A dissimilatory nitrate reductase from Neurospora crassa

466

BIOCFIIMICA ET BIOPHYSICA ACTA

BBA 25 038

A DISSIMILATORY

NITRATE

REDUCTASE

FROM

NE UROSPORA CRASSA D. J. D. N I C H O L A S * AND P. J. W I L S O N

Chemical Microbiology Department, Long Ashton Research Station, University of Bristol, Bristol (Great Britain) (Received S e p t e m b e r 23rd, 1963)

SUMMARY

A dissimilatory nitrate reductase (cytochrome :nitrate oxidoreductase, EC 1.9.6.I) has been purified 35-fold from submerged felts of Neurospora crassa. I t requires iron as well as molybdenum for its activity and is thus similar to the dissimilatory enzyme found in denitrifying bacteria. Reduced methyl or benzyl viologen was the most effective hydrogen donor and under these conditions there is no evidence for a flavin requirement. NADPHz, an effective hydrogen donor for the assimilatory nitrate reductase (NADH~: nitrate oxidoreductase, EC 1.6.6.1 and NAD(P)Hi :nitrate oxidoreductase, EC 1.6.6.2), did not function for the purified dissimilatory enzyme. Although iron concentrated in purified fractions of the enzyme the most purified preparation contained less iron than did less pure ones. It is shown that the purified enzyme contains a molybdenum-protein component which m a y also be common to the assimilatory enzyme. Assimilatory and dissimilatory nitrate reduction systems differ only in the penultimate electron transfer sequence to the terminal nitrate reductase which is a molybdenum-containing protein. There is no close parallel between the activity of the dissimilatory B V H : n i t r a t e oxidoreductase and that of the NADPH~ : cytochrome c oxidoreductase, (EC 1.6.2.3) in the most purified fractions as is known to occur with the assimilatory N A D P H , : n i t r a t e reductase and cytochrome c reductase. The purified B V H : n i t r a t e reductase does not utilize NADPH~ or F A D H 2 as hydrogen donors and has negligible NADPH2:cytochrome c oxidoreductase activity.

INTRODUCTION

Two types of nitrate reductase enzymes that reduce nitrate to nitrite have been characterized in microorganisms. The assimilatory one concerned in the first step reduction of nitrate to cell nitrogen is a flavoprotein containing molybdenum as a functional constituent 1-4. The other, the dissimilatory enzyme found when nitrate serves as an alternative hydrogen acceptor to oxygen, contains iron in addition to Abbreviations : BVH, benzyl viologen (N,N'-dibenzyl-4,4'-dipyridyl) ; MVH, m e t h y l viologen (N,N'-dimethyl-4,4'-dipyridyl). * P r e s e n t address : D e p a r t m e n t of Biochemistry, W a i t e Institute, University of Adelaide, South Australia.

Biochim. Bioph3,s. Acta, 86 (1964) 466-476

DISSIMILATORY NITRATE REDUCTASE FROM Neurospora crassa

467

molybdenum 5. I t has been shown that the two types occur in the same bacterium when grown under different oxygen pressures with nitrate as the sole source of nitrogen e. WALKER AND NICI-IOLAS7 reported that in submerged felts of Neurospora crassa a dissimilatory enzyme which required iron and molybdenum for its activity was formed but in the aerial hyphae the assimilatory enzyme only was present. In this paper further properties of the dissimilatory nitrate reductase (cytochrome : nitrate oxidoreductase, EC 1.9.6.1 ) from Neurospora are discussed. METHODS

Enzyme. source N. crassa macroconidial Wild type 5297 a, was grown in a modified FRIES medium with sodium nitrate as the only source of nitrogen s. The fungus was grown at 28 ° in the dark for about 2 days. At this stage, the felts, which were still submerged, were collected in a 13uchner funnel, washed well with de-ionised water, squeezed dry and stored at --17 ° . Extracts of the frozen felts were prepared b y grinding them in a pestle and mortar with five times their weight of cold o.I M phosphate buffer (pH 7.4) containing io -~ M glutathione and homogenized finally in a Ten Broeck glass macerater. The homogenate was centrifuged at 15 ooo × g for 30 min to remove cell debris. The supernatant fraction was used as the crude source of the enzyme. Removal of mineral micronutrients from culture media The following coprecipitation methods were used to remove trace metals from a solution of the macro-salts and sugar; copper and molybdenum with acid copper sulphide and iron, zinc and manganese with alkaline calcium phosphate*, 9. After the appropriate purification of the culture solution, spectrographically checked trace metals (Johnson Matthey, Hatton Gardens, London, E . C . I . ) were added in rag/l: 2, Fe as FeCI~; 2, Zn as Zn(NO)3; 0.8 Cu, as CuSO,; 0.2, Mo as (NHd)eMoTOz,; 0.2, Mn as MnSOd. Cofactors and other reagents N A D P (95 % pure), FAD (98 % pure) and cytochrome c (horse heart Type III) were obtained from Sigma Chemical Company (U.S.A.). NADPH~ was prepared enzymically with the isocitrate dehydrogenase (Ls-isocitrate :NADP oxidoreductase (decarboxylating), EC I.I. 1.42) enzyme purified from acetone powder of pig heart TM,1~. Radioactive iron (59FeCls) was supplied by the Atomic Energy Research Establishment, Amersham, Great Britain. Assay methods for nitrate reductase and cytochrome c reductase Two methods were used to assay nitrate reductase: I. In open tubes; o.I ml extract; o.I ml lO-1 M NaNOa; 0.7 ml lO-1 M phosphate buffer (pH 7-4)- Reaction started b y adding o.I ml 2. IO-~ M N A D P H v 2. Thunberg tubes: o.I ml extract; o.I ml lO-1 M NaNO~; 0.2 ml phosphate (pH 7.4) and in the sidearm: 0.2 ml io -~ M benzyl viologen; o. 4 ml phosphate and o.I mg 5 % palladised asbestos. The tubes were alternatively evacuated and filled with pure hydrogen gas until the benzyl viologen was fully reduced. The reaction was started b y tipping the reduced dye into the well and retaining the pellet of palladised asbestos in the sidearm. The reaction mixture was incubated at 3 °0 for IO rain Biochim. Bioph~Vs.Acta, 86 (1964) 466-476

468

D.J.D.

NICHOLAS, P. J. WILSON

and then 0.2 ml zinc acetate and 1.8 ml 98 % (w/v) ethanol added. The resulting precipitate was centrifuged at 3000 × g for 5 min and nitrite determined in a suitable aliquot of the supernatant solution using the sulphanilamide-a naphthylenediamine reagents 10. Cytochrome c reductase (NADPH,:cytochrome c oxidoreductase, EC 1.6.2.3) activity was determined by following increase in absorbancy at 550 m/z in a light path of I cm in a Unicam SP 500 spectrophotometer in the following reaction mixture : 0.05 ml aqueous 2 To cytochrome c, 0.03 ml lO-4 M FAD, enzyme (o.oi to o.Io ml depending on the activity of the preparation), o.I M phosphate buffer (pH 7.5) and 0.02 ml lO-3 M N A D P H 2. The latter was added to start the reaction which was followed at I5-sec intervals for 5 min. Enzyme dilution was such that an absorbancy change not greater than o.Io between 15 and 75 sec was obtained after adding NADPH2. Under these conditions the density change was proportional to the protein concentration. Protein was determined by the Folin method 11. RESULTS

Distribution of the enzyme Most the crude 200000 × the pellet

of the activity was found in the supernatant fraction after centrifuging extract at 200o0 >~ g for 30 min. When this fraction was centrifuged at g for I h, 55 To of the activity in the crude supernatant fraction was in whereas the assimilatory enzyme is non-particulate.

Fractionation of the enzyme The enzyme was purified 35-fold from the crude extract as shown in Table I. The purified fraction retMned 60% of its activity after storage at --17 ° for 3 months.

Substrate saturation T h e K m for the nitrate-enzyme complex using fractionVI was 5" lO-5 M. Concentrations of nitrate as high as I . lO-8 M did not depress its activity.

Temperature effects Enzyme activity of fraction VI was determined between 5 ° and 55 °- There was a sharp optimum at 3°0 and a rapid inactivation at 55 °. Preincubation of the enzyme at 5 °0 for 5 min resulted in a 7° % reduction in activity and after 15 rain at 4 °0 activity was reduced by one half. It was, however, comparatively stable when kept at 20 ° for 16 h.

pH optimum The p H optimum of the purified enzyme (fraction VI) in o.I M phosphate buffer was 7.5. The activity was reduced to 3o % at p H 6 and 9. Similar results were obtained with the following buffers at the same p H : o.i M Tris-HC1; 0.05 M cacodylate or o.I M barbitol. Pyrophosphate buffer (o.I M), over the p H range 6.8 to 8.4 increased enzyme activity by about 20 %. The purified enzyme (fraction V) was found to be active only in a phosphate buffer. The addition of phosphate to this fraction which had been taken up in o.I M pyrophosphate buffer (pH 7-5), restored enzyme activity but there was no reactivation by phosphate when the purified enzyme was contained first in the Tris-HC1 buffer. Biochim. Biophys. Acta, 86 (1964) 466-476

OF N I T R A T E

REDUCTASE

FROM

Neurospora

6

6

IO

20

45

T~Pt~

5297a, GROWN

2390

3600

3900

7000

IO 500

15 ooo

Total uftits (ml*molesNO, Ixo mini.a)

WILD

Volume (rot)

crassa

0.98

2.1

3.0

26

7°

21o

2440

175o

13oo

270

15o

7°

Specific activity

SEMI-ANAEROBIC

Total protein (mg)

IN

Specific a c t i v i t y of t h e e n z y m e : m t , moles NO.o-/io m i n / m g protein.

VI. F r a c t i o n V p a s s e d t h r o u g h a S e p h a d e x c o l u m n (G-75) a n d eluted with o.i M p h o s p h a t e (pH 7.4)

V. Ppt. from 0-60 % s a t u r a t i o n w i t h a m m o n i u m s u l p h a t e of fraction IV dissolved in 0.05 M p h o s p h a t e (pH 7-4)

IV. 3 ° m g C y gel stirred into fraction I I I a n d after s t a n d i n g for 15 m i n c e n t r i f u g e d a t IOOO × g for 5 rain. E n z y m e t h e n w a s h e d w i t h increasing c o n c e n t r a t i o n s of O.l-O. 5 M p h o s p h a t e (pH 7.6) a n d finally eluted w i t h 0.2 M p y r o p h o s p h a t e (pH 7.5)

III. P p t . from 20-45 % s a t u r a t i o n w i t h a m m o n i u m s u l p h a t e of fraction II dissolved in o.i M p h o s p h a t e (pH 7.4)

II. P p t . from 0-45 % s a t u r a t i o n w i t h a m m o n i u m s u l p h a t e of fraction I dissolved in o.I M p h o s p h a t e (pH 7.4)

I. S u p e r n a t a n t solution after c e n t r i f u g i n g t h e h o m o g e n a t e a t 2oooo x g for 3o m i n

Fraction

PURIFICATION

TABLE I

16

22

26

46

68

IOO

% of recovery enzyme

CULTURES

35

24

I8

4

2

i

Purification

E

.~

,~

~.

© N

t~

t~

,-t

,4 0 ,.~

~ ~ >

470

1). J. D. NICHOLAS, P. J. WILSON

Incubation time Nitrite production was linear with time over the first IO rain, but the rate decreased after 15 min.

Electron donors Reduced forms of benzyl and methyl viologen were found to be the most effective hydrogen donors. The Km for reduced benzyl viologen was 1.2.1o -4 M. Reduced NAD was only 5 % as effective as the reduced viologens. Reduced forms of cytochrome c, FMN and ferredoxin (the latter prepared from Clostridium pasteurianum) 14 did not substitute for the reduced viologens. The addition of FMN, or boiled pig heart had no effect on the purified enzyme. Flavin was not detected in concentrated preparations of fraction V using fluorometric, spectrophotometric or ionophoretic techniques.

The effect of inhibitors The extent of inhibition by a number of compounds is shown in Table II. The fact that cyanide, potassium thiocyanate and dithiol inhibited the enzyme indicates that a metal is probably required for its activity. The last two are known to complex readily with molybdenum. Thiocyanate also reacts with ferric iron. The inhibition by p-chloromercuribenzoate and its complete reversal b y glutathione suggests an - S H requirement for enzyme action. The fact that quinine hydrochloride and mepacrine did not restrict enzyme activity indicates that no free flavin is required. T A B L E II E F F E C T OF SOME I N H I B I T O R S ON E N Z Y M E A C T I V I T Y (FRACTION

¥I)

No i n h i b i t o r y effects were o b s e r v e d w i t h =,='-dipyridyl, o - p h e n a n t h r o l i n e , s o d i u m e t h y l e n e diaminetetraacetate, mepacrine and carbon monoxide. F i n a l concentration o f i n h i b i t o r ( M )

Inhibitor

Potassium cyanide Potassium thiocyanate I)ithiol (4-methyl-~ ,2-dimercapto-benzene) p-Chloromercuribenzoate I o d o a c e t i c acid Quinine hydrochloride

~ "IO -~

IO ~

90 80

75 65 80 76* 38 5

5 "IO 4

47 62 48

10-4

.t 0 ~

42 24

8 o

3°

o

* I n h i b i t i o n c o m p l e t e l y reversed b y a d d i t i o n of lO -3 M glutathione.

When the purified enzyme (fraction VI) was dialyzed for 6 h against IO-~ M KCN in phosphate buffer (pH 7.5) in a sac previously soaked in IO-~ M glutathione, it was inactivated but when it was dialyzed further for 16 h in fresh phosphate buffer containing IO-~ M glutathione, enzyme activity was restored.

Metals and enzyme activity A deficiency of iron in the culture medium depressed enzyme activity in extracts of 2-day-old felts but at a later stage when the hyphae were no longer submerged, there was no such effect as shown in Fig. I. Increased amounts of iron, up to 500/,g/l, Biochirn. Biophys. Acta, 86 (1964) 466-476

DISSIMILATORY NITRATE REDUCTASE FROM Neurospora crassa

471

increased the dryweights and enzyme activity in extracts of the felts which were grown under semi-anaerobic conditions, as shown in Table rlI. When iron was returned aseptically and in vivo to felts after 4 8 h growth in iron-deficient medium, the enzyme was restored to the normal levels found in submerged felts of the control

k 30 E 4C

• •

._c

E

o~ ~o

/

"1~

~

~

I~

I~

o

:~

_~ 0

k

•

o

8

~

~

~

!

4

6

I

•

,0~

•o

2

E

:3.

•

/\

A 0_~

~

I

0

Time (days)

:~.

~o

.,.. ",.

•

I¢~

o

./\ ~o

(48)

6~

-o o \ ! I

0

30

(96)

E Time (h) Fig. 2. Effect of adding iron to iron-deficient cultures on the n i t r a t e reductase activity in e x t r a c t s of the felts. After 48 or 96 h growth, 4 ° # g Fe/2oo ml was added aseptically in vivo to some of the cultures a n d the felts collected after the s t a t e d incubation periods. E n z y m e a c t i v i t y and p r o t e i n c o n t e n t were determined in s u p e r n a t a n t fractions after centrifuging a t 150o0 × g (see METHODS). A - - A , 4 ° /*g Fe/2oo ml; 0 - - 0 , Fe omitted.

Fig. I. Effect of the iron c o n t e n t of the culture m e d i u m on n i t r a t e reductase activity of Neurospora. The f u n g u s w a s g r o w n in an irondeficient m e d i u m a n d in cultures containing 20o/~g Fe/1. The felts were h a r v e s t e d a t v a r i o u s times a n d e x t r a c t s (see M E T H O D S ) w e r e centrifuged at 15 ooo × g. The e n z y m e activity a n d p r o t e i n c o n t e n t were determined in t h e supern a t a n t fraction. O, 2o0/~g Fell; O, Fe omitted.

TABLE III EFFECT ENZYME

OF THE

ACTIVITY

IN

IRON

CONTENT

EXTRACTS

OF THE

OF ~EUROSPORA

CULTURE GROWN

MEDIUM UNDER

ON

GROWTH

AND

SEMI-ANAEROBIC

l*g Fe,llitm culture solution

Dry we. as % of optimum groa~,th

Enzyme activity az % of optimum

o IOO 200

38 58 95

34 92 96

500

IOO

IOO

IOOO

IOO

IOO

CONDITIONS

cultures as shown in Fig. 2. After this period, however, when the felts had emerged from the culture solution and were now growing in a more aerobic environment there was no longer an iron requirement for the enzyme. When the experiment was repeated using older felts (96 h growth) there was only a small response to iron (Fig. 2) since these felts were no longer growing submerged under semi-anaerobic conditions. The results of two independent experiments in F i g : 3 confirm that iron concentrates in purified fractions of the enzyme up to fraction V but a further purification Biochim. Biophys. Acta, 86 (1964) 466-476

472

D.J.D.

NICHOLAS, P. J. WILSON

on a Sephadex column yielded fractionVI which had only one-third of the iron content of fraction V. A deficiency of molybdenum was more severe at the early stage of growth when the dissimilatory enzyme was most active (Fig. 4). Molybdenum concentrated in the purified enzyme even after the Sephadex treatment (fraction VI).

Effect of oxygen pressure in the culture medium on enzyme production The results in Table IV show that enzyme activity increased markedly at low oxygen tensions when nitrate was rapidly dissimilated resulting in increased pro-

• A

"--2 0 IA A

1

t

I

O~

I

I

200 40O 60O rnl~moles NO~ produced/lOmin/rng protein

]Fig. 3. R e l a t i o n b e t w e e n t h e iron c o n t e n t s of v a r i o u s p r o t e i n fractions of N e u r o s p o r a a n d t h e i r n i t r a t e r e d u c t a s e activities. T h e f u n g u s w a s g r o w n u n d e r s e m i - a n a e r o b i c c o n d i t i o n s in c u l t u r e s o l u t i o n s c o n t a i n i n g 2 m g iron labelled ~vith 1.4 # C 59Fe/1. T h e e n z y m e w a s f r a c t i o n a t e d in felts collected a f t e r t h r e e d a y s g r o w t h as s h o w n in T a b l e I. E x p t . i, A ; E x p t . 2, @.

E E ~o ~o

6

•

•

~v'~" 5~ . "1~

~v .

P 4c e~

;

~ ~c

~

o @

o

o

I 1

I ~

D~ys

o

~ ~

~ I 3

I 4

•

~

~ O 0=

O

~ ~c

I 5

~ 1

~ 2

~ 3 Doys

o

•

~ 4

~ 5

Fig. 4. Effect of m o l y b d e n u m deficiency o n d r y wt. a n d n i t r a t e r e d u c t a s e a c t i v i t y in felts collected a t v a r i o u s t i m e periods. T h e f u n g u s g r o w n in c u l t u r e s deficient in m o l y b d e n u m a n d in t h o s e cont a i n i n g 20 # g Mo/1, were s h a k e n on a reciprocator (IOO oscillations/min) a n d a g a s m i x t u r e c o n t a i n i n g 1 % o x y g e n in n i t r o g e n w a s s p a r g e d t h r o u g h t h e m e d i u m b y m e a n s of No. 2 p o r o s i t y glass sinters. E n z y m e a c t i v i t y w a s d e t e r m i n e d in t h e s u p e r n a t a n t fraction a f t e r c e n t r i f u g i n g at 15oo0 x g (see METI~ODS). O , 2O /*g Mo/1; O, Mo o m i t t e d .

Biochim. Biophys. Acta, 86 (1964) 466-476

DISSIMILATORY

NITRATE

REDUCTASE

FROM

Neurospora crassa

473

duction of free nitrite in the culture medium. At low oxygen pressores (I-IO % 02) there was better growth with ammonia than with nitrate, which m a y in part result from the inhibition b y nitrite formed from nitrate as shown in Table V. The effects of oxygen supply in the medium on nitrite, nitric oxide and hydroxylamine reductases (NAD(P)H2, : nitrite oxidoreductase, EC x.6.6.5; nitrogen: (acceptor) oxidoreductase, EC x.7.99.2 and axnmonia:(acceptor) oxidoreductase, EC 1.7.99.I respectively) in extracts of the felts grown with nitrate or ammonium as the sole nitrogen source are given in Tables V]~ and VH. The enzymes were more active in felts grown with nitrate. Nitrate reductase activity and to a lesser extent nitric oxide uptake increased at low oxygen pressures. Ammonia production from nitrate, nitrite, or hydroxylamine respectively were independent of oxygen supply. TABLE

IV

EFFECT OF OXYGEN PRESSURE IN THE CULTURE SOLUTION ON YIELD, NITRITE PRODUCTION AND NITRATE REDUCTASE ACTIVITY The fungus was grown as the nitrogen source, cil]ations/min) at 25% solutions by No.

in 5o0 ml of medium, with either sodium nitrate or ammonium chloride d i s p e n s e d i n 1.5-1 e r l c n m c y c r f l a s k s a n d s h a k e n o n a r e c i p r o c a t o r ( I o o osO 2 and N 2 gases mixed in rotameters were dispersed through the culture 2 porosity glass sinters. The felts were harvested after three days.

Nitrogen source*

% Oa in N2

Yield (g[dry wt.)

Nitrite in medium after 3 days growth (ml~molesNO2-[mI)

Nitrate reductase activity (ml~molesNO~ling protein)

Nitrate

i 5 Io 20

o.8 2.9 5.2 9.4

128o 348 45 o

54 35 IO i .4

Ammonium

I 5 IO 20

3:2 4-9 6. 4 8. 7

o o o o

3.2 o o o

* NaNO3 or NH4C1 each at 3 g]l culture solution. TABLE

V

TOXIC EFFECTS OF ADDED NITRITE ON FELTS GROWN IN CULTURE SOLUTIONS WITH AMMONIUM AS SOLE NITROGEN SOURCE T h e f u n g u s w a s g r o w n f o r 24 h a t 25 °, w h e n v a r i o u s c o n c e n t r a t i o n s o f N a N O 2 w e r e a d d e d a s e p t i c a l l y i n vivo a n d t h e p H of t h e m e d i u m a d j u s t e d t o 8 . o w i t h i N N a O H . A f t e r a f u r t h e r 24-h incubation the felts were harvested, dried and weighed. Nitrite and protein were determined i n c r u d e h o m o g e n a t e s o f t h e felts. M NaNO~ added to felts

Increase in dry wt. between24 and 48 h ( g)

% reduction in yield of felts comparedwith controls

o

i .2o

o

o

I • lO -4

i .20

o

o

3° 48 85 90

i • io -a

1.05

IO

5 "Io-3 1 " 1 0 -2 5 "1o-2

0.75 0.48 0.30

35 60 75

Nitrite in extracts of felts (ml~molesNO,-[mg protein)

B i o c h i m . B i o p h y s . A c t a , 86 (1964) 4 6 6 - 4 7 6

474

D.J.D.

NICHOLAS, P. J. W I L S O N TABLE

THE

EFFECT OF DIFFERENT ACTIVITIES OF NITRATE,

OXYGEN NITRITE,

VI

PRESSURES IN THE CULTURE SOLUTION ON NITRIC OXIDE AND HYDROXYLAMINE REDUCTASES •

T h e f u n g u s w a s g r o w n a n d h a r v e s t e d a s i n T a b l e V. R e a c t i o n m i x t u r e s : (a) n i t r a t e r e d u c t a s e , see METHODS, (b) n i t r i t e r e d u c t a s e : o . i m l e x t r a c t ; o . i m l 1 . 1 o -3 M N a N O 3 ; o . i m l 2 . I o -2 M N A D H ~ ; o.7 m l i . IO -1 M p h o s p h a t e ( p H 7-3), see ref. IO, (c) h y d r o x y l a m i n e r e d u c t a s e : o.2 m l e x t r a c t ; o . i m l 2. i o -a M N A D P H ~ ; o . i m l 2. i o -~ M N H 2 O H . HC1; o.6 m l I - i o -1 M p h o s p h a t e ( p H 7.4), see ref. 15, (d) n i t r i c o x i d e r e d u c t a s e i n W a r b u r g a p p a r a t u s a t 3 o ° ; v e s s e l : i m l e x t r a c t ; I.O m l o . i M p h o s p h a t e ( p H 7.4); s i d e - a r m : o . i m l 2. i o -~ M N A D P ; o.2 m l c o n t a i n i n g o . i m g c r y s t a l l i n e y e a s t a l c o h o l d e h y d r o g e n a s e ; o. i m l 5 ~o e t h a n o l ; c e n t r a l w e l l : o.2 m l 2o % (w/v) K O H . After flushing the apparatus with p u r e N~ for 3 ° m i n t o r e m o v e t h e l a s t t r a c e s of o x y g e n i t w a s g a s s e d w i t h 25 ~o (v/v) N O / N 2 . R e a d i n g s w e r e t a k e n a t 5 r u i n i n t e r v a l s u p t o i h. Enzyme activity/2o min/mg protein Nitrogen so,roe* N O 3NHI+

NO, reductaze NO~ reductase NO reductase NH20H reductase % 02 in N~ (re#molesNOa-formed) (mamolesNO, reduced) (ml*molesNO uptake) (ml~m°les NH20H reduced) I 20 I 20

59.0 6.8 3.4 o

lO. 3 12.6 2. 4 o

IO.O 5.9 1.6 o

9.7 9.0 3.6 2.8

* N a N O a o r NH~C1 e a c h a t 3 g / l i t r e . TABLE AMMONIA

VII

PRODUCTION FROM NITRATE, NITRITE, AND HYDROXYLAMINE RESPECTIVELY" I N E X T R A C T S O F F E L T S GRO~vVN A T I A N D 2 0 ~o O X Y G E N P R E S S U R E S

T h e f o l l o w i n g r e a c t i o n m i x t u r e s w e r e u s e d : (a) R e d u c t i o n of n i t r a t e : 0. 4 m l e x t r a c t ; 0.2 m l 2. lO -2 M N A D H z ; 0.2 m l 2. i o -3 M N A D P H ~ ; o . i m l i . i o -1 ~V[ N a N O a ; 0 . 6 m l i . lO -1 Y[ p h o s p h a t e ( p H 7.4). (b) R e d u c t i o n of n i t r i t e a n d h y d r o x y l a m i n e : 0. 4 m l e x t r a c t ; o. 3 m l 2" lO -3 M N A D P H a ; o.15 m l 2 - l O -3 M N a N O ~ o r N H ~ O H - H C 1 ; 0.55 m l i . lO -1 M p h o s p h a t e ( p H 7.4). T h e r e a c t i o n m i x t u r e s w e r e i n c u b a t e d a t 280 for 3 ° m i n a n d t h e r e a c t i o n s t o p p e d b y a d d i t i o n of 0.2 m l i 6 % (w/v) t r i c h l o r o a c e t i c a c i d . A m m o n i a p r o d u c e d w a s d e t e r m i n e d i n a MARKHAM a p p a r a t u s 1.. % O~in N~

I 2o

t,moles NH3 produced/3omin/mg protein NO~-

NO~-

NH~OH

3.I 2.1

2.9 3.o

3.o 3.2

Cytochrome c reductase

Since NADPH~-cytochrome c reductase has been shown to parallel the assimilatory NADPH~:nitrate reductase (NADPH~:nitrate oxidoreductase, EC 1.6.6.3) activity during purification in Neurospora by KINSKY AND MCELROY ~0, it was of interest to determine whether this occurred with the dissimilatory nitrate reductase. An active cytochrome c reductase was present in the crude extract but during purification it decreased by one-fifth whereas the dissimilatory BVH :nitrate reductase increased 4e-fold. Thus the ratio of BVH:nitrate reductase to NADPH~-cytochrome reductase increased from 3 in fraction I to 35 in fraction VI. It is of interest that the NADPH~:nitrate reductase paralleled the cytochrome c reductase as far as fraction III then both were markedly reduced in subsequent fractions. By repeating passage of fraction VI through the Sephadex column (Table I) a preparation with high BVH:nitrate reductase activity but with negligible NADPH~-cytochrome c reductase activity was produced. Biochim. Biophys. Acta, 86 (1964) 4 6 6 - 4 7 6

DISSIMILATORY NI'IRATE REDUCTASE FROM Neurospora crasse

475

DISCUSSION

Nitrate has two distinct functions in bacterial metabolism; it is either assimilated into cell nitrogen or it acts as an alternative hydrogen acceptor to oxygen during dissimilation4-S, 17. The two types of nitrate reductases involved have been characterized. Thus the assimilatory enzyme is a flavoprotein containing molybdenum 1-4, whereas the dissimilatory one has an additional iron requirement usually in the form of cytochromes~-~, 17-19. It is established that both enzymes occur in the same bacterium grown with nitrate as sole nitrogen source. The dissimilatory one predominates when oxygen is limiting in the medium ~-9. Although the assimilatory nitrate reductase was first characterized in Neurospora~, 3 it was only recently that the dissimilatory type was found in felts grown at low oxygen pressures 7. I t was observed that cytochromes b and c increased about 4-fold in felts grown at I To oxygen pressure compared with those at 2o %. The dissimilatory enzyme was much reduced in iron-deficient felts, in agreement with the results for the comparable bacterial system v. I t has been suggested that all nitrate reductases contain the same terminal component, that is the molybdenum-protein, but the penultimate hydrogen donating system can vary markedly ~& It is usual to find that the dissimilatory p a t h w a y to nitrate involves an iron component, usually cytochromes, whereas the assimilatory system with its slower turnover does not. When the reduced viologens were the hydrogen donors the dissimilatory nitrate reductase activity in Neurospora was much reduced in crude extracts of iron-deficient felts. The most purified enzyme preparation (fraction VI), however, was not inhibited b y iron-chelating agents, contained less iron than did less pure preparations and no detectable cytochromes or ravin. Since NADPH2 which was about 3o % as effective as the reduced viologens for nitrate reduction in crude preparations did not function for the purified enzyme the link to nitrate was lost during purification. Thus the reduced viologens in crude extracts operate via an iron system but do not do so in the purified-enzyme, Qn_e interpretation of the data is that the reduced dye operates at two sites in the electron transfer: ]~VH ---> Fe s y s t e m - - > Mo - - > N O 3(crude extracts) ]" BVI-I (purified e n z y m e )

It is likely that the purified enzyme (fraction VI) contains the terminal nitrate reductase, that is ~he molybdenum-protein which is also part of the assimilatory enzyme. KINSKY AND MCELROY2° have shown that cytochrome c reductase and assimilatory nitrate reductase, both linked to N A D P H 2 in Neurospora are intimately associated during purification; the ratio of the two activities remaining constant up to a seventy-fold concentration of nitrate reductase. Since both activities were shown to be induced in parallel fashion by nitrate they concluded that either one enzyme was involved or the two activities possess a common intermediate step. SORGER~1 also found that sucrose density gradient preparations of crude extracts of Neurospora had nearly identical activity profiles for the two activities. From genetic studies with mutants of Neurospora and Aspergillus it was concluded that there was one genetic Biochim. Biophys. Acta, 86 (1964) 466-476

476

D.J.D.

NICHOLAS, P. J. WILSON

control locus for NADPH 2 to flavin and another locus for the reduced flavin to the molybdenum. T h e w o r k r e p o r t e d h e r e i n is c o n s i s t e n t w i t h t h e h y p o t h e s i s p u t f o r w a r d e a r l i e r t h a t t h e t r u e n i t r a t e r e d u c t a s e is t h e m o l y b d e n u m - c o n t a i n i n g p r o t e i n d e v o i d of o t h e r e l e c t r o n c a r r i e r s 6. W h e n N A D P H 2 w a s u s e d as t h e d o n o r f o r e n z y m e f r a c t i o n a t i o n then the electron transfer system to nitrate in the purified enzyme required flavin a n d i t also h a d a c y t o c h r o m e c r e d u c t a s e a c t i v i t y . O n t h e o t h e r h a n d w h e n B V H was the donor neither NADPH2 nor flavin was required by the purified enzyme which is a m o l y b d e n u m - p r o t e i n w i t h n e g l i g i b l e N A D P H z : c y t o c h r o m e c r e d u c t a s e a c t i v i t y . In t h i s c o n n e c t i o n i t is of i n t e r e s t t h a t TANIGUCHI AND ITAGK122 h a v e s e p a r a t e d t h e enzyme that transfers electrons from cytochrome b to the molybdenum-protein and t h e n c e t o n i t r a t e f r o m t h e r e m a i n d e r of t h e e l e c t r o n t r a n s f e r c h a i n i n E. coli. T h u s the results reported herein are consistent with the view that in both assimilatory a n d d i s s i m i l a t o r y s y s t e m s n i t r a t e r e d u c t a s e is a m o l y b d e n u m - c o n t a i n i n g protein o n l y . E v i d e n c e i n t h i s p a p e r p o i n t s t o t h e f a c t t h a t i t is p o s s i b l e t o s e p a r a t e t h e m o l y b d e n u m - p r o t e i n f r o m t h e r e s t of t h e e l e c t r o n t r a n s f e r c h a i n .

REFERENCES

1 ~). j . D. NICHOLAS, A. NASON AND W. D. MCELROY, J. Biol. Chem., 207 (1954) 341. 2 D. J. D. INIcHOLAS AND A. NASON, J. Biol. Chem., 207 (1954) 353. a D. J. D. NICHOLAS AND A. NASON, J. Biol. Chem., 211 (1954) I83. 4 D. J. D. NICHOLAS, Ann. Rev. Plant Physiol., 12 (1961) 63. ~i C. A. FEWSON AND ~). J. D. NICHOLAS, Biochim. Biophys. Acta, 49 (1961) 335~ C. A. FEWSON AND D. J. D. NICHOLAS, Nature, 19o (1961) 2. ? G. W. WALKER AND D. J. D. NICHOLAS, Nature, 189 (1961) I41. s D. J, D. NICHOLAS AND A. H. FIELDING, J. Hort. Sci., 26 (1951) 125. 9 D. J. D. NICHOLAS, Analyst, 77 (1952) 629. 10 D. J. D. NICHOLAS, ANTONIA MEDINA AND O. T. G. JONES, Biochim. Biophys. Acta, 37 (196o) 468. 11 O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (195i) 265. 12 G. W. RAFTER AND S. P. COLOWlCK, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. 3, Academic Press, New York, 1957, p. 887. 13 S. OCHOA, in S. P. COLOWlCK AND N. O. •APLAN, Methods in Enzymology, Vol. I, Academic Press, New York, 1955, p. 699. 14 L. E. MORTENSON, ~_. C. VALENTINE AND J. E. CARNAHAN, Biochem. Biophys. Res. Commun., 7 (1962) 448. 1~ M. ZLICKER AND A. NASON, J. Biol. Chem., 233 (1955) 463 • 19 R. MARKHAM, Biochem. J., 36 (1942) 790. IT ~D. J. ~D. NICHOLAS, Biol. Rev. Cambridge Phil. Soc., 38 (1963) 53 o. 18 I~. IIDA AND S. TANIGUC~II, J. Biochem. Tokyo, 46 (1959) lO41. 1~ S. TANIGUCHI, ~R. SATO AND E. EGAMI, in W. D. MCELROY AND B. GLASS, Inorganic Nitrogen Metabolism, Johns Hopkins Press, Baltimore, I96O, p. 87. 20 G. C. KINSKY AND W. D. MCELROY, Arch. Biochem. Biophys., 73 (1958) 466. 21 G. J. SORGER, Biochem. Biophys. Res. Commun., 12 (1963) 39522 S. TANIGOCHI AND E. ITAGAKI, Biochim. Biophys. Acta, 31 (1959) 294.

Biochim. Biophys. Acta, 86 (1964) 466-476