonstrating that the initial rate of cytochrome oxidation was proportional to the amount of enzyme present. As shown in Fig. 2, a replot of the absorbance change per unit time as a function of enzyme concentration from a set of typical spectrophotometric traces reveals
I
I
I/vCrMe’
I
300.~ mln) zoo.-
25
0
25 l/[cyt
50
C] CmM-‘1
FIG. 3. Determination of the K, for reduced cytochrome c for the nitrite reductase from Achromobacfer cycloclastes. The K,,, for cytochrome c was determined from a Lineweaver-Burk double reciprocal plot. A. cycloclustes nitrite reductase (0.54 rg) was assayed in a total assayvolume of625 ~1 in a 2-mm path length anaerobic cuvette. The concentration of nitrite was I .7 mM. The Km for reduced cytochrome c in the presence of saturating levels of nitrite is 86 * 5 f.tM.
DISSIMILATORY
-020
I 0
-0 IO IdNO;]
1 010
NITRITE
I 0 20
CPM-‘1
FIG. 4. Determination of the K, for nitrite for the nitrite reductase from Achromobacter cycloclastes. The K,,, for nitrite was determined from a Lineweaver-Burk double reciprocal plot. A. cvc(oc(aste~ nitrite reductase (0.22 pg) was assayed in a total volume of 625 r.d in a 2-mm path length anaerobic cuvette. The concentration of cytochrome c was 280 ELM.The K, for nitrite in the presence of saturating levels of cytochrome c is 5.63 + 0.03 PM.
REDUCTASES
ASSAY
425
ited. As a result, only minimal data are available on the steady-state kinetics properties of the various nitrite reductases. Some of these data have been obtained using artificial electron donors and rather cumbersome and tedious techniques. The data presented here for the copper enzyme from A. cycloclastes and the heme cd enzyme from P. stutzeri demonstrate that precise and convenient kinetics measurements are possible with this system and that the assay can be utilized to study any nitrite reductase that is capable of oxidizing mammalian cytochrome c. Detailed mechanistic studies of the A. cycloclastes copper enzyme and the P. stutzeri heme cd enzyme are in progress. ACKNOWLEDGEMENTS
Studies on the cd Enzymefrom
P. stutzeri
To demonstrate the generality of the assay, it was used to determine the Michaelis constants for extracts containing the heme cdcontaining nitrite reductase from P. sfutzeri. Assays performed at pH 7.0 yielded values of 260 + 60 and 1.8 + 0.8 PM for the apparent K,,,‘s of reduced cytochrome c and nitrite, respectively (data not shown). Michaelis constants for nitrite ranging from 5.3 to 53 prvt have been reported for the cd enzyme from P. aeruginosa (22). Values for the K,,, of reduced cytochrome I~ have not been reported previously for the cd enzyme system. That the mammalian cytochrome is relatively inefficient as an electron donor, however, is suggested by the lower rate of electron transfer between equine heart cytochrome c and the cd system relative to the endogenous reductants (5). CONCLUSIONS
Although various cytochromes have been reported to act as electron donors and their kinetics parameters have been determined for the nitrite reductases isolated thus far, their utilization in spectrophotometric assays and steady-state kinetics studies has been lim-
We thank E. Weeg-Aerssens for helpful discussions and D. L. Fitzgerald for providing partially purified extracts of the P. stufzeri nitrite reductase. This research was supported by the USDA Competitive Research Grants Office (83-CRCR- 1292) and the NSF Chemistry of Life Processes Program (CHE-8607681). B.A.A. was an Alfred P. Sloan Foundation Fellow, I98 I - 1985.
REFERENCES I. Yamanaka, T., and Okunuki, K. (1983) B&him. Blophys. Acta 67,379-393. 2. Komama, T. (1970) Plant CellPhyssiol. 11,231-239. 3. Pichinoty. F. (1969) Arch. Microbial. 69,3 14-329. 4. Newton, N. (1969) Biochim. Biophys. .4cta 185, 316-331. 5. Timkovich, R.. Dhesi. R., Martinkus. K. J., Robinson. M. K., and Rea, T. M. ( 1982) Arch. Biochem. Blophys. 215,47-58. 6. Iwasaki, H., and Matsubara. T. (197 1) J. Biochem (Tokyo) 69,847-857. 7. Iwasaki, H., and Matsubara, T. (1972) J. Biochem (Tokyo) 71,645-652. 8. Iwasaki, H., Noji, S., and Shidara, S. (1975) J. Biothem (Tokyo) 78,355-36 I. 9. Masuko. M.. Iwasaki, H., Sakurai, T., Suzuki, S., and Nakahara, A. (1984) J. Biochem. (Tokyo) 96, 447-454. 10. Sawada, E., Satoh, T.. and Kitamura. H. (1978) Plant Ceil Physiol. 19, 1339- I35 1. I I. Michalski, W., and Nicholas, D. J. D. (1985) Biochim. Biophys. Acta 828,130- 137. 12. Katutani, T., Watanabe, H., Arima, K.. and Beppu, T. (1981) J. Biochem. (Tokyo) 89,453-461.
426
HULSE,
TIEDJE,
13. Payne, W. J. (198 1) Denitrification. pp. 54-67. Wiley. New York. 14. Tiedje, J. M. (1982) in Methods ofSoil Analysis. Part 2: Chemical and Microbiological Properties (Page, A. L., Miller, R. H., and Keeney. D. R., Eds.), Agronomy Monogr. No. 9, pp. 10 II- 1026. Amer. Sot. Agron., Madison, WI. 15. Bessieres, P., and Henry, Y. (1984) Biocltirnic 66, 313-318. 16. Tordi, M. G.. Silvestrini. M. C.. Colosimo. A., Tuttobello. L., and Brunori. M. (1985) Biochetn. J. 230.797-805. 17. Ranaweera, S. S., and Nicholas, D. J. D. (I 985) Biochew. IN. lo,41 5-423. 18. Robinson. M. K., Martinkus, K.. Kennelly, P. J..
AND
AVERILL and Timkovich, 392 l-3926.
R.
(1979)
Bioclrc~mhlq~
18,
19. Liu. M.-Y., Liu, M.-C., Payne, W. J.. and LeGall, ( 1986) J. Bac,leriol. 166.604-608.
J.
20. Hames. B. D. ( 198 1) in Gel Electrophoresis of Proteins: A Practical Approach (Hames, B. D.. and Rickwood, D., Eds.), pp. 30-31, IRL Press, London. 2 1. Nicholas, D. J. D.. and Nason. A. (1957) in Methods of Enzymology (Colowick. S. P., and Kaplan, N. 0.. Eds.). Vol. 3. pp. 981-984, Academic Press, New York. 22. Henry. Y.. and Bessieres, 259-289.
P. (1984)
Biochitnic66,