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Plant Physiol. (1998) 117: 303-309
In Vitro Reconstitution of Electron Transport
from
Glucose-6-Phosphate and NADPH to Nitrite1
Tie Jin,
Heather C. Huppe2, and
David H. Turpin*
Department of Biology, Queen's University, Kingston, Ontario,
Canada K7L 3N6
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ABSTRACT |
An
NADPH-dependent NO2 -reducing system was
reconstituted in vitro using ferredoxin (Fd) NADP+
oxidoreductase (FNR), Fd, and nitrite reductase (NiR) from the green
alga Chlamydomonas reinhardtii.
NO2 reduction was dependent on all protein
components and was operated under either aerobic or anaerobic
conditions. NO2 reduction by this in vitro
pathway was inhibited up to 63% by 1 mm NADP+.
NADP+ did not affect either methyl viologen-NiR or Fd-NiR
activity, indicating that inhibition was mediated through FNR. When
NADPH was replaced with a glucose-6-phosphate dehydrogenase
(G6PDH)-dependent NADPH-generating system, rates of
NO2 reduction reached approximately 10 times
that of the NADPH-dependent system. G6PDH could be replaced by either
6-phosphogluconate dehydrogenase or isocitrate dehydrogenase,
indicating that G6PDH functioned to: (a) regenerate NADPH to support
NO2 reduction and (b) consume
NADP+, releasing FNR from NADP+ inhibition.
These results demonstrate the ability of FNR to facilitate the transfer
of reducing power from NADPH to Fd in the direction opposite to that
which occurs in photosynthesis. The rate of G6PDH-dependent NO2 reduction observed in vitro is capable of
accounting for the observed rates of dark NO3
assimilation by C. reinhardtii.
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INTRODUCTION |
NO3 assimilation in
plants consists of three processes. Initially
NO3 is reduced to
NO2 by
NO3 reductase. This is followed by the
subsequent reduction of NO2 to
NH4+ by NiR. Finally, the
resulting NH4+ is assimilated
into amino acids by glutamine synthatase/glutamate synthase (Beevers
and Hageman, 1980 ; Sivarsankar and Oaks, 1996). The Chlamydomonas
reinhardtii NO3
assimilatory system is similar to that of higher plants (Barea and
Cárdenas, 1975). When grown under
NO3-sufficient conditions,
green algae depend on light for
NO3 assimilation, whereas
cells grown under NO3-limited
conditions are capable of NO3
assimilation in the light and in the dark (Syrett, 1981). The onset of
dark NO3 assimilation in
N-limited cells stimulates the respiration of starch, thereby providing
carbon skeletons for amino acid synthesis and the reductant required
for NO3 reduction (Turpin et
al., 1997). The onset of NO3
assimilation coincides with the Fd-thioredoxin-dependent activation of
G6PDH, the key regulatory enzyme of the OPP pathway (Huppe et al.,
1992, 1994; Farr et al., 1994). Physiological and biochemical studies
indicated that the source of reductant for
NO3 reduction in the dark is
the OPP pathway (Vanlerberghe et al., 1992; Huppe et al., 1994). If
this is the case, electrons from NADPH must be able to reduce Fd, which
is the electron donor to NiR. To date, however, no direct evidence
exists to support this hypothesis.
The relationship between the OPP pathway and
NO2 reduction has also been
investigated in nonphotosynthetic tissues of higher plants (Oaks and
Hirel, 1985), where it has also been reported that carbohydrate
oxidation via the OPP pathway provides reducing power for
NO2 reduction (Emes and
Fowler, 1983; Oji et al., 1985; Bowsher et al., 1989; Borchert et al.,
1993). However, NiR in roots is also a Fd-dependent enzyme, which
cannot utilize directly the NADPH generated by the OPP pathway. This
implied the presence of FNR-like proteins that mediate the electron
transfer from NADPH to Fd (Oji et al., 1985; Suzuki et al., 1985).
Recently, several FNRs and Fds have been purified from
nonphotosynthetic plant tissues (Wada et al., 1989; Hirasawa et al.,
1990; Morigasaki et al., 1990a, 1990b; Bowsher et al., 1993), but there
is still no direct evidence to show FNR and Fd mediating electron
transfer from NADPH to NO2
reduction.
The purpose of this study was to test the hypothesis that electrons
from NADPH may support NO2
reduction via a FNR- and Fd-mediated electron transfer pathway from
NADPH to NO2. We report the
purification of NiR, FNR, and Fd from the green alga C. reinhardtii and the reconstitution of an in vitro
electron-transfer system from NADPH to NiR for
NO2 reduction via FNR, Fd, and
NiR. Furthermore, we have isolated G6PDH from the same source and
coupled G6PDH-dependent NADPH generation to
NO2 reduction, providing
direct evidence for the potential of G6PDH to support
NO2 reduction in the
dark.
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MATERIALS AND METHODS |
Chlamydomonas reinhardtii cc-1183 was grown in
NO3-sufficient chemostat
cultures as previously described (Huppe and Turpin, 1996). Cells were
harvested daily, frozen in liquid N2, and then stored at  80°C until use.
Reagents and Enzymes
Chemical reagents were obtained from commercial sources and were
of the highest quality available. Q-Sepharose (fast flow) and
phenyl-Sepharose CL-4B were from Pharmacia; Blue-Cellulofine was from
Seikagaku-kogyo Company, Ltd. (Tokyo, Japan); Butyl-Toyopearl was from
Tosoh Company (Tokyo); DEAE-Fractogel was from EM Science (NJ); Glc
oxidase (Aspergillus niger), catalase (bovine liver), and
G6P (yeast) were purchased from Sigma; and 6PGDH (yeast) and ICDH
(porcine heart) were from Boehringer Mannheim. Fd-Sepharose 4B was
prepared by purified C. reinhardtii Fd and CNBr-Sepharose 4B
(Pharmacia) according to the protocol provided by the manufacturer.
Isolation of Enzymes
Twenty milliliters of frozen cells (approximately 20 g in
fresh weight) was thawed at room temperature. One hundred and eighty milliliters of buffer A containing 50 mm Tris-HCl (pH 7.8),
5 mm EDTA, 5 mm benzamidine, 5 mm
6-aminocaproic acid, 1 mm PMSF, 0.04% (v/v) chemostatin, 2 mm 2-mercaptoethanol, and 3 g of insoluble PVP was
added with stirring during sample thawing. The thawed extract
homogenate was centrifuged (39,000g for 30 min) and 20 mL of
2% (w/v) protamine sulfate was added to the supernatant (180 mL) to
precipitate DNA. The sample was clarified by centrifugation as above
and applied to a Q-Sepharose column (1.7 × 20 cm) preequilibrated with buffer A. The column was washed until the
A280 returned to baseline and then eluted
with a linear gradient of NaCl (0-1.0 m) in buffer A. Enzymes were assayed and the fractions containing NiR, Fd, FNR, and
G6PDH activities were pooled separately.
NiR Purification
(NH4)2SO4
was added to the NiR fraction from the Q-Sepharose to a final
concentration of 15%. The sample was centrifuged (39,000g for 15 min) and the supernatant was applied to a phenyl-Sepharose column (1.7 × 20 cm) as described by Romero et al. (1987). The column was developed by a 135-mL linear gradient of
(NH4)2SO4 (15-0% saturation) in buffer B containing Tris-HCl (pH 7.8), 1 mm EDTA, and 2 mm 2-mercaptoethanol. NiR
activity was pooled and brought to 20% saturation with
(NH4)2SO4
and centrifuged as above. The supernatant was applied to a
butyl-Toyopearl column (1.7 × 20 cm) preequilibrated with buffer
B containing 20% saturated (NH4)2SO4.
The column was eluted with the same buffer (buffer B containing 20%
saturated
[NH4]2SO4).
The active fractions were pooled and dialyzed overnight against 50 mm Tris-HCl (pH 7.8) and 1 mm EDTA (buffer C).
The dialyzed sample was applied to a DEAE-Fractogel column (1 mL) and
eluted with a 0 to 0.3 m linear gradient of NaCl (12.5 mL).
Fractions with NiR activity were pooled, concentrated by
ultrafiltration with a Centricon-50 concentrators (Amicon, Beverly,
MA), frozen in liquid N2, and stored at 80°C.
FNR Purification
FNR eluted from the Q-Sepharose column as a broad peak that also
contained G6PDH activity. FNR was purified as in Jin et al. (1994)
using a Blue-Cellulofine and Fd-Sepharose chromatography. Purified FNR
was desalted and concentrated by ultrafiltration with a Centricon-30
concentrators (Amicon) and stored at 80°C. Mung bean leaf FNR was
purified as previously described in Jin et al. (1994).
G6PDH Isolation
G6PDH and FNR coeluted from a Blue-Cellulofine column (1.7 × 20 cm) by a linear gradient of NaCl (0-1.0 m) in buffer B. After dialyzing against buffer B overnight, the sample was applied to a
Fd-Sepharose column (1.4 × 6 cm) to separate the activities. FNR
was bound on the column and G6PDH was passed through. The G6PDH sample
was brought to 20% saturation with
(NH4)2SO4
and centrifuged (39,000g for 15 min). The supernatant was
applied to and eluted from a phenyl-Sepharose column (1.7 × 20 cm) as previously described for NiR except buffer B containing 20%
(NH4)2SO4. The active fractions were pooled, concentrated, and desalted on Centricon-10 concentrators (Amicon); frozen in liquid
N2; and stored at 80°C.
Fd Purification
Fd eluted from the Q-Sepharose column was further purified by
butyl-Toyopearl chromatography according to the method of Jin et al.
(1994), except that the concentration of
(NH4)2SO4
in the linear gradient was 50 to 0%. Purified Fd was concentrated and desalted on Centricon-10 concentrators and stored at 4°C. Spinach leaf Fd was purified according the method described in Jin et al.
(1994).
Rapid Isolation of NiR
One liter of freshly harvested cells (about 1 g in fresh
weight) was frozen in liquid N2. The cells were
thawed in 2 mL of homogenizing buffer A and centrifuged
(18,200g, 3 min). The supernatant was desalted on a PD-10
column (Pharmacia) and the desalted sample was applied to a
Blue-Cellulofine column (1.4 × 10 cm) equilibrated with buffer A. The column was washed with buffer A and the fractions showing NiR
activity were pooled, concentrated on a Centricon-50 unit (Amicon),
frozen in liquid N2, and stored at 80°C.
Assay Methods: NiR
Four methods were developed to assay NiR activity.
MV-NiR Assay
The reaction mixture (1 mL) contained 50 mm
Tris-HCl (pH 7.8), 2 mm NaNO2, 0.4 mm MV, and 23 mm
Na2S2O4 freshly prepared in 0.5 mm NaHCO3. The enzyme sample was added and the
reaction was carried out at 30°C for 10 min.
Fd-NiR Assay
The reaction was carried out as described above, except that MV in
the reaction mixture was replaced by 20 µm purified
C. reinhardtii Fd.
NADPH-Dependent NO2 Reduction Assay
The reaction mixture (0.5 mL), containing 50 mm
Tris-HCl (pH 7.8), 0.4 mm NaNO2, 20 nm purified C. reinhardtii FNR, 20 µm purified C. reinhardtii Fd, 10 mm Glc, 100 µg/mL Glc oxidise, 50 µg/mL catalase, and
0.1 unit of NiR (activity calculated by MV-NiR assay), was mixed in a
rubber-capped glass chamber (3 mL). To ensure anaerobicity, the mixture
was bubbled with N2 for 1 min, and then the
reaction was started by injecting NADPH to a final concentration of 1 mm. The assay continued for 15 min at 30°C.
NADPH-dependent NO2 reduction
was assayed aerobically in the same reaction mixture except that no
Glc, Glc oxidase, or catalase were included.
G6PDH-Coupled NO2 Reduction Assay
Reactions were carried out under aerobic conditions and the
reaction mixture (0.5 mL) contained 50 mm Tris-HCl (pH
7.8), 2 mm NaNO2, 0.35 unit of G6PDH,
6 mm G6P, 0.1 mm NADP+
(or NADPH), 20 nm purified C. reinhardtii FNR,
20 µm C. reinhardtii Fd, and 0.1 unit of NiR
(calculated by MV-NiR activity). The reaction was performed at 30°C
for 10 min.
For all of the assays, the disappearance of
NO2 was used to calculated
enzyme activity and 1 unit of NiR activity catalyzed 1 µmol
NO2 reduction per min.
NO2 concentration was measured
as described by Hirasawa et al. (1989), and a mean of at least three
independent measurements was used to calculate NiR activity. The
se associated with the reported values was always less than
3%.
Other Enzyme Assays
Fd and FNR activity were measured by the reduction of Cyt
c in the presence of NADPH, essentially as described by
Morigasaki et al. (1990b). The assay mixture contained 50 mm Tris-HCl (pH 7.8), 40 µm Cyt c,
100 µm NADPH, and 5 µm purified C. reinhardtii Fd for the FNR assay or 20 nm purified
C. reinhardtii FNR for the Fd assay. G6PDH activity was
measured by monitoring the formation of NADPH in the presence of G6P as
described by Farr et al. (1994), except that no 2-mercaptoethanol was
added in the reaction mixture.
Other Methods
Gels (10%) were prepared according to the method of Laemmli
(1970) and electrophoresed at 100 V for 2 h on a Bio-Rad mini protein gel system. The gel was stained with Coomassie brilliant blue.
Protein concentrations were determined by the method of Bradford (1976)
using BSA as a standard. All enzyme assays and absorbances were
measured with a spectronic array (model 3000, Milton Roy, Spokane, WA).
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RESULTS |
NiR, Fd, and FNR were separated from the same cell extract using
Q-Sepharose. Fd and FNR were further purified by standard methods to
yield homogeneous proteins (Fig. 1).
G6PDH activity copurified with FNR on Q-Sepharose and the affinity
column, but was separated by chromatography on Fd-Sepharose. Following
this step G6PDH contained no interfering FNR or Fd activity (data not shown). NiR eluted from the Q-Sepharose column as a sharp peak near the
beginning of the NaCl gradient. NiR bound to phenyl-Sepharose at 15%
(NH4)2SO4,
whereas the enzyme did not bind to a different hydrophobic matrix,
butyl-Toyopearl, at 20%
(NH4)2SO4.
The specific activity of NiR increased significantly after
chromatography on these two hydrophobic columns (data not shown). A
final chromatography step using DEAE-Fractogel yielded NiR, which
appeared as a single band on SDS-PAGE (Fig. 1). Purified NiR has
absorption maxima at 281, 385, and 544 nm and the ratio of
A281/A385 was
1.6 (data not shown). The specific activities of MV-NiR and Fd-NiR were 39 and 14 units mg1 protein, respectively,
which yields a Fd-NiR/MV-NiR ratio of 0.36 (Table
I).
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| Figure 1.
SDS-PAGE of NiR, FNR, and Fd purified from
C. reinhardtii. The gel was stained with Coomassie
brilliant blue. M, Molecular mass standard. Lane 1, One microgram of
NiR; lane 2, 1 µg of FNR; and lane 3, 1 µg of Fd.
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Table I.
Specific activity of purified and rapidly isolated
NiR
Chemically reduced MV and Fd were used in respective NiR assays. For
fresh protein, the assay was carried out immediately after isolation;
for frozen protein, the assay was carried out after storage at
80°C.
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A rapid method was developed to isolate NiR, which exhibited higher
relative Fd-NiR activity. Desalted extracts from freshly harvested
cells were chromatographed on Blue-Cellulofine, an adenosine-affinity matrix. NiR eluted from the column slightly after the major nonadherent protein peak passed off the column. To improve purification in this
step, only fractions containing NiR activity that eluted after the
major protein peak were pooled (data not shown). This sample was
separated from FNR, G6PDH, or Fd because FNR and G6PDH bound to the
Blue-Cellulofine column, and Fd eluted in the main, nonadherent protein
peak. The ratio of Fd-NiR/MV-NiR was approximately 1 immediately after
isolation; however, it decreased to 0.43 after storage at 80°C
(Table I).
The ability of NADPH to reduce
NO2 to
NH4+ was examined by
reconstituting a NO2-reducing
pathway using the algal FNR, Fd, and the rapidly isolated NiR.
NO2 was reduced only in the
presence of all components of this reaction (Table
II).
NO2 reduction was observed
under both aerobic and anaerobic conditions, but the reaction was
somewhat more effective under anaerobic conditions (Table II). Kinetic
studies of the dependency of activity on each of the protein components
of the electron-transfer chain under anaerobic conditions revealed that
any one could be a limiting factor for the reaction (Fig.
2).
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Table II.
Examination of NADPH-dependent
NO2 reduction reactions
The complete reaction mixture contained NADPH, FNR, Fd, rapidly
isolated NiR, NaNO2, and all other components of the
NADPH-dependent NO2 reduction reaction as
described in ``Materials and Methods''. The assays were carried out
under aerobic and anaerobic conditions in the absence () of the
component indicated.
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Under anaerobic conditions the presence of NADP+
in the reaction mixture decreased the rate of
NO2 reduction when the
electron donor was NADPH (Table III).
NO2 reduction was inhibited by
27% when the NADPH:NADP+ ratio was 10 (1 mm NADPH-0.1 mm NADP+).
At a ratio of 1 (1 mm NADPH and 1 mm
NADP+), inhibition rose to 63%. The inhibitory
effect of NADP+ was not pronounced when
chemically reduced MV or Fd was the electron donor (Table III).
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Table III.
Effect of NADPH/NADP+ on NADPH-, Fd-,
and MV-dependent NO2 reduction
The effects of the indicated NADPH/NADP+ ratios on NADPH-,
Fd-, and MV-dependent NO2 reduction under
anaerobic conditions. Conditions were as described in "Materials and
Methods," except that 0.1 unit (as MV-NiR activity) of rapidly
isolated NiR was included in each reaction mixture.
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In plant cell chloroplasts, NADPH can be generated either by
photosynthetic electron transport or via chloroplastic G6PDH, the key
regulatory protein of the OPP pathway. In the presence of partially
purified G6PDH from C. reinhardtii, G6P could support in
vitro NO2 reduction at a rate
of 0.49 unit mg1 protein (Table
IV), approximately 10 times the
NADPH-dependent rate (Tables II and IV). G6PDH-coupled
NO2 reduction did not occur in
the absence of G6P or NADP+, the substrates of
G6PDH, or if any other components of the electron-transfer pathway (FNR
and Fd) were absent (Table IV). High rates of
NO2 reduction were also
observed when NADP+ was replaced by NADPH, but
only in the presence of G6PDH and G6P (Table IV).
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Table IV.
Dependence of G6PDH-coupled
NO2 reduction on reaction components
The dependence of G6PDH-dependent NO2
reduction was examined using a complete reaction mixture containing
NADP+, G6P, G6PDH, FNR, Fd, rapidly isolated NiR,
NaNO2, and all components of the G6PDH-coupled
NO2 reduction reaction, as described in
``Materials and Methods''. Subsequent experiments were carried out
with (+) or without () the indicated components in the reaction
mixture.
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G6PDH-dependent NO2 reduction
also showed inhibition with increasing concentrations of
NADP+, even though this compound is a substrate
of G6PDH. The reaction rate of 16.6 nmol
NO2
min1 as supported by 0.1 mm
NADP+ declined to 2.6 nmol
NO2
min1 when 1.0 mm
NADP+ was used (Table
V).
NO2 reduction was observed
when the components of this G6PDH coupled reaction were replaced with
enzymes from different sources (Table VI). Yeast G6PDH was as effective in this
reaction as the C. reinhardtii enzyme; however, replacement
of either FNR or Fd with enzymes from leaf sources decreased the
reaction rate. 6PGDH and ICDH could replace G6PDH and mediate
NO2 reduction in this reaction
system (Table VI).
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Table V.
Effect of NADPH/NADP+ on
G6PDH-coupled NO2 reduction
The effects of the indicated NADPH/NADP+ ratios on G6PDH-
coupled NO2 reduction under aerobic
conditions, as described in ``Materials and Methods''. Rapidly isolated NiR (0.1 unit of MV-NiR activity) was included in the reaction
mixture.
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Table VI.
Effects of heterologous components and different
dehydrogenases on related NO2 reduction
The effects of replacing the protein components of G6PDH-coupled
NO2 reduction reaction with heterologous
proteins and different dehydrogenases. The complete reaction mixture
contained NADP+, G6P, G6PDH, FNR, Fd, rapidly isolated NiR,
NaNO2, and all components of the G6PDH-coupled
NO2 reduction reaction, as described in
``Materials and Methods''. The respective C. reinhardtii component was replaced by an identical concentration of G6PDH from
yeast; FNR from mung bean leaf, and Fd from spinach leaf. In 6PGDH- and
ICDH-coupled reactions, G6PDH and G6P were replaced by the same amount
of 6PGDH (porcine heart) and 6-phosphogluconate, ICDH (yeast), and the
isocitrate.
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DISCUSSION |
NADPH can support in vitro
NO2 reduction in the presence
of NiR, FNR, and Fd (Table II). Removal of NiR, FNR, or Fd from the reaction mixture prevented NO2
reduction. The rate of NO2
reduction could also be limited by the concentration of each of these
proteins (Fig. 2). Analysis showed that in a crude extract of C. reinhardtii, the concentrations of Fd, FNR, and NiR were approximately 4.5, 0.10, and 0.14 µm, respectively (data
not shown). These data suggested that the concentration of Fd may be
the limiting factor for the rate of
NO2 reduction in vivo. The
failure of NADPH to support
NO2 reduction in the absence
of Fd confirms that the algal NiR is a Fd-dependent enzyme and cannot
use NADPH directly. The dependency of electron transport on FNR
demonstrates for the first time, to our knowledge, that FNR can couple
Fd reduction with the oxidation of NADPH. This reaction proceeds only
slightly better under anaerobic conditions, indicating that the
oxidation of components of this pathway with atmospheric
O2 may not be a significant factor in vivo.
During the purification of NiR, comparison of MV- and Fd-dependent
activity revealed that Fd became a relatively less-effective electron
donor as the enzyme was purified (Table I). To maintain high Fd-NiR
activity required the rapid separation of enzyme from fresh cells. It
has been suggested that NiR may contain a subunit with a molecular mass
of 24 kD that is specifically involved in binding Fd (Romero et al.,
1987; Hirasawa et al., 1989); therefore, the drop in the ratio of
Fd-NiR/MV-NiR activity could result from the loss of this subunit.
However, our purified NiR retained substantial Fd-NiR activity, and no
second subunit for this protein appeared on SDS-PAGE even when the gels
were overloaded (data not shown). This result is consistent with that
reported by Pajuelo et al. (1993), but the ratio of Fd-/MV-NiR activity
reported in this study is somewhat lower than the value of 0.82 that
they reported for C. reinhardtii NiR.
Several reports of work on heterotrophic tissues have indicated that
NADPH must be the electron donor for dark
NO2 reduction (Oji et al.,
1985; Suzuki et al., 1985; Bowsher et al., 1989); however, it has been
suggested that high concentrations of NADPH would be necessary to
support the reduction of Fd by FNR (Bowsher et al., 1989).
NADPH-dependent NO2 reduction
was extremely sensitive to the presence of
NADP+, dropping almost 30% even at a
NADPH:NADP+ ratio of 10:1 (Table III). As
NADP+ did not affect either MV- or Fd-NiR
activity (Table III), it can be concluded that
NADP+ was not inhibiting the activity of either
NiR or Fd. The inhibitory effect of NADP+ must
therefore be mediated through FNR directly. Additional support for this
conclusion is the observation that the reduction of Cyt c by
NADPH via FNR and Fd is also inhibited in the presence of NADP+ (data not shown).
The sensitivity of the reconstituted NADPH-dependent system to
NADP+ levels supports the idea that high ratios
of NADPH/NADP+ would have to be maintained for
NADPH to be able to serve as an electron donor to Fd. Measurements of
pyridine nucleotides in N-limited algae have shown that the ratio of
NADPH/NADP+ ranges from approximately 2 in the
light to approximately 4 in the dark (Huppe et al., 1992, 1994;
Vanlerberghe et al., 1992), which is within the range yielding high NiR
activity in vitro (Table V).
The onset of NO3 assimilation
in N-limited algae has been shown to result in increased activity of
the OPP pathway resulting from G6PDH activation (Huppe et al., 1992,
1994; Vanlerberghe et al., 1992). Previous studies demonstrated that
the addition of G6P to broken plastids allowed NADPH to serve as an
electron donor for NO2
reduction (Oji et al., 1985). These investigators suggested that G6PDH
served to regenerate NADPH from NADP+, thereby
maintaining a high NADPH/NADP+ ratio, favoring
NO2 reduction. In our
reconstituted system NADPH generated by G6PDH supported
NO2 reduction at rates nearly
10-fold greater than that when NADPH was added alone (Tables II and
IV). When G6PDH-dependent NADP+ recycling was
prevented by withholding G6P in the presence of NADPH, the rate of
NO2 reduction dropped to the
NADPH-dependent rate (Tables II and IV). This demonstrated that the key
factor in allowing G6PDH-enhanced rates of
NO2 reduction was the role of
G6PDH in scavenging NADP+. This role for G6PDH
could be replaced by G6PDH from other sources or by 6PGDH or ICDH, both
of which consume NADP+ and produce NADPH (Table
VI). These results indicated that the OPP pathway supports dark
NO2 reduction in the
chloroplast in at least two ways: (a) it generates the NADPH that is
necessary to reduce Fd and subsequently
NO2, and (b) it consumes the
NADP+ produced by FNR in the reduction of Fd,
thereby releasing FNR from NADP+ inhibition.
Although substitution of a nonplant G6PDH did not affect the rate of
NO2 reduction, replacement of
either Fd or FNR did lower the rate of reaction (Table VI). Fd has also
been noted to interact more favorably with another reductase,
Fd-thioredoxin, when both proteins were from similar sources (Huppe et
al., 1990).
The potential for G6PDH-coupled
NO2 reduction must be
considered in the context of the observed rates of dark
NO3 assimilation rates in
C. reinhardtii of approximately 30 µmol mg1 chlorophyll
h1 (L.K. McPhee, personal
communication). Given the observed G6PDH-NiR/MV-NiR ratio of 0.175 (0.49 unit mg1 protein/2.8 units
mg1 protein; Tables I and IV) and a MV-NiR
activity in the crude extract of 260 µmol
NO2
mg1 chlorophyll h1
(T. Jin, unpublished data), the G6PDH-coupled system could
support in vitro assimilation of (260 × 0.175) at approximately
45 µmol NO2
mg1 Chl h1. As a
result, the observed in vitro rate of G6PDH-dependent
NO2 reduction is sufficient to
account for the observed rates of dark
NO3 assimilation.
The results of this study demonstrate for the first time, to our
knowledge, that NADPH may reduce Fd via FNR and thereby support NO2 reduction. The reversible
character of the reaction catalyzed by FNR is the key point of this
reconstituted system. The equilibrium between reduced/oxidized Fd and
NADPH/NADP+ drives the FNR reaction in the
appropriate direction. Therefore, to have Fd serve as an efficient
electron donor to NiR, the ratio of NADPH/NADP+
must be very high. When G6PDH is used to generate NADPH and to consume
NADP+, NiR activity increased approximately
10-fold primarily due to the consumption of
NADP+, thereby releasing FNR from
NADP+ inhibition. The rates of
NO2 reduction supported in
this way account for the observed rates of whole-cell
NO3 assimilation in the dark.
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FOOTNOTES |
1
This work was supported by the
Natural Sciences and Engineering Research Council of Canada.
2
Present address: Global Bureau Environment
Center, U.S. Agency for International Development, RRB 3.08, 1300 Pennsylvania Avenue, NW, Washington, DC 20007-3800.
*
Corresponding author; e-mail turpind{at}post.queensu.ca; fax
1-613-545-6617.
Received November 20, 1997;
accepted February 10, 1998.
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ABBREVIATIONS |
Abbreviations:
FNR, Fd NADP+ oxidoreductase.
G6P, Glc-6-P.
G6PDH, G6P dehydrogenase.
ICDH, isocitrate dehydrogenase.
MV, methyl viologen.
NiR, nitrite reductase.
OPP, oxidative pentose
phosphate.
6PGDH, 6-phosphogluconate dehydrogenase.
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Abstract
Introduction
