TRIPHOSPHOPYRIDINE NUCLEOTIDE-NITRATE REDUCTASE IN NEUROSPORA* BY ALVIN ...

20 Pages·2003·1.07 MB·English

TRIPHOSPHOPYRIDINE NUCLEOTIDE-NITRATE REDUCTASE
IN NEUROSPORA*
BY ALVIN NASON AND HAROLD J. EVANSt
(From the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore,
Maryland)
(Received for publication, September 30, 1952)
Although it had been well established that many species of microor-
ganisms and higher *plants are able to reduce nitrate to nitrite, it remained
for Green, Stickland, and Tarr (l), working with Escherichia co%, to show
that nitrate reduction could be demonstrated in cell-free extracts. Yama-
gata (2) also obtained a cell-free preparation of the enzyme, designated as
nitrate reductase, from E. coli, and reported that it failed to oxidize re-
duced coenzyme I and was strongly inhibited by cyanide. Subsequently,
a number of investigators have prepared the enzyme from this source in a
partially purified soluble state and have further described its properties.
Some of these workers claim that the enzyme system is a sulfhydryl pro-
tein identical with cytochrome b, inhibited by carbon monoxide, and hav-
ing a flavin requirement (3-5), whereas others (6) have been unable to
demonstrate an effect of -SH reagents and carbon monoxide and report
that dyes and flavoprotein were active as carriers between hydrogenase
and nitrate reductase. However, the nature of the immediate natural
electron donor still remains obscure, and in the above enzymatic studies
reduced dyes were used as the electron source.
The present paper’ deals with the purification and properties of a TPN-
nitrate reductase in Neurospora, already reported from this laboratory (7),
and its identification as a flavoprotein with FAD as the prosthetic group.
Materials and Mefhods
Cell-free extracts were prepared from the mats of the wild type fungus,
Neurospora crassa (5297a), grown in the Fries basal medium.2 The me-
+ Contribution No. 32 of the McCollum-Pratt Institute. This investigation was
supported in part by a research grant from the National Institutes of Health, United
States Public Health Service.
t E. F. Johnson Postdoctoral Fellow, on leave from the North Carolina State Col-
lege of the University of North Carolina.
1 The following abbreviations are used: TPN and TPNH, unreduced and reduced
triphosphopyridine nucleotide, respectively; DPN and DPNH, unreduced and
reduced diphosphopyridine nucleotide, respectively; DPNase, diphosphopyridine
nucleotidase; FAD, flavin-adenine dinucleotide; FMN, flavin mononucleotide or
riboflavin phosphate; Tris, tris(hydroxymethyl)aminomethane.
* In units per liter, NH4 tartrate 5 gm., NHaNOa 1 gm., KH,P04 1 gm.,
655
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656 TPN-NITRATE REDUCTASE
dium was dispensed in 125 ml. amounts in 500 ml. Erlenmeyer flasks or in
10 liter amounts in 20 liter bottles with forced aeration for the large scale
runs, inoculated, and incubated for 4 to 5 days in the dark at 28 to 30”.
The mycelial mats were collected on a Biichner funnel, washed with
triply distilled water, and frozen for 1 to 3 hours at -15”. They were
then homogenized in a TenBroeck glass homogenizer in 3 times their weight
of cold 0.1 M KzHP04 and centrifuged in the Servall centrifuge at 20,000
X g for 10 minutes at +4”. The supernatant solution, which was cell-free
and turbid, was used as the crude extract for studies of the enzyme content
of mycelia, as well as for purification. Preliminary experiments showed
that 85 per cent or more of the nitrate reductase activity of the homogenates
was present in the extracts.
Micronutrient Dejiciencies and Growth-For the phase of the work dealing
with the effects of deficiencies of micronutrients on the activity of the
enzyme, deficiencies of zinc, iron, manganese, and magnesium were ob-
tained and growth was measured as described previously (8).
CofactorsTPN of 75 per cent purity was prepared from sheep liver by
a procedure involving ion exchange chromatographyP TPNH was pre-
pared enzymatically as follows: To 40 mg. of TPN dissolved in 15.4 ml. of
0.1 M phosphate buffer, pH 7.5, were added 0.8 ml. of 0.1 M MgC12 and
3.0 ml. of 0.05 M d-isocitrate. The reaction was started by the addition of
0.8 ml. of a phosphate extract at pH 7.5 of washed acetone-dried pig heart
containing isocitric dehydrogenase (9). The reaction was allowed to con-
tinue at room temperature for 30 to 45 minutes, when the optical density
at 340 rnp had reached a maximum and minimal TPN remained as tested
by the cyanide reaction (10). At the end of the reaction, the incubated
mixture was adjusted to pH 9.0 to 9.5 and placed in a boiling water bath
for 3 minutes. The resulting suspension was centrifuged for 5 minutes
and the supernatant solution, which could be stored for months at -15’,
was used as a TPNH source. The absorption at 340 rnp was confirmed to
be due almost entirely to enzymatically active TPNH, by preliminary tests
with partially purified glutathione reductase from peas (11). The con-
centration of TPNH (2 PM per ml.) was determined spectrophotometrically
with 6.24 X lo6 sq. cm. X mole-’ as the extinction coefficient at 340 rnH
(12). TPNH was also enzymatically prepared in essentially the same
manner as above, except that 13 units of purified yeast phosphogluconic
dehydrogenase (13) and 100 PM of phosphogluconic acid, kindly supplied
by Dr. B. L. Horecker, were used in place of the isocitric dehydrogenase
system.
MgSOa-7HzO 0.5 gm., NaCl 0.1 gm., CaCl% 0.1 gm., biotin 5 y, sucrose 20 gm., Na
tetraborate 8.8 X 10-b gm., (NHI)GMo1021 6.4 X 10-s gm., FeC13.6H20 9.6 x lo-4
gm., ZnS04-7HzO 8.8 X 10d3 gm., CuCls 2.7 X lo--1 gm., MnC12.4H20 7.2 X lo-6 gm.
* Kornberg, A., and Horecker, B. L., unpublished.
A. NASON AND H. J. EVANS G57
DPN of 65 per cent purity was obtained from the Sigma Chemical Com-
pany, and DPNH was prepared enzymatically as described elsewhere (14).
FAD of approximately 60 per cent purity, prepared from yeast by the
method of Warburg and Christian (15), was kindly furnished by Dr. L. A.
Heppel. FAD was also obtained from the Sigma Chemical Company, and
FMN from the Nutritional Biochemicals Corporation. The concentration
of FAD solutions was determined spectrophotometrically with 1.13 X 10’
sq. cm. X mole-l as the extinction coefficient at 455 rnp (15). Boiled pig
heart extract was prepared by heating at 100” for 5 minutes and centrifug-
ing the extract of acetone-dried pig heart.
Cytochrome c was obtained from the Sigma Chemical Company.
Other Substances-The sodium salt of d-isocitric acid of 70 per cent purity
(30 per cent citric acid) was prepared by ion exchange chromatography
(16). The protein of n-amino acid oxidase was prepared from pig kidney,
according to a modified procedure4 of Negelein and Brbmel (17). Snake
venom, used as a source of a nucleotide pyrophosphatase (18), was ob-
tained from Ross Allen’s Reptile Institute.
Determination of Activi@-Enzymatic activity was determined colori-
metrically by testing for nitrite by use of the sulfanilamide and N-(l-
naphthyl)ethylenediamine hydrochloride reagents (19). The test proced-
ure consisted of adding 0.05 ml. of enzyme at zero time to give a final
reaction mixture of 0.5 ml., containing 0.1 ml. of 0.1 M KN03, 0.05 ml. of
1.5 X low5 M FAD, 0.04 ml. of 2 X 10T3 M TPNH, and 0.26 ml. of 0.2 M
pyrophosphate buffer, pH 7.0. After 5 minutes incubation at 23-28”, 0.9
ml. of Hz0 and 0.5 ml. of sulfanilamide reagent were added to stop the
reaction, followed by 0.5 ml. of the naphthylethylenediamine reagent to
develop the color. After 10 minutes, the density of the color was read on
a Klett calorimeter with a 540 rnp filter. Control tubes lacking TPNH
were used to correct for the turbidity caused by the enzyme. The enzyme
exhibited no difference in activity when the assay was conducted under
anaerobic compared to aerobic conditions. There was no chemical reduc-
tion of nitrate to nitrite by TPNH.
1 unit of nitrate reductase is defined as that amount of enzyme which
results in the formation of lo5 pM of nitrite under the above conditions of
the assay.6
* Frisell, W., unpublished.
6 At times nitrite reductase activity was present in some fresh crude extracts
under the conditions of the nitrate reductase assay. In these cases it was found that
the determination of nitrate reductase activity could be made by the use of KCN
and FMN, in place of FAD, at 5 X 10e6 M and 9 X 10m6 M final concentrations, re-
spectively. These inhibited 85 to 95 per cent of the nitrite reductase activity and
only 10 to 20 per cent of the nitrate reductase. Nitrite reductase was removed in
the first step of the enzyme purification procedure and was therefore absent in Frac-
tion II (Table I).
658 TPN-NITRATE FLEDUCTASE
Fig. 1 illustrates that the quantity of nitrite formed is directly propor-
tional to the enzyme concentration.
Specific activity is expressed as units per mg. of protein. The protein
contents of the crude extracts from the deficiency series and the nitrogen
source series were determined by the biuret procedure of Robinson and
Hogden (20). In all other cases protein was measured by the Folin phenol
reagent according to Lowry et al. (21). Partially purified nitrate reductase
fractions with a protein content of 200 to 600 y per ml. were used in these
studies.
20r
0 29 4.0 6.0
MICROGRAMS PROTEIN
FIG. 1. Proportionality of concentration of enzyme to nitrite formation. Stand-
ard conditions of assay.
Results
Purification of Enzyme-All steps of the purification procedure were
carried out at O-4’; centrifugations were performed at approximately 3000
X g; and ammonium sulfate was used only as the saturated solution,
20-25”, adjusted with sodium hydroxide to pH 7.0 to 7.5.
In the following purification procedure 75 ml. of the saturated am-
monium sulfate were added to 100 ml. of crude extract (Fraction I, Table
I) to give a final concentration of 43 per cent saturation. After standing
for 15 minutes, followed by centrifugation, the supernatant solution was
discarded and the precipitate was dissolved in 40 ml. of cold 0.1 M phos-
phate buffer, pH 7.0. The dissolved precipitate (Fraction II) contained
50 per cent of the initial activity with a 2-fold increase in purity (Table I).
To Fraction II (40 ml.) at 0” were added 12.5 ml. of the saturated
ammonium sulfate solution. After 15 minutes the precipitate (24 per cent
saturation) was collected by centrifugation and discarded. The super-
natant solution was treated with 21.5 ml. of the saturated ammonium
sulfate solution, and after 15 minutes the precipitate was collected by
A. NASON AND H. J. EVANS 659
centrifugation and dissolved in 16 ml. of cold 0.1 M phosphate buffer, pH
7.0. The activity in Fraction III (24 to 46 per cent ammonium sulfate
saturation) represented 20 per cent of the units in the crude starting mate-
rial and a 7-fold purification.
Fraction III (16 ml.) was treated with 7.2 ml. of calcium phosphate gel
(22), aged 9 months or longer (11 mg. of dry weight per ml.). After 15
minutes the precipitate was collected by centrifugation, washed twice with
5 ml. portions of cold 0.1 M phosphate buffer, pH 7.5, and eluted once with
4.0 ml. of cold 0.1 M pyrophosphate buffer, pH 7.0. The pyrophosphate
eluate (Fraction IV) showed about 10 per cent of the total activity of the
original crude extract and an over-all purification of 40 to 50 times. The
previous phosphate washings as well as subsequent pyrophosphate elutions
TABLE I
Summary of Purijication of Neurospora Nitrate Reductase
Fraction I. Crude extract. . . . . . . 24,800 414 60
‘I II. O-43% ppt., ammonium sulfate. . 12,700 105 121 51.2
“ III. 24-46s ppt., ammonium sulfate.. 5,000 12 417 20.0
“ IV. Calcium phosphate gel eluate.. . 2,420 0.9 2690 9.8
“ V. O-SO’% ppt., ammonium sulfate. . 2,408 0.6 4000 9.7
of the precipitate after the first pyrophosphate elution showed only slight
enzyme activity.
To Fraction IV (4.0 ml.) were added 6.0 ml. of the saturated ammonium
sulfate solution to give 60 per cent saturation. After standing for 15
minutes at O”, the precipitate was collected by centrifugation and dissolved
in 2.0 ml. of cold 0.1 M phosphate buffer, pH 7.0. The protein in this final
fraction (No. V) represented 10 per cent of the units in the crude starting
material and an over-all purification of 60- to ‘IO-fold.
1 mg. of the purified protein is capable of reducing 48 PM of nitrate to
nitrite per hour. In terms of turnover number this corresponds to the
reduction of 80 moles of nitrate per mole of protein per minute, assuming
a molecular weight for the enzyme of 100,000.
Stability of Enzyme-Of the five fractionation steps which constitute the
purification procedure, Fraction II is most stable, losing only 10 to 20
per cent of its activity after a month at - 15’. The optimal pH for storage
of this fraction is 7.0. On the other hand storage of Fraction III overnight
at - 15” or +4’ resulted at least in a 50 per cent loss in activity. The
calcium phosphate eluate (Fraction IV) loses about half of its activity
660 TPN-NITRATE REDUCTASE
within a week at -15”. Fraction V is quite unstable, losing at least half
of its activity when stored overnight at -15”. With reference to heat
stability the latter loses 50 per cent of its activity at pH 7.0 at 40” in 5
minutes, and all of its activity at 50” in 5 minutes, the shortest time
interval tried.
All attempts to dialyze the enzyme either as the crude extract or as
Fractions II and III against phosphate buffer at pH 7.0, running tap water,
or triply distilled water resulted in a complete loss of activity within 1
hour, the shortest time interval tried. When the enzyme was exposed to
washed cellophane dialyzing membrane (5.0 ml. of enzyme + 0.5 gm. of
shredded membrane) for 2 minutes, 95 per cent of the original activity was
lost. Attempts to restore activity by the addition of boiled crude extract,
ashed crude extract, glutathione, or the inorganic ions Mg++, MoOP, B401’,
Zn+f, Cu++, Mn++, Fe++, or Fe* (5 X 1O-6 M final concentration) were
unsuccessful. Attempts to elute an active enzyme from the dialyzing
membrane by use of phosphate and pyrophosphate buffers also failed.
pH Optimum-The effect of pH on enzymatic activity was determined
by the use of various buffers of suitable pH values. Fig. 2 indicates that
there is a striking rise in activity with highly purified enzyme (Fraction V)
when pyrophosphate is present, and that a sharp maximal peak exists at
pH 7.0, a negligible activity remaining below pH 6.0 and above pH 9.0.
Increased rates were also obtained in the absence of pyrophosphate if
Versene was added to the reaction mixture in final concentrations varying
from 1O-3 to 10eK moles per liter. Triphosphate was less effective than
Versene or pyrophosphate. These results suggest the presence of a metal
inhibitor in Fraction V which is removed by chelating agents such as
pyrophosphate, Versene, or triphosphate.
Nitrate Afinity-The dependence of the reaction rate on substrate con-
centration is shown in Fig. 3. The dissociation constant (K,) of the
enzyme-nitrate complex, estimated from the saturation curve, is 1.4 X
1O-3 in moles per liter.
Prosthetic Flavin Group-In the early phases of this work it was observed
that only a small fraction of the original nitrate reductase activity present
in the crude Neurospora extract could be recovered after the first am-
monium sulfate purification step. However, the addition of boiled pig
heart extract to the reaction mixture resulted in an approximately 5-fold
increase in activity. This reactivating effect by boiled pig heart extract
could be replaced in full by FAD and in part by FMN in this and all
subsequently purified enzyme fractions. The separation of nitrate reduc-
tase into its protein moiety and prosthetic flavin group was to a lesser
extent facilitated by treatment with calcium phosphate gel. The reactiva-
tion of a purified protein fraction as a function of FAD and FMN concen-
A. NASON AND H. J. EVANS 661
trations is shown in Fig. 4. The dinucleotide was considerably more effec-
tive than the mononucleotide in increasing the residual activity of the
enzyme with respect to both the extent of maximal activation and the
concentration required for saturation. An approximate determination of
the dissociation constants from the data indicates that half of maximal
activation was obtained with FAD at about 3.2 X lo-’ and with FMN at
about 30 X lo-’ in moles per liter.
00 44 88 1122 1166 2200
NNIITTRRAATTEE CCOONNCCEENNTTRRAATTIIOONN
MMOOLLEESS PPEERR LLIITTEERR XX IIOO33
PH
FIQ. 2 FFIIGG 33
FIG. 2. Effect of pH on nitrate reductase activity. All buffers present in a final
concentration of 10-l M. Each sample contained 30 units of enzyme (Fraction V).
A., pyrophosphate; A, phosphate; 0, acetate; l , Tris.
FIG. 3. Effect of nitrate concentration on nitrate reductase activity. 20 units
of enzyme.
Although the more effective action of FAD in reactivating the enzyme
does not necessarily identify this nucleotide as the prosthetic group of
nitrate reductase, the following experiments establish the identity of the
flavin associated with purified enzyme as FAD. The flavin was liberated
from nitrate reductase by heating at 100” for 5 minutes in the dark. A
separate 0.1 M pyrophosphate solution at pH 7.0 containing 3.8 X 1O-3 PM
of FAD was also heated in the same manner. The supernatant solutions
were collected by centrifugation and analyzed fluorometrically by the method
of Burch el al. (23), based on the increase in fluorescence which accompanies
the splitting of FAD to FMN and adenylic acid. Snake venom containing
pyrophosphatase was used to hydrolyze the dinucleotide present in the
662 TPN-NITRATE REDUCTASE
supernatant solutions. A marked increase in fluorescence due to flavins
was obtained by hydrolyzing the sample (Table II). The snake venom
showed no fluorescence due to flavins, and control solutions incubated
without snake venom did not change in fluorescence. Since the fluores-
cence of FAD is 0.14 times that of FMN, the per cent of FAD in a mixture
16 32 48 64 80 96 112
FLAVI N CONCENTRATION
MOLES PER LITER X IO7
FIG. 4. Effect of flavin concentration on nitrate reductase activity. Preincu-
bated for 5 minutes at 4” with all components of the reaction mixture except TPNH,
prior to the usual nitrate reductase assay. The flavin concentrations are the final
concentrations in the test.
of FAD and FMN is given by Equation 1 where fi and fz are the fluoro-
‘$&FAD =‘$$ x 100
. 2
metric readings before and after hydrolysis, respectively. As shown in
Table II, almost all the flavin in the supernatant solutions of the boiled
enzyme preparation and the known FAD solution can be accounted for as
FAD.
Additional evidence for the identity of the flavin of Neurospora nitrate
A. NASON AND H. J. EVANS 663
reductase as FAD was provided by its activity with the protein of the
FAD specific n-amino acid oxidase. In this experiment 2.6 ml. of the
boiled enzyme preparation, adjusted to pH 8.5, and 0.2 ml. of the apo-
enzyme of n-amino acid oxidase were placed in the main compartment of
a Warburg vessel. The side arm contained 0.2 ml. of 0.33 M nn-alanine
and the center well KOH. The mixture was preincubated for 30 minutes
at 4’, and oxygen uptake was determined at 30”. Other reaction mixtures
containing up to 2.5 X 10” PM of FAD in place of the boiled enzyme
preparation were run at the same time in order to estimate the FAD
content of the unknown sample. The boiled enzyme preparation contained
1.6 X lo4 PM of FAD per ml. as determined by this method. This com-
TABLE II
Fluorometric Analysis of Nitrate Reductase Flavin
-
Galvanometer readings
FAD Enzyme flavin
Before hydrolysis cfi). . . . . . . . 11 3
After “ (f2) . . . . . . . . . 36 14
% FAD*. . . . . . . . . . . . . . . . . . . . . . . . . 81 91
0.1 M pyrophosphate solutions, pH 7.5, containing 3.8 X 10m3 PM of FAD and
boiled nitrate reductase supernatant solutions, respectively, were hydrolyzed by
incubating in the dark with 0.3 ml. of snake venom (containing nucleotide pyro-
phosphstase) for 1 hour at 37” in a final volume of 5.0 ml. made up with 0.1 M phos-
phate, pH 7.5. The control solutions (before hydrolysis) were incubated as above
but without snake venom. For the fluorometric analysis 2.1 ml. and 3.0 ml. aliquots
of the above FAD and enzyme supernatant solutions, respectively, were used.
* Calculated from Equation 1.
pares favorably with the value of 2.4 X 10e4 PM of FAD per ml. calcu-
lated from the fluorescence data of the same preparation with a known
sample of riboflavin as a standard.
TPNH As Electron Donor-The activity of the enzyme in catalyzing the
reduction of nitrate to nitrite as a function of pyridine nucleotide concen-
tration (Fig. 5) indicated a marked specificity for TPNH as the electron
donor. When the nitrate reductase concentration was increased 6-fold,
however, the specificity for TPNH was shown to be relative, since DPNH
under these conditions also acts as an electron source, although much less
efficiently, as shown in Fig. 5. The maximal rate of activity achieved
with TPNH was about 20-fold that obtained with DPNH. An estimate of
the dissociation constants from the curves indicates that one-half maximal
activity with TPNH was obtained at about 7 X lo+’ and with DPNH at
about 1.4 X lo4 in moles per liter. DPNH at the higher concentrations
appears to inhibit the activity of the enzyme.
664 TPN-NITRATE REDUCTASE
TPNH preparations made enzymatically by use of isocitric dehydrogen-
ase and phosphogluconic dehydrogenase were equally effective. TPNH,
prepared by chemical reduction with hydrosulfite (24), appeared to be as
effective as enzymatically prepared TPNH, although it was less stable
when stored at -15”.
48
t I
54 ENZYME UNITS
54 ENZYME UNITS
’ DPNH, 324 ENZYME UNITS
0 14 28 42 56 70 84 98
COENZYME CONCENTRATlON
MOLES PER LITER X I05
FIG. 5. Effect of concentration of reduced pyridine nucleotides on nitrate reduc-
tase activity. The coenzyrne concentrations are the final concentrations in the
test.
Succinate, cysteine, glutathione, and ascorbic acid in final concentrations
ranging from 5 X 1O-3 to 8 X 1OW moles per liter failed to replace TPNH
under anaerobic or aerobic conditions as the electron donor in the en-
zymatic reduction of nitrate to nitrite.
Xtoichiometry of Enzymatic Reaction-In order to determine the stoichi-
ometry of the nitrate reductase reaction, experiments to measure con-
comitant disappearance of TPNH and production of nitrite were carried
out in spectrophotometric cuvettes containing approximately the same
proportion of constituents that was used in the regular determination of