Isolation and properties of the NAD(P)H-cytochrome c reductase subunit of Chlamydomonas reinhardii NAD(P)H-nitrate reductase

12 Biochimica et Biophysica A cta, 745 (1983) 12-19 Elsevier

BBA 31604

ISOLATION AND PROPERTIES OF THE NAD(P)H-CYTOCHROME c REDUCTASE SUBUNIT OF CHLAMYDOMONAS REINHARDII NAD(P)H-NITRATE REDUCTASE

EMILIO FERNANDEZ a., and JACOBO CARDENAS b.**

a Departamento de Bioqutmica, Facultad de Biologia y C.S.I.C, Universidad de Sevilla, Sevilla, and b Departamento de Bioquimica, Facultad de Ciencias, Universidad de Ctrdoba, Ctrdoba (Spain)

(Received December 6th, 1982)

Key words: Nitrate reductase subunit; NAD(P)H-cytochrome c reductase; NAD(P)H-nitrate reductase; (C. reinhardiO

Chlamydomonas reinlmrdii wild strain 6145c as well as the mutant strain 104, which lacks NAD(P)H-nitrate reductase (EC 1.6.6.2) activity and molybdenum-containing cofactor, exhibit an ammonia-repressible NAD(P)H-cytochrome c reductase activity of identical molecular size, 44 500. This diaphorase activity, which in both strains produces a fast-moving band on electrophoresis, has been separated from other constitutive diaphorases unrelated to nitrate reduction by means of Blue-agarose affinity chromatography and characterized as the sole NAD(P)H-diaphorase able to reconstitute the native NAD(P)H-nitrate reductase complex by complementation with extracts of mutant 305, which only has reduced flavin or viologen-nltrate reductase activity. The ammonia-repressible NAD(P)H-cytochrome c reductase possesses FAD and a b-type cytochrome, and its kinetic and enzymatic parameters are similar to those previously reported for the diapborase activity of the whole complex, which indicates that the NAD0P)H-cytochrome c reductase of 104 mutant is a true subunit of the Chlamydomonas reinhardii NAD0P)H-nitrate reductase native enzyme.

Introduction

NAD(P)H-nitrate reductase (EC 1.6.6.2) from Chlamydomonas reinhardii is an enzyme complex which utilizes NAD(P)H to reduce nitrate [1]. It also displays two partial activities: an NAD(P)H- diaphorase which reduces several electron acceptors such as ferricyanide, cytochrome c or tetrazolium salts, and a reduced flavin or viologen- nitrate reductase which mediates the reduction of nitrate to nitrite [2-4]. Mutants of C. reinhardii affected in their capability of reducing nitrate have been isolated and characterized [5-9], and by in vitro complementation between a mutant strain uniquely containing the NAD(P)H-diaphorase ac-

* Present address: Departamento de Bioquimica, Facultad de Ciencias, Universidad de C6rdoba, Cbrdoba, Spain.

** To whom correspondence should be addressed.

tivity (mutant 104) and another mutant, which only exhibits reduced flavin or viologen-nitrate reductase activity (mutant 305), reconstitution of the whole NAD(P)H-nitrate reductase native complex has been recently achieved [4].

In fungi and higher plants, ammonia-repressible NAD(P)H-cytochrome c reductases of low molecular weight have been found and proposed as true subunits of their nitrate reductase complex [ 10-12]. On the other hand, flavin and heine have been long since described as prosthetic groups of nitrate reductase, although their exact localization on the possible subunits of the entire complex has not yet been found [1,13]. Moreover, C. reinhardii cells exhibit many NAD(P)H-cytochrome c reductases unrelated to nitrate reduction in addition to the ammonia-repressible diaphorase [7,9].

In the present work, the isolation and purification of a low molecular weight ammonia-repressi-

0167-4838/83/$03.00 © 1983 Elsevier Science Publishers B.V.

ble NAD(P)H-cytochrome c reductase f romthe mutant strain 104 of C. reinhardii are described. This diaphorase activity is also found in wild cells of C. reinhardii grown on nitrate, in addition to the diaphorase activity of the nitrate reductase complex. It contains FAD and cytochrome b557, and is the only diaphorase activity capable of reconstituting the whole nitrate reductase enzyme by in vitro complementation with extracts of derepressed 305 cells. Therefore we propose that this NAD(P)H-cytochrome c reductase is the diaphorase subunit of C. reinhardii nitrate reductase.

Experimental procedure

Materials. NADH, NADPH and horse heart cytochrome c were purchased from Boehringer, 4-nitroblue tetrazolium chloride and benzidine from Serva, Blue 2-agarose and FAD from Sigma and Sephadex G-25, Sephadex G-100 and DEAE- Sephacel from Pharmacia.

Culture methods. C. reinhardii 6145c and mutants 104 and 305 obtained from this wild type [5] were grown in 10 mM NH4C1 liquid media [4]. When required, the cells were collected at the exponential phase of growth, derepressed on 4 mM KNO 3 for 5.5 h, harvested, frozen in liquid nitrogen and stored until use.

Preparation o f extracts and purification procedure. Step 1. Cell pellets from liquid nitrogen were broken by thawing in 50 mM Tris-HC1 buffer (pH 7.5)/0.1 mM dithioerythritol/0.1 mM EDTA/20 /~M FAD/50 mM NaC1 (4 ml /g wet weight) (buffer 1) [7]. The resulting homogenate was centrifuged at 30000 × g, 15 min.

Step 2. The supernatant was applied to a column (3 x 7.2 cm) of DEAE-Sephacel, previously equilibrated with buffer l, and the column was washed with 400 ml of the same buffer. Cyto- chrome c reductase activity was eluted with a continuous gradient of 50-300 mM NaC1 in buffer 1 at about 0.15 M NaC1.

Step 3. The most active fractions were Com- bined and precipitated with ammonium sulfate 30-60% saturation. The suspension was spun down at 27000 x g, 20 min, and the precipitate, dis- solved in 40 mM potassium phosphate buffer (pH 7.0)/0.1 mM dithioerythritol/0.1 mM EDTA/10 #M FAD (buffer 2), was applied to a Sephadex

13

G-100 column (3 x 22 cm) equilibrated with buffer 2, and eluted with the same buffer. 6-ml fractions were collected.

Step 4. The most active fractions were pooled and passed through a Blue-agarose column (1.5 x 4.5 cm) equilibrated with buffer 2, at a flow rate of 10 m l / h per cm 2. 4-ml fractions were collected.

Step 5. NAD(P)H-cytochrome c reductase, ammonia-repressible, eluted from step 4, was precipitated with ammonium sulfate up to 60% saturation and, after centrifugation at 27 000 x g, 15 rain, resuspended in 10 mM potassium phosphate buffer (pH 6.5)/0.1 mM dithioerythritol/0.1 mM EDTA/10 #M FAD (buffer 3). The resulting solution was desalted in a Sephadex G-25 column (1.5 x 6.5 cm) previously equilibrated with buffer 3.

Step 6. The desalted solution was applied to a Blue-agarose column (1.5 x 4.5" cm) equilibrated with buffer 3 at a flow rate of 7 ml /h per cm 2 and, after washing the column with 50 ml buffer 3, the enzyme was eluted with the same buffer 100 mM in KC1.

Enzyme assays. NAD(P)H-cytochrome c reductase activity was measured spectrophotometri- cally by following the reduction of cytochrome c at 550 nm in a 1 ml final volume mixture containing 100 nmol cytochrome c, 400 nmol NADH or NADPH, 100 #mol Tris-HC1 buffer (pH 7.5), and appropriate amounts of enzyme. NAD(P)H-nitrate reductase activity was assayed by determining the nitrite formed by enzymatic reduction of nitrate [14]. Units of activity are expressed as #mol sub- strate transformed per rain, and specific activity as units per mg protein.

Electrophoresls. Gel electrophoresis was performed at 4°C in 7.5% polyacrylamide gels according to the method of Jovin et al. [15]. Heme was located on the gels as described by Solomonson et ai. [16], and NAD(P)H-diaphorase activity as by Wang and Raper [17]. Determination of molecular weights by disc-gel electrophoresis was carried out as described by Hedrick and Smith [18] using as standards: ovalbumin (M r 43000), bovine serum albumin (67000), lactate dehydrogenase (EC 1.1.1.27) (140000) and catalase (EC 1.11.1.6) (232000).

Complementation procedure. Complementation with nitrate derepressed 305 mutant extracts was

14

performed as described elsewhere [4]. Analytical methods. Flavin was analyzed fluo-

rimetrically as previously described [16]. Protein was estimated according to the method of Brad- ford [19], using bovine serum albumin as standard. Nitrite was determined by the diazotization procedure of Snell and Snell [20]. Spectrophotometric determinations were performed in a Pye-Unicam SP 8-100 spectrophotometer, spectra were recorded in a DW-2a TM UV-VIS Aminco spectrophotometer, and fluorimetric analysis was carried out in an Aminco-Bowman spectrofluorimeter.

Stokes radius and sedimentation coefficient determination. Stokes radii were determined as described by Siegel and Monty [21] by using a Bio- Gel A-1.5m column (2.5 × 50 cm) under conditions detailed elsewhere [4]. Ovalbumin (2.75 nm), bovine serum albumin (3.5 nm), alcohol dehydrogenase (EC 1.1.1.1) (4.6 nm) and catalase (5.2 nm) were used as standards.

Sedimentation coefficients were determined by sucrose-density-gradient centrifugation according to Martin and Ames [22]. 0.2-ml samples containing the enzyme a n d / o r standard proteins were applied to a linear gradient 5-20% (w/v) sucrose in 50 mM Tris-HC1 buffer, pH 7.5, with 0.1 mM dithioerythritol/0.1 mM E D T A / 1 0 #M FAD, and centrifuged at 4°C for 14 h at 45000 rpm in a Spinco L2-65B ultracentrifuge with an SW-56 Ti rotor. After centrifugation, 3-drop fractions were collected. Cytochrome c (1.85 S), alcohol dehydrogenase (7.6 S) and catalase (11.3 S) were used as standards.

Results

Purification of ammonia-repressible diaphorase of the 104 mutant

T h e p re sence of several cons t i tu t ive NAD(P)H-diaphorase activities unrelated to nitrate reductase represents a serious hindrance to the isolation and character izat ion of the NAD(P)H-cytochrome c reductase repressible by ammonia [9]. Two kinds of control were used to follow this diaphorase activity along the purification procedure: its R F = 0.58 in 7.5% polyacrylamide gels [7,9] and its singular ability to reconstitute the whole NAD(P)H-nitrate reductase native complex by in vitro complementation with

extracts of derepressed 305 cells [4,8]. All the diaphorases present in partially purified

preparations (step 3) of mutant 104 were adsorbed when applied on a Blue-agarose column (1.5 × 4.5 cm) equilibrated with 40 mM potassium phosphate buffer (pH 6.5)/0.1 mM dithioerythritol/0.1 mM E D T A / 1 0 #M FAD at a flow rate of 8 m l / h per cm 2. In these conditions all the diaphorases, the ammonia-repressible and those unrelated to nitrate reductase, were eluted together when the column was washed with the same buffer as above except for 1 mM N A D P H (results not shown). When diaphorase preparations at the same step of purification were applied to a Blue-agarose column in buffer 2, at a flow rate of 10 m l / h per cm 2, only the ammonia-repressible NAD(P)H-cytochrome c reductase was obtained, as detected on polyacrylamide gel electrophoresis (Fig. 1). By washing the column with the same buffer but 1 mM in NADPH, the other NAD(P)H-cytochrome c reductases unrelated to nitrate reduction were subse- quently eluted. These unrelated diaphorases were incapable of reconstituting the NAD(P)H-nitrate reductase complex when mixed with derepressed 305 extracts. After ammonium sulfate precipita-

HH D w

12o

= 4 ~ e ~ 1 mM

- / \ ~ 8 0 NADPH

-,- _ / ,,, I . . ,

z 0 5 10 15 20

FRACTION NUMBER

Fig. 1. Isolation of the ammonia-repressible NAD(P)H-diaphorase of mutant 104 from C. reinhardii. A partially purified preparation from mutant 104 (step 3) was subjected to affinity chromatography in a Blue-agarose column under conditions described in Experimental procedure. Where indicated by the arrow the column was washed with buffer 2, I mM in NADPH. Samples of fractions 11, 13 and 18 were subjected to electrophoresis on 7.5% polyacrylamide gels. Gels stained for NADPH-diaphorase activity are shown over their corresponding fractions.

tion, the preparation of ammonia-repressible diaphorase was desahed through a Sephadex G-25 column equilibrated with buffer 3 and, finally, adsorbed on a second Blue-agarose column at a flow rate of 7 ml /h per cm 2. The enzyme was eluted, after washing the column with the same buffer but 100 mM in KC1. The preparation obtained is not homogeneous (five bands of proteins in 7.5% polyacrylamide gels) although it contains a unique NAD(P)H-cytochrome c reductase protein of high activity (15.1 U/rag) which represents 70% of the total protein and is able to complement with the partially purified reduced benzyl viologen- nitrate reductase from mutant 305 (results not shown).

Molecular properties The purified enzyme has a Stokes radius of 3.0

nm and a sedimentation coefficient of 3.55 S (Ta- ble I). From these data, and assuming a specific volume of 0.725 cm 3. g-i , a molecular weight of 44000 has been calculated for this diaphorase by the method of Siegel and Monty [21]. A frictional ratio of 1.28 has been also calculated, which indicates a near-spherical shape for this protein. A very similar molecular weight value was obtained by using the method of Hedrick and Smith [18] with polyacrylamide gels of 4.5, 5.5, 6.5 and 7.5% (w/v) total acrylamide concentration (Table I).

Flavin and heme content In order to establish the nature of the possible

prosthetic groups present in the ammonia-repressible NAD(P)H-cytochrome c reductase, five sam- pies of purified enzyme (step 6), each containing 150 mU, were run separately and simultaneously in 7.5% polyacrylamide gels. After electrophoresis completion, one gel was sliced into 0.6-cm por-

TABLE I

MOLECULAR PROPERTIES OF THE AMMONIA- REPRESSIBLE DIAPHORASE

Stokes radius (a) 3.0 nm Sedimentation coefficient (s20.,.) 3.55 S Molecular weight from a and s20.w 44000 Molecular weight from electrophoretic data 45 000 Frictional ratio 1.28

15

tions which were used to detect NADPH-cytochrome c reductase activity. As shown in Fig. 2, the peak of activity was coincidental with the activity band detected directly on the gels run in parallel in the same experiment. Another gel was stained specifically for heme. The band corresponding to heme iron was that containing the NAD(P)H-cytochrome c reductase activity (Fig. 2). When another gel from the same experiment was sliced into 0.4-cm portions and analyzed spec- trofluorimetrically, only the portions with NAD(P)H-cytochrome c reductase exhibited a fluorescence spectrum typical of flavin (Fig. 3).

Absorption spectra The absorpt ion spectrum of purified

NAD(P)H-cytochrome c reductase related to nitrate reductase from mutant 104 in oxidized state shows a maximum at 413 nm (Fig. 4A). Upon reduction with NADPH, two new absorption bands at 557 nm (a) and 527 nm (fl) ap- peared together with a shift of the peak at 413 nm to 424 nm (Soret) accompanied by an increase in absorption. The same results were obtained with NADH or dithionite (results not shown). The difference spectrum of reduced minus oxidized enzyme shows more clearly the absorption bands of

u

UJ ~E

"I- n . O.

A I • m 1

C .... .... ~ " i ~ ' ° ~ . . . . . . . . . . . . . . . . ~: '

Z

0 2 4 6 8

MIC.fl~ATION DISTANCE (cm}

Fig. 2. Heme presence in the ammonia-repressible NAD(P)H- cytochrome c reductase of mutant 104 from C. reinhardii. 7.55 polyacrylamide gels, each containing 150 mU of purified enzyme (step 6), were subjected to electrophoresis. Lower part: gels were specifically stained for NADH-diaphorase (A), NADPH-diaphorase (B) and heme (C). Upper part: NADPH- cytochrome c reductase, expressed as mU/fraction, was de-

tected by sficing the gel into 0.6-cm discs. Arrow indicates the tracking dye.

16

100:

v w

z

ul

~. so . i u .

w

ul

~ 0 ' ' ' 600 WAVELENQTH (nm)

Fig. 3. Fluorescence spectra of ammonia-repressible NAD(P)H-diaphorase. 150 mU of purified enzyme (step 6) were subjected to electrophoresis on 7.5% polyacrylamide gels. 0.4-cm slices were ground and extracted to determine flavin as indicated in Experimental procedure. A: emission spectrum of 0.5 nmol FAD. B: emission spectrum of the slice corresponding to the ammonia-repressible diaphorase. The sensitivity in B is 10-fold higher than in A. The excitation wavelength was 434 n m .

a b-type cytochrome (peaks at 557, 527 and 424 nm in reduced state) and an absorption minimum at 450 nm and a shoulder at 480 nm typical of the flavin presence (Fig. 4B).

Kinetic properties The ammonia-repressible NAD(P)H-cy to -

chrome c reductase from 104 can use either N A D H or N A D P H as electron donor, and cytochrome c, FAD, oxidized viologens, dichlorophenolin- dophenol, tetrazolium salts and ferricyanide as electron acceptors. Routine assays were performed with either donor and cytochrome c as acceptor. The apparent K m values for N A D H and N A D P H at cytochrome c saturation, calculated from the corresponding Woolf's plots, were 270 and 150 #M, respectively. The values for cytochrome c at N A D H and N A D P H saturation, calculated from the same type of representation, were 40 and 25 /~M, respectively. The mechanism suggested by the initial-velocity data representation according to Cleland [23] is of the class hexa uni ping pong (results not shown).

LU C.) Z o

/~ A

I t t

/ AA 10,01 B

4 0 0 450 500 550

WAVELENOTH (nm) Fig. 4. Absorption and difference spectra of ammonia-repressible NAD(P)H-diaphorase. A: Solid line: absorption spectrum of ammonia-repressible NAD(P)H-cytochrome c reductase (0.1 mg) dialyzed against 40 mM potassium phosphate buffer, pH 7.0. Dashed line: absorption spectrum of the enzyme reduced with small amounts of solid NADPH. B: difference spectrum of the reduced enzyme minus the oxidized enzyme. Spectra of reduced enzyme were recorded immediately after the addition of NADPH at room temperature.

Inhibition Treatment with 10 #M p-hydroxymercuri-

benzoate or incubation of the enzyme at 45°C, 5 min, abolished the NAD(P)H-cytochrome c reductase activity. Added N A D H or N A D P H (concentrations higher than 100 #M) protected against inactivation by the sulfhydryl reagent. Similarly, exogenous FAD (concentrations higher than 10 #M) protected against heat inactivation (results not shown).

Discussion

The ammonia-repressible NAD(P)H-cytochrome c reductase related to the nitrate reductase complex has been isolated from the 104 mutant of Chlamydomonas reinhardii. The procedure of sep- aration from other NAD(P)H-cytochrome c reductases unrelated to nitrate reduction and present in the cell extract took advantage of the lower affinity of the nitrate-inducible diaphorase for the chromophore groups of Blue-agarose in compari- son with that shown by the other constitutive diaphorases. A unique NAD(P)H-diaphorase with high activity (15.1 U/mg) is obtained which is able to complement with partially purified preparations of reduced benzyl viologen-nitrate reductase of mutant 305 from C. reinhardii. This diaphorase activity appears also as a rapidly migrating band of R F - - 0 . 5 8 o n 7 .5~ polyacrylamide gels and it has been found in derepressed 301, 102 and 307 mutants as well as in 6145c wild cells of C. reinhardii [7-9].

The molecular size of this diaphorase (44 500) is notably less than that of the whole complex (241000) [24]. The repressible diaphorase of 6145c wild strain has the same molecular weight and is also capable of reconstituting the whole nitrate reductase complex when complemented with derepressed 305 extracts [8,24].

17

NAD(P)H-diaphorases repressible by ammonia and smaller than the NAD(P)H-nitrate reductase complex have been described in several organisms, as summarized in Table II. In Neurospora crassa, Aspergillus nidulans and spinach, specific reconstitution of nitrate reductase activity from derepressed diaphorases of low molecular weight has led to the conclusion that these diaphorases are true subunits of the nitrate reductase complex [ 1,12,37,38]. Similar conclusions have been reached for repressible NAD(P)H-cytochrome c reductases from barley, maize and Nicotiana tabacum [30,32-36].

Mutant 104 has no molybdenum cofactor and thus lacks nitrate reductase and xanthine dehydrogenase activities [6]. Its ammonia-repressible diaphorase has, however, flavin and heme iron. Al- though the type of flavin has not been determined, the requirement of FAD (but not of FMN or other flavins) for activity along the purification steps and the specific protecting effect of this nucleotide against heat denaturation point to FAD as the flavin coenzyme of the ammonia-repressible NAD(P)H-cytochrome c reductase. FAD as a con- stituent of nitrate reductases has been described in Chlorella vulgaris [16], A nkistrodesmus braunii [39] and N. crassa [40], and its protecting effect against thermal denaturation has been reported in the same enzyme from Chlorella fusca [41], spinach

TABLE II

NAD(P)H-DIAPHORASES RELATED TO NITRATE REDUCTASE FROM DIFFERENT ORGANISMS

The electron acceptor used was always cytochrome c. Size values in the first column correspond to the diaphorase activity associated with the nitrate reductase complex.

Organism Size Electron Ref. (S) donor

A. nidulans 7.6-7.8 NADPH cnx from A. nidulans 7.6 4.5 NADPH N. crassa 6.8-7.9 NADPH nit-i from IV. crassa 4-4.5 NADPH Barley shoots a 7.7-8.8 3.7-3.8 NAD(P)H Spinach leaves 8. I 3.7 NADH Maize scutellum 8.0 4.0 NADH N. tabacum 7.6 4.1 NADH cnx-68/2 from N. tabacum 7.6 4.1 NADH C. reinhardii 8.8 3.55 NAD(P)H 104 from C. reinhardii 3.55 NAD(P)H

25,26 26 10,27,28 28,29 30-33 12 34 35.36 36 24

a NADH-cytochrome c reductases associated with proteins of 3.1 S, 5.6 S and 6.8 S have been reported in aged leaves of barley [33].

18

[42] and C. reinhardii [3,43]. The absorption spectrum of ammonia-repressi-

ble diaphorase from 104 shows clearly the ex- istence of a cytochrome/,557 with absorption bands at 413 nm in its oxidized form, and at 424 (Soret), 527 (a) and 557 (fl) nm in its reduced state. Purified nitrate reductase of the parental wild strain 6145c exhibits the same spectral properties [24]. All the nitrate reductases of eukaryotes studied until now have a cytochrome b of the same class [1,13].

The presence of heme has been also reported in the middle-sized nitrate reductase of mutant nit-I of N. crassa with only NADPH-cytochrome c reductase activity [44] and in the high molecular weight nitrate reductase complex of the cnx-68/2 mutant of N. tabacum devoid of nitrate reductase activity and exhibiting only NADH-cytochrome c reductase activity [36]. These facts notwithstand- ing, the precise conditions of heme b incorporation into the polypeptide complex formed by aggrega- tion of equal or different subunits, as well as its location and functional role within the whole nitrate reductase complex, are challenging prob- lems which are still unresolved [1].

On the other hand, the kinetic mechanism hexa uni ping pong, the K m values for donors and substrates and the specific protection afforded by pyridine nucleotide or FAD against inactivation of the ammonia repressible diaphorase are very similar to the corresponding properties of the partial diaphorase activity of the nitrate reductase complex from parental wild strain [43], although higher K m values than those previously reported [45] have been obtained for NADH and NADPH, which would explain the low affinity for the chromophore groups of Blue-agarose exhibited by the ammonia-repressible diaphorase.

All these facts taken together force us to propose that this NAD(P)H-cytochrome c reductase repressible by ammonia is a true subunit of the NAD(P)H-nitrate reductase complex from C reinhardii. This subunit bears the diaphorase activity of the complex, contains FAD and cytoehrome b557 as prosthetic groups, and connects, through a molybdenum cofactor, the electron transport re- sponsible for assimilatory nitrate reduction with the terminal moiety of the whole nitrate reductase.

Acknowledgements

The authors thank Professor M. Losada for provision of facilities. This work was supported by Grants from Comisi6n Asesora de Investigaci6n (Spain) and Phihps Research Laboratories (The Netherlands). One of us (E.F.) thanks the Minis- terio de Educaci6n y Ciencia (Spain) for a fellow- ship.

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Documents

Isolation and properties of the NAD(P)H-cytochrome c reductase subunit of Chlamydomonas reinhardii NAD(P)H-nitrate reductase