From the Departamento de Bioquímica y
Biología Molecular y Celular, Facultad de Ciencias, Universidad
de Zaragoza, 50009 Zaragoza and
Grupo de Cristalografía
Macromolecular y Biología Estructural, Instituto
Química-Física Rocasolano, Consejo Superior de
Investigaciones Científicas, Serrano 119,
28006 Madrid, Spain
Received for publication, October 11, 2000, and in revised form, November 21, 2000
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ABSTRACT |
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On the basis of sequence and three-dimensional
structure comparison between Anabaena PCC7119
ferredoxin-NADP+ reductase (FNR) and other reductases from
its structurally related family that bind either NADP+/H or
NAD+/H, a set of amino acid residues that might determine
the FNR coenzyme specificity can be assigned. These residues include
Thr-155, Ser-223, Arg-224, Arg-233 and Tyr-235. Systematic
replacement of these amino acids was done to identify which of them are
the main determinants of coenzyme specificity. Our data indicate that all of the residues interacting with the 2'-phosphate of
NADP+/H in Anabaena FNR are not involved to the
same extent in determining coenzyme specificity and affinity. Thus, it
is found that Ser-223 and Tyr-235 are important for determining
NADP+/H specificity and orientation with respect to the
protein, whereas Arg-224 and Arg-233 provide only secondary
interactions in Anabaena FNR. The analysis of the T155G FNR
form also indicates that the determinants of coenzyme specificity are
not only situated in the 2'-phosphate NADP+/H interacting
region but that other regions of the protein must be involved. These
regions, although not interacting directly with the coenzyme, must
produce specific structural arrangements of the backbone chain that
determine coenzyme specificity. The loop formed by residues 261-268 in
Anabaena FNR must be one of these regions.
During the last decades, the understanding of protein function
and, more specifically, the role of the individual amino acid residues
involved in substrate binding and in the catalytic action have achieved
considerable progress. Among the most relevant enzymes studied are
those involved in electron transfer processes due to their practical
importance. Now, the opportunity to design novel proteins is becoming
more feasible, especially due to the increased detailed knowledge of
the three-dimensional structure of many proteins. As a first step in
this direction recent investigations have been aimed to redesign
already existing proteins, so that they can produce a function
different to that for which they were naturally synthesized (1, 2).
Following this direction, a lot of effort is being made in the
description of the determinants of coenzyme specificity for NAD(P)
+/H-dependent redox enzymes (3-5). In biological
systems NAD+/H is almost exclusively used by enzymes that
catalyze oxidative exergonic reactions, whereas reductive endergonic
reactions are generally catalyzed by enzymes that utilize
NADP+/H (6). However, the only structural difference
between them is the presence of a 2'-P group bound to the AMP moiety of
the coenzyme in NADP+/H, and it is the presence or the
absence of this phosphate group that permits the enzymes to make the
distinction between these two coenzymes. Moreover, among the
structurally related enzymes of the FNR family, members with preference
either for NADP+/H or NAD+/H can be found.
Crystallographic studies have demonstrated that discrimination between
these coenzymes does not result from the presence of different
structural domains in these enzymes (7-9).
We describe the introduction of point mutations in the coenzyme binding
domain of ferredoxin-NADP+ reductase
(FNR,1 EC 1.18.1.2) from the
cyanobacterium Anabaena PCC7119 to probe the determinants of
its coenzyme specificity and also as an initial attempt to alter the
coenzyme specificity. This enzyme consists of a soluble single
polypeptide chain that contains a noncovalently bound FAD group that is
the cofactor involved in the redox reaction. Several points prompted us
to choose FNR. During photosynthesis FNR accepts electrons from
ferredoxin and uses them to convert NADP+ into NADPH (10).
This process is highly specific for NADP+/H
versus NAD+/H (11-13). Extensive biochemical
characterization of FNR from different sources and, in particular, from
Anabaena has been carried out (11-23), and several
three-dimensional structures of FNR forms are available (24-26).
Moreover, the structural arrangement of FNR has been proposed to be the
prototype of a family of flavin oxidoreductases that interact
specifically with either NADP+/H or NAD+/H (8,
25, 27). Finally, considering the high economical value of the reduced
form of NADP+/H, the development of an in vitro
system using FNR to generate NADPH is of high interest. Work is already
under progress in this direction, and we foresee this development
occurring in the near future (28). If we also achieve change of
coenzyme specificity for FNR, generation of NADH could also be obtained
with the same system. Moreover, due to the much cheaper value of NADH
versus NADPH, the system could be used in the opposite
direction, with NADH as reducing power, to produce reduced proteins
(like ferredoxin, flavodoxin, hydrogenase, cytochrome P450 reductase,
etc.).
Different crystallographic approaches on a variety of FNR forms from
different sources have provided a picture of how the NADP+
substrate must bind to FNR (24-26). Thus, the studies of spinach and
Anabaena FNR revealed the importance of the side chains of residues Arg-100, Ser-223, Arg-224, Arg-233, Tyr-235, and Gln-237 (Anabaena FNR numeration) in the stabilization of the
complex with NADP+/H by making contacts to its adenine
ring, its 2'-P, and its 5'-phosphoryl (24, 25). The negative charge of
the 2'-P group is apparently stabilized by the lateral chains of two
positive-charged arginine residues, Arg-224 and Arg-233 (Arg-235 and
Lys-244 in the spinach enzyme). The 2'-P of NADP+/H might
also form hydrogen bonds with Ser-223 and Tyr-235 (in this case a
stacking interaction is also formed with the adenine moiety of
NADP+/H) (Fig.
1A). The sequence and
three-dimensional structure of FNR at the site of NADP+
interaction have been compared with those of several NADP+
and NAD+ reductases within the FNR family (Table
I, Fig. 1). Conservation of
residues interacting with the 2'-P group of NADP+/H was
observed. Thus, Ser-223, Arg-224, Arg-233, and Tyr-235 are conserved or
show conservative substitutions in all the
NADP+/H-depending enzymes (Fig. 1B, Table I).
However, in the NAD+/H-dependent enzymes, these
residues are not conserved, which interrupts the stabilization of 2'-P
group and probably modifies the stacking interaction with the adenine
moiety of NADP+/H by changing Tyr-235 by Phe (Fig.
1C). Sequence and structure analysis suggest that other
regions of the protein might also account for FNR coenzyme specificity
(Table I, Fig. 1, D-F). Thus, most of the members of the
FNR family that bind NADP+/H show the sequence T(P)GTGXAP
(residues 155-161 in Anabaena FNR; whereas in those
interacting with NAD+/H, the corresponding sequence is
GGXGXTP (Table I). These residues form the loop between Oligonucleotide-directed Mutagenesis--
Anabaena
FNR mutants were prepared using a construct of the petH gene
previously cloned into the expression vector pTrc99a as a template
(30). The FNR mutants T155G, S223D, S223G, R224Q, R233A, Y235F, and
Y235A were produced using the Transformer site-directed mutagenesis kit
from CLONTECH in combination with suitable
synthetic oligonucleotides. The pTrc99a vectors with the desired
mutation were used to transform the Escherichia coli Pasteur
collection strain 0225 (17).
Purification of the FNR Mutants--
FNR mutants were purified
from isopropyl-1-thio- Spectral Analysis--
Ultraviolet-visible spectral analyses
were carried out either on a Hewlett-Packard diode array 8452 spectrophotometer, a Kontron Uvikon 860 spectrophotometer, or a Kontron
Uvikon 942 spectrophotometer. Circular dichroism was carried out on a
Jasco 710 spectropolarimeter at room temperature in a 1-cm path length
cuvette. Protein concentrations were 0.7 µM for the far
UV and 3 µM for the aromatic and visible regions of the
spectrum. Photoreduction of different FNR forms was performed at room
temperature in an anaerobic cuvette containing 32-65 µM
FNR samples and 3 µM 5-deazariboflavin in 50 mM Tris/HCl buffer, pH 8. The solutions were made anaerobic
by repeated evacuation and flushing with O2-free Ar. The
spectra were recorded in a HP8452 diode array spectrophotometer before
and after irradiating the samples with a 300-W light source for
different times. Dissociation constants of the complexes between
oxidized FNR mutants and either NADP+ or NAD+
were measured by differential spectroscopy using a double beam spectrophotometer at 25 °C as previously described (12, 17).
Enzymatic Assays--
Diaphorase activity, assayed with DCPIP as
artificial electron acceptor was determined for all the FNR mutants as
described previously (17). Both NADPH and NADH were assayed as coenzyme electron donors to each of the different FNR mutants. Unless otherwise stated, all the measurements were carried out in 50 mM
Tris/HCl, pH 8.0. In all measurements, direct reduction of DCPIP by the coenzyme was subtracted from that of the enzyme-coenzyme mixture. The
kinetic results obtained from the diaphorase activity were interpreted
using the Michaelis-Menten kinetic model. In the case of the diaphorase
reactions studied using NADH, high enzyme concentrations (0.5-9
µM) were required to detect and follow their activity. Therefore, in some of these cases the coenzyme concentration used was
only 100 times higher than that of the corresponding enzyme. This was
also the case for the S223D FNR form with NADPH, where the enzyme
concentration in the cuvette was 1 µM. When assaying the
reaction of the other FNR enzymes with NADPH, enzyme concentrations ranging from 3 to 25 nM were used.
Stopped-flow Kinetic Measurements--
Fast electron transfer
processes between NADPH or NADH and the different FNRox
mutants were studied by stopped-flow methodology under anaerobic
conditions using an Applied Photophysics SX17.MV spectrophotometer
interfaced with an Acorn 5000 computer using the SX.17MV software of
Applied Photophysics as previously described (17). The observed rate
constants (kobs) were calculated by fitting the
data to a mono- or bi-exponential equation. Samples were made anaerobic
(in specially designed tonometers that fit the stopped-flow apparatus)
by successive evacuation and flushing with O2-free Ar in 50 mM Tris/HCl, pH 8.0. Final FNR concentrations were kept
between 6 and 11 µM, whereas, unless otherwise stated, NADPH final concentrations were in the range of 160-200
µM, and NADH was used at final concentrations in the
range of 250-300 µM or at 2.5 mM. The same
methodology was also applied to the study of the reduction of
NADP+ by T155G FNRrd. The time course of the
reactions was followed at 460 nm, although other wavelengths were also
analyzed (340 and 600 nm).
Crystal Growth, Data Collection, and Structure
Refinement--
Crystals of the T155G FNR mutant were grown by the
hanging drop method. The 5-µl droplets consisted of 2 µl of 25.9 mg
of protein/ml of solution buffered with 10 mM Tris/HCl, pH
8.0, 1 µl of unbuffered
These crystals were mounted in glass capillaries and screened on a Mar
Research (Germany) image plate area detector for intensity, resolution, and mosaic spread using graphite-monochromated
CuK
The T155G structure was solved by molecular replacement using the
program AmoRe (33) on the basis of the 1.8-Å resolution native FNR
model (24) without the FAD cofactor. An unambiguous single solution for
the rotation and translation functions was obtained. This solution was
refined by the fast rigid-body refinement program FITING (34). The
model was subjected to alternate cycles of conjugate gradient
refinement with the program X-PLOR (35) and manual model building with
the software package O (36). The crystallographic R and
Rfree (37) values converged to values of 0.16 and 0.24, respectively for reflections between 9.0- and 2.4-Å
resolution (Table II). The final model
contains 2335 nonhydrogen protein atoms and 1 FAD, 1 SO Expression and Purification of the Different FNR Mutants--
The
level of expression in E. coli of all the mutated FNR forms
was judged to be similar to that of the recombinant WT. All the mutants
were obtained in homogeneous form and in amounts suitable to perform
the demanding characterization studies described herein. Mutants at the
position of Ser-223 interacted weakly with the Cibacron blue column,
which binds specifically those proteins with a NAD(P)+/H
interaction site, requiring the use of a fast protein liquid chromatography Mono-Q column for purification.
Spectral Properties--
No major differences were detected in the
UV-visible absorption and CD spectra of any of the FNR forms (Fig.
2). Therefore, no major structural
perturbations appear to have been introduced by the mutations, and the
extinction coefficient of Anabaena WT FNR (9.4 mM Steady-state Kinetics of the Different FNR Forms--
The
steady-state kinetic parameters of the different FNR mutants were
analyzed for the DCPIP-diaphorase reaction using either NADPH or NADH
as electron donor (Table III). T155G,
R224Q, and Y235F yielded similar values for kcat
in the NADPH-dependent reaction to the WT enzyme. This
parameter decreased by a factor of 4, 2, and 4 for S223G, R233A, and
Y235A, respectively, and was up to 200 times smaller in the case of the
S223D FNR form. With regard to the Km value for
NADPH, T155G, R224Q, and Y235F showed moderated increments with regard
to the WT enzyme value (about 4-, 13-, and 7-fold), whereas S223G,
R233A, and Y235A yielded much higher Km values for
NADPH (about 50-, 36-, and 67-fold, respectively), with S233D being the
mutant with the highest Km value (about 125-fold
larger) (Table III). Taking into account the kinetic parameters
obtained, all the mutants showed a significantly decreased catalytic
efficiency (kcat/Km) with
regard to the WT FNR. The introduction of an aspartic acid residue at
position 223 was the mutation that most affected the catalytic
efficiency of the enzyme with NADPH (Fig.
3A).
The DCPIP-diaphorase activity of the WT and all the FNR mutants was
also assayed with NADH as electron donor (Table III). The data indicate
that, in terms of both kcat and
Km, NADH is a very poor reductant for WT FNR
(kcat decreased 38-fold, and Km for the coenzyme increased by a factor of 133 with respect to NADPH). Therefore, Anabaena FNR has a very
low catalytic efficiency with NADH (Table III), and the specificity of
the enzyme for NADPH, expressed as the ratio between the catalytic
efficiency for NADPH and NADH, was found to be 67,000 times higher
(Fig. 3). Although all the mutants presented a
kcat value for the NADH-dependent reaction within a factor of 10 with regard to that of WT FNR, R224Q,
R233A, and Y235F show a clear increase of this value (4-7-fold), whereas T155G and Y235A show a decrease (about 7-8-fold) (Table III).
It is noteworthy that these two mutants, T155G and Y235A, are the only
ones showing a decrease in the Km value for NADH
(about 4- and 2-fold respectively), whereas the rest of the mutants
show an increment for this parameter to within a factor of 4 of the WT
value. As is also shown in Table III, the catalytic efficiency of all
these mutants with NADH is within a factor of 10 that of the WT enzyme
with this coenzyme and for all of them is considerably smaller than
that observed with NADPH (Fig. 3). However, it is noteworthy that when
Ser-223 is replaced by an aspartic acid, the catalytic efficiency with
NADPH approximates that obtained with NADH. Thus, the S223D single
mutation decreases the enzyme specificity for NADPH from 67,000 times
in the WT to only 8 times in the mutant (Fig. 3A).
Interaction of FNR Mutants with NADP+ and
NAD+--
The interaction of the different FNR forms with
either NADP+ or NAD+ was investigated by
differential spectroscopy (Fig. 4). When NADP+ binds to oxidized WT FNR, the visible spectrum of the
bound flavin undergoes a perturbation, yielding the difference spectrum
shown in Fig. 4A. This has been shown in the case of the
Anabaena FNR to be due to the interaction of the 2',5'-ADP
moiety of the cofactor with the reductase (12). The spectral
perturbations observed for oxidized R224Q, R233A, and Y235F FNRs upon
NADP+ binding were weaker but very similar in shape to
those observed for the WT FNR, and only minor displacements of the
minima (around 392 and 502 nm) and maxima (around 354, 458, 480, and
522 nm) were detected (Fig. 4, A and B). The
difference spectra obtained at different coenzyme concentrations
allowed the determination of the dissociation constants and binding
energies for the corresponding complexes (Fig. 4F, Table
IV) (12). Thus, Y235F, R224Q, and R233A bind NADP+ 35-, 95-, and 210-fold weaker than the WT
FNR. Therefore, although binding of NADP+ to either Y235F,
R224Q, or R233A FNRs produce equivalent structural perturbations around
the flavin ring as those observed for the WT, apparently the positive
side chains of Arg-224 and Arg-233 play an active role in positioning
the coenzyme by making direct contacts with it. Interestingly, binding
of NADP+ to T155G FNR elicited spectral changes at
different wavelengths than those reported for WT FNR (Fig.
4D), with minima at 396 and 440 nm and maxima around 476 and
512 nm, whereas the binding was estimated to be only 17-fold weaker
than that of the WT (Table IV). This result indicates that, although
Thr-155 might not be directly involved in the interaction with the
coenzyme, the replacement of Thr-155 by Gly produced slight structural
changes in the protein that have an important influence in the
arrangement of the flavin environment when the coenzyme is bound.
Finally, when oxidized S223D, S223G, and Y235A FNR forms were titrated
with NADP+, no difference spectra were detected in the
flavin region of the spectra as shown in Fig. 4E for Y235A.
These mutants were only characterized by a loss of absorbance peaking
in the 334-338 nm range, which allowed estimation of very high values
for the dissociation constants in the case of these complexes (Table
IV), indicating the importance of positions Ser-223 and Tyr-235 in coenzyme recognition and binding.
It has already been shown that NAD+ is not able to produce
any spectral perturbation in the flavin absorption range when added to
WT FNR, presumably due to the absence of the 2'-P group, which is
essential for NADP+ binding to Anabaena FNR
(12). When the mutants were titrated with NAD+, only T155G
elicited a weak difference spectrum in the flavin region with maxima at
420 and 505 nm (Fig. 4C). These data indicate that
replacement of Thr-155 by Gly produced some changes in the protein that
allow NAD+ to perturb the FAD environment of FNR. No
difference spectra were obtained with any other FNR mutant in the
flavin region of the spectra.
Fast Kinetic Studies of the Reduction of FNR Mutants by NADPH and
NADH--
The fast kinetic reaction of oxidized Anabaena
FNR forms with either NADPH or NADH was determined by following the
flavin spectral changes at 340, 460, and 600 nm under anaerobic
conditions. As already reported (17), WT Anabaena FNR
reacted rapidly with NADPH, producing a decrease in the absorption at
460 nm that was best fit by two processes that have been attributed to
the production of the charge-transfer complex
[FNRox-NADPH] (kobs > 500 s
In the case of the T155G FNR form, the kinetics of reoxidation of the
enzyme by NADP+ has also been studied by stopped-flow. An
increase in absorbance at 460 nm due to FNR reoxidation and an increase
at 340 nm due to NADPH formation were observed (Fig.
6). The traces were well fit by a single
exponential having a rate constant of at least 350 s
When analyzing the reaction of WT FNR with NADH by stopped-flow a very
slow decrease in the absorption at 460 nm was observed that was best
fit by two processes having kobs values of 0.35 (~60% amplitude) and 0.005 (~40% amplitude)
s Three-dimensional Structure of the T155G FNR Mutant--
After
observing the difference spectra for the T155G FNR mutant in the
presence of NADP+, which indicated a slightly different
structural arrangement of the flavin environment in the presence of the
coenzyme, we decided to determine the three-dimensional structure of
the T155G FNR form by x-ray diffraction. The first eight residues in
the sequence were not included in the model due to the poor electron density map in this region. The overall folding of the T155G FNR mutant
shows no significant differences with respect to the native structure,
as shown by the low root-mean-square deviations (0.4 Å) of the C Effect of the Single Mutations at the 2'-Phosphate Interaction Site
of NADP+--
The importance of a stabilizing interaction
between the side chain of Ser-223 and the 2'-P of NADPH has been proven
by replacement of this residue by a Gly, which lacks the OH group and,
therefore, the hydrogen binding capability, and by an Asp, the residue
that is highly conserved at the equivalent location in
NADH-dependent enzymes (Table I; Fig. 1, A and
C). Replacement of Ser-223 by either Gly or Asp produced
enzymes that had only 0.5 and 0.005% of the catalytic efficiency of
the WT FNR with NADPH (Table III, Fig. 3A), and the
kobs values for the reaction with NADPH were at
least 30- and 1500-fold, respectively, slower than that of the WT
enzyme when analyzed by stopped-flow (Fig. 5, Table V). However, no
gross differences were detected for the catalytic parameters with NADH
when compared with those of the WT enzyme (Fig. 3B). Thus,
steady-state and fast kinetic studies suggest that removal of the
Ser-223, and/or introduction of a negatively charged residue at this
position produces an enzyme that lacks its ability to discriminate
between NADP+/H and NAD+/H. This behavior must
be mainly due to the low affinity of both Ser-223 mutants for either
NADP+/H or NAD+/H, as suggested from the fact
that neither of them showed a difference spectrum in the presence of
any of the oxidized coenzymes (Table IV). Thus, our studies confirm
that Ser-223 side chain is a crucial determinant of FNR coenzyme
specificity for NADPH. The very low degree of interaction between S223G
and the coenzyme suggests that stabilization of NADPH binding must be
due to a hydrogen bond or a charge-dipole interaction between the
Ser-223-OH group and the 2'-P of NADP+/H. Such interactions
cannot be formed in this mutant. Moreover, introduction of a negative
charge at position 223 almost completely prevents the
FNR-NADP+/H interaction. As mentioned above, the
NAD+/H-dependent enzymes of the FNR family have
an aspartate residue at this position (Table I, Fig. 1). Several
studies have already established the importance of such a negatively
charged residue in NAD+/H -dependent enzymes
from different families as well as the strong alteration of the
affinity for NAD+/H when hydrogen bonds between this
carboxylate group and the adenosine 2'- and 3'- hydroxyl positions of
the coenzyme are not able to be formed (9, 40-42). It has also been
shown that the introduction of such a negative charge at the residue
that occupies the position spatially similar in some
NADP+/H-dependent enzymes prevents the
interaction with the 2'-P of the coenzyme and might even enhance
NAD+/H affinity (43, 44). However, this latest effect has
not been shown by our S223D mutant (Fig. 3B, Table V). Thus,
it can be proposed that in the case of Anabaena FNR, the
introduction of a negatively charged residue at position 223 produces a
change in the catalytic behavior of the enzyme that is mainly due to the electrostatic repulsion between the negative side chain and the
negatively charged 2'-P of NADP+/H, which prevents its
binding rather than allowing a favorable interaction of the Asp-233
with the 2'-OH group of NAD+/H.
Previous studies suggest that the positively charged side chains of
Arg-224 and Arg-233 might be involved in FNR coenzyme discrimination
(15, 16, 24-26, 45-47). To test this hypothesis, the R224Q and R233A
FNR mutants were produced by removing the positive charge. These
enzymes were found to have 7.5 and 1.5%, respectively, the catalytic
efficiency of the WT FNR with NADPH (Table III, Fig. 3A),
and these low efficiencies are mainly due to the high
Km values for NADPH for these mutants. When analyzing the catalytic parameters with NADH for these mutants, both of
them showed only a moderate increment in the
kcat value with regard to the WT FNR.
Combination of these two factors produced a slight increment in their
catalytic efficiencies with NADH (Table III, Fig. 3B). Both
of these mutants elicited changes in their visible spectra upon
NADP+ addition (Fig. 4), indicating coenzyme binding. R224Q
and R233A were estimated to bind NADP+ 90- and 210-fold
weaker than the WT FNR (Table IV). However, no affinity for
NAD+ was detected (Table IV). When analyzing the fast
kinetic parameters of the reaction of NADPH with R224Q and R233A,
different behaviors were detected for both mutants. R224Q shows a
pattern of behavior similar to that of the WT enzyme but had
kobs1 and kobs2 values at
least 3 and 10 times smaller, respectively (Fig. 5, Table V). However,
the reaction with R233A is more than 500-fold slower than that of the
WT enzyme, even at high NADPH concentration. Finally, when these
mutants were analyzed for the reaction with NADH, both reactions were
best fit to monoexponential processes, with the observed rate
constants for both of the mutants slightly higher than those observed
for the WT enzyme (Table V). The obtained results indicate that
replacement of Arg-224 by a Gln produces an enzyme that is still able
to form a productive complex with NADPH, although considerably weaker,
and to accept electrons efficiently from the coenzyme. This indicates
that the function played by the guanidinium group of Arg-224 in
stabilizing an interaction with the 2'-P could be also supported in a
considerable degree by the Gln side chain. Moreover, a Gln residue at
this position also slightly improves some of the processes studied with
NADH. Analyzing sequence homology at this position allows the behavior observed for the R224Q mutant to be easily understood. As expected, all
the NADP+/H -dependent enzymes of the FNR
family possess a positively charged residue at this position (Table I).
Moreover, it is noteworthy that this is also the case for some of the
NAD+/H-dependent enzymes, and it is evident
that in those enzymes that do not have negatively charged residues at
this position, residues having side chains (Gln or His) capable of
forming hydrogen bonds or dipole-charge interactions with the 2'-P are
located at the equivalent position. Therefore, we can conclude that,
although Arg-224 might be involved in an electrostatic interaction with the 2'-P that allows tighter binding to the substrate, it must be
mainly involved in stabilization of the coenzyme binding through hydrogen bonds or charge-dipole interactions. However, this residue is
not involved in determining FNR specificity for NADP+/H
versus NAD+/H. Replacement of Arg-233 by an Ala
produces a more drastic effect on the interaction between the enzyme
with its natural coenzyme, NADP+/H, which therefore affects
the fast electron transfer process. This indicates that the side chain
of the Ala is not able to provide some interactions that apparently
allow an appropriate orientation of the coenzyme in its FNR binding
region to allow subsequent electron transfer. Also, taking into account
the conservation of a positively charged residue in all the
NADP+/H-dependent enzymes, and the fact that none
of the NAD+/H-dependent enzymes have a
positively charged side chain at this position, our results
suggest that the Arg-233 side chain provides stabilization of the 2'-P
group and allows optimal NADP+/H binding. However, removal
of this interaction only slightly improves the affinity of the
FNR for NAD+/H. Positively charged residues equivalent to
the aforementioned have already been shown to play a role in
determining NADP+/H specificity in other enzymes, such as
cytochrome P-450 reductase or isocitrate dehydrogenase (48, 49).
The three-dimensional structural analysis of the complexes of FNR with
NADP+ also indicates a close interaction between the
adenine moiety of the NADP+ and the aromatic side chain of
Tyr-235, which is conserved as a Tyr in all
NADP+/H-dependent enzymes and as a Phe in the
NAD+/H-dependent enzymes of the family (Fig. 1,
Table I) (24, 26). The residue at this position of Anabaena
FNR has been replaced either by a Phe or by an Ala. The diaphorase
activities with NADPH for the Y235F and Y235A FNR enzymes were,
respectively, 28% and just 0.3% of the WT catalytic efficiency (Table
III, Fig. 3A). These results are mainly due to a 7-fold
increment in the Km of the Y235F form and to a
70-fold for the Y235A mutant, which was also accompanied by a 4-fold
decrease of its kcat. A slightly increment in
the kcat value of Y235F with NADH was also
observed (Table III, Fig. 3B). On the contrary, Y235A was
4-fold less efficient with NADH than the WT. Moreover, whereas Y235F
binds NADP+ 35-fold weaker than the WT FNR (Fig. 4), no
binding of this coenzyme was detected for Y235A. None of the mutants
elicited a difference spectrum in the presence of NAD+,
indicating a very low affinity for this coenzyme (Table IV). Fast
kinetic parameters of the reactions of either NADPH or NADH with Y235F
and Y235A also show different behaviors for both mutants. Although
Y235F behaved similarly to the WT enzyme, with only slightly slower
observed rate constants (Figs. 5 and 7, Table V), the destabilizing
effect introduced by the Ala at position 235 clearly resulted in a
drastic decrease of the observed rate constant values of the processes.
Therefore, our results clearly indicate that replacing Tyr-235 for a
nonaromatic residue noticeably reduced the ability of FNR to interact
efficiently with NADP+/H, and it even produced a decrease
in the already low affinity of the enzyme for NAD+/H. This
confirms that the stacking interaction between the aromatic residue at
position 235 of Anabaena FNR and the adenine ring of NAD(P)+/H is required to provide an adequate orientation
between the protein and the coenzyme, which places the nicotinamide
ring in a position capable of accepting electrons from the
isoalloxazine ring. Our data also show that a Tyr at position 235 is
much more efficient than a Phe for NADP+/H binding and
electron transfer, confirming the importance of the hydrogen bond
between the Tyr-OH and the 2'-P of NADP+/H in complex
formation and orientation. The lack of this hydrogen bond in the Y235F
mutant still allows NADP+/H binding but results in a weaker
and less productive complex. On the other hand it is noticeable that
this latter mutant appears to accommodate NAD+/H more
efficiently. This might also be expected, taking into account that the
NADH-dependent members of the enzyme have a Phe at this
position (Table I, Fig. 1D).
Effect of the Single Mutation T155G--
Thr-155 of
Anabaena FNR was replaced by a Gly, the residue present at
the equivalent position in all NAD+/H
-dependent members of the family (Fig. 1, Table I), to test if this residue and the region that contains it is involved in coenzyme
specificity and if NAD+/H binding could be induced. This
mutation produced only a slight effect on the catalytic properties of
the enzyme when its reactivity was assayed with NADPH either by
steady-state or by fast kinetic methods (Table III and V, Fig. 3). The
Km for NADPH and Kd for the
T155G-NADP+ complex suggest that these changes must be due
to a decrease in the affinity for the coenzyme (Tables III and IV). The
latest analysis also indicates that this mutant accommodates the
NADP+/H coenzyme in a different orientation with regard to
the flavin ring than the WT (Fig. 4). Analysis of the T155G FNR
three-dimensional structure indicates that the introduced mutation
produces an alteration of a hydrogen bond network (Fig. 9), which
produces a slight displacement of the backbone in the NADP+
binding domain (Fig. 8). In particular, important structural differences are observed in the loop comprising residues 261-268. Interestingly, the three-dimensional structural analysis of this region
in the different members of the FNR family shows that in the case of
NAD+/H-dependent enzymes a different
organization of the hydrogen bond network (Fig. 9) and a hairpin-like
region rich in proline residues (Table I, Fig.
10) are present. This analysis also
shows that the conformation of such a region rich in proline residues in the NAD+/H members would not be compatible with the
presence of a Thr (or Pro) at the position equivalent to 155 of
Anabaena FNR in the NADP+/H-members due to
steric hindrance (Fig. 10). Replacement of Thr-155 in
Anabaena FNR by a Gly produces a slight retraction of the
loop that might explain some of the behaviors observed for this mutant such as the increase in its affinity for NAD+/H, as
indicated by the decrease observed in the Km for NADH and by the fact that of all the FNR forms assayed, T155G was the
only one that showed a difference spectra upon NAD+ binding
(Fig. 4C). However, the mutant did not enhance reactivity with NADH, as shown by the very small values obtained by
kcat and kobs when
studied by steady-state and by fast kinetic methods (Table III and V).
This indicates that, although the structural modifications induced by a
Gly at position 155 enhance the enzyme affinity for NAD+/H,
the resulting complex does not provide an orientation conducive to
electron transfer. This was expected, since the other regions of the
protein that determine the coenzyme specificity are involved in
NADP+/H recognition.
General Conclusions--
The analysis of the determinants of
coenzyme specificity in Anabaena FNR indicates that all the
residues interacting with the 2'-P group of NADPH, Ser-223, R224,
Arg-233, and Y235, are not involved to the same extent in determining
coenzyme affinity and specificity. Thus, the side chain of Ser-223 is
crucial in determining NADP+/H binding, with the presence
of a negatively charged residue at this position preventing its
binding. Although the presence of a positively charged residue at
positions 224 and 233 of Anabaena FNR are not crucial in
determining coenzyme specificity, these residues are involved in
providing a stronger interaction between the enzyme and
NADP+/H. The importance of an aromatic residue at position
235 of Anabaena FNR for the interaction with both coenzymes,
NADP+/H and NAD+/H, has been demonstrated, as
has the fact that a hydrogen bond between the coenzyme and the OH group
of Tyr-235 is also involved in determining the NADP+/H
versus NAD+/H specificity in FNR. Finally, our
results also indicate that the determinants of coenzyme specificity of
FNR are not only situated in the 2'-P binding region, but that other
regions of the protein must be involved. Thus, the arrangement of the
backbone chain in the coenzyme binding domain around the loop that
connects strand 4 and the
INTRODUCTION
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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1 and
-helix A as well as the first residues of this
-helix, whose
N-terminal end might stabilize an interaction with the negative
pyrophosphate of the coenzyme (Fig. 1D). A similar motif has
also been shown to exist in the case of the flavoenzyme glutathione
reductase, which is not a member of the FNR family and to be involved
in coenzyme specificity (3). To confirm the importance of the
interactions with the 2'-P group of NADP+ and of the
TGTGXAP FNR motive in Anabaena FNR coenzyme specificity, the
T155G, S223G, S223D, R224Q, R233A, Y235F, and Y235A Anabaena FNR mutants have been constructed and characterized by a variety of
techniques. The choice of the introduced mutations has been made taking
into account the residues that occupy the equivalent positions in the
NAD+/H-dependent members of the FNR family and
trying to simulate a potential change in cofactor specificity (Table
I). The rest of the mutations have been analyzed as controls. Moreover,
after a careful analysis of the three-dimensional structure recently reported for a complex between FNR and ferredoxin (29), none of these
residues is expected to be involved in ferredoxin binding or electron
transfer.
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Fig. 1.
Three-dimensional structure comparison of the
FNR family in the regions involved in the coenzyme binding.
Anabaena FNR is shown in A and D, and
NADPH cytochrome P450 reductase is shown in B and
E. NAD+/H-dependent reductases are
shown in C and F, and they are represented by
corn nitrate reductase (orange) and NADH-cytochrome
b5 reductase (green). The region
interacting with the 2'-phosphate of NADP+ is compared in
A, B, and C. In the FNR and other
NADP+/H-dependent enzymes, this region has two
(FNR) or three (NADPH cytochrome-P450 reductase) positively charged
residues, respectively, stabilizing the negative charge of the
phosphate group; this group is also hydrogen-bonded to Ser-223 and a
Tyr-235 residues (A and B). In the case of
NAD+/H enzymes (C) these residues are not
conserved. The region interacting with the pyrophosphate of the
coenzyme presents a different consensus sequence depending on the
coenzyme, NADPH (D and E) or NADH (F).
In NADP+/H-dependent enzymes Thr-155 (Pro),
Thr-157, and Ala-160 (Anabaena FNR numbering) are always
conserved (D and E). In those interacting with
NAD+/H, the equivalent residues are Gly, X, and
Thr (F). The FAD cofactor and the coenzyme (if present) are
drawn as sticks. Flavin cofactor are colored
orange, and NADP+/H analogues are colored
yellow. This figure was drawn using MOLSCRIPT (61) and
RENDER (62).
Sequence alignment of different members of the FNR family in three of
the conserved sequence regions involved in coenzyme binding
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-D-galactopyranoside-induced LB
cultures as described previously (17, 30). Some of the mutants were not
retained by the Cibacron blue gel and were purified by fast protein
liquid chromatography using a Mono-Q column. UV-visible spectra and
SDS-polyacrylamide gel electrophoresis were used as purity criteria.
-octylglucoside at 5% (w/v), and 2 µl
of reservoir solution containing 18% (w/v) polyethylene glycol 6000, 20 mM ammonium sulfate, and 0.1 M sodium
acetate, pH 5.0. The droplet was equilibrated against 1 ml of reservoir
solution at 20 °C. Under these conditions crystals grew within 1-7
days up to a maximum size of (0.8 × 0.4 × 0.4 mm) in the
presence of a phase separation caused by the detergent.
radiation generated by an Enraf-Nonius rotating anode generator. X-ray data for the T155G FNR were collected at 20 °C to a maximum resolution of 2.4 Å. Crystals belong to the P65 hexagonal
space group with the following unit cell dimensions: a = b = 88.13 Å and c = 97.25 Å. The
VM is 3.0 Å3/Da with one FNR molecule
in the asymmetric unit and 60% solvent content. The x-ray data set was
processed with MOSFLM (31) and scaled and reduced with SCALA from the
CCP4 package (32).
Data collection and refinement statistics
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1 cm
1
at 458 nm) (38) has been assumed herein for all the FNR mutants. Illumination of the FNR forms in the presence of 5-deazariboflavin caused the reduction of the protein to the neutral FNR semiquinone form
with maxima in the range of 520 and 588-595 nm for all the FNR mutants
(Fig. 2B). As for the WT enzyme, isosbestic points are also
detected around 364 and 507 nm for the oxidized-semiquinone transition
for all the mutants. Under the assayed conditions WT FNR stabilizes
only 22% semiquinone. However, although a similar amount of
semiquinone form is stabilized by most of the mutants, S223G and S223D
showed an unexpected increment in the proportion of the radical
stabilization (34 and 43%, respectively).
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Fig. 2.
Spectral properties. (A)
Superposition of the absorption spectra of WT FNR (dotted
bold) and the different mutated FNR forms in the visible region.
(B) Photoreduction of S223D FNR in the presence of 3 µM dRf. The inset shows the production of the semiquinone
form absorbance (600 nm) vs that of the oxidized and reduced
protein (458 nm). Absorption spectra were recorded in 50 mM Tris/HCl, pH 8.0 at room temperature. Circular dichroism
spectra of WT (dotted bold) and mutated FNR forms in the
(C) far-ultraviolet and the (D) near-ultraviolet
and visible regions of the spectrum. All the CD spectra were recorded
in 1 mM Tris/HCl, pH 8.0 at room temperature.
Steady-state kinetic parameters for the diaphorase activity with DCPIP
of wild-type and mutated FNR forms from Anabaena
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Fig. 3.
Comparison of the DCPIP-diaphorase activity
kinetic parameters of WT and mutated FNR forms. A,
catalytic efficiency (µM 1
s
1, black columns) and specificity
for NADPH (gray columns). B, catalytic efficiency
(µM
1
s
1, black columns) and specificity
for NADH (gray columns). Specificity for a coenzyme is
defined as the ratio between its catalytic efficiency and that for the
other coenzyme.
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Fig. 4.
Spectroscopic characterization of the
complexes between the FNRox forms and the coenzymes
NADP+ or NAD+. Difference absorbance
spectra elicited by binding of (A) WT FNR (25 µM) to NADP+ (60 µM),
(B) Y235F FNR (20 µM) to NADP+ (500 µM), (C) T155G FNR (19 µM) to
NAD+ (4.5 mM) and in grey WT FNR (20 µM) to NAD+ (4.5 mM) (D) T155G
FNR (24 µM) to NADP+ (740 µM)
and (E) Y235A FNR (23 µM) to NADP+
(2 mM). The spectra were recorded at 25 °C in 50 mM Tris/HCl, pH 8.0. (F) Spectrophotometric
titration of selected FNR forms with NADP+. Open
circles, WT FNR; filled squares, Y235F FNR;
filled circles, T155G FNR.
Dissociation constants and free energy for complex formation of
different Anabaena PCC 7119 FNR forms in the oxidized state with
NADP+ and NAD+
1) followed by the hydride transfer
from NADPH to FAD (kobs ~ 200 s
1), resulting in the equilibrium mixture of
both charge-transfer complexes, [FNRox-NADPH] and
[FNRrd-NADP+] (39). The time course for the
kinetics of the reaction of T155G, R224Q, and Y235F FNR forms with
NADPH were similar to that of the WT enzyme observed at 460 nm (Fig.
5A), and fitting of the
experimental kinetic traces showed only slightly slower
kobs values for charge-transfer complex
formation and hydride transfer with NADPH than those observed for the
WT enzyme reaction (Table V). However,
when analyzing the reaction of NADPH with S223G, S223D, R233A, or Y235A
FNR forms, much longer time scales were required to complete the
reaction and to reach an equivalent decay amplitude of the FNR
absorbance at 460 nm (Fig. 5B). Moreover, the observed
kinetics for these mutants were best fit to mono-exponential processes
having kobs values that were lower than those of
the WT enzyme by 30-fold for Y235A and S223G, 330-fold for R233A, and
1500-fold for S223D (Table V). Taking into account the
Kd values reported in Table IV, complex formation
must be the rate-limiting process for these FNR forms, since this
reaction is much slower than the subsequent electron transfer reaction
whose rate constant, therefore, cannot be estimated. Measurements were
also carried out at 340 and 600 nm to get a better knowledge of the
observed processes (not shown). It is noteworthy that, although only
negligible absorbance changes were observed at these wavelengths for
the kinetics of the reaction of WT, T155G, R224Q, and Y235F FNR forms with NADPH corresponding to the kinetics observed at 460 nm, much slower processes can be detected at 340 and 600 nm, with similar amplitudes and time scales for all the FNR forms assayed. For the
moment we do not have an explanation for these observations, but
additional work is being done in the general characterization of the
catalytic mechanism of this system that exceeds the scope of the
present study.
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Fig. 5.
Time course of the anaerobic reactions of the
different FNR forms with NADPH observed at 460 nm. A, 6 µM WT FNRox (bold line) reacted
with 170 µM NADPH, 9 µM T155G
FNRox (closed squares) reacted with 160 µM NADPH, 9 µM R224Q FNRox
(dotted line) reacted with 190 µM NADPH, 6 µM Y235F FNRox (open circles)
reacted with 170 µM NADPH. B, 9 µM S223G FNRox (open circles)
reacted with 240 µM NADPH, 11 µM S223D
FNRox (open squares) reacted with 160 µM NADPH, 6.5 µM R233A FNRox
(closed squares) reacted with 2.5 mM NADPH, 15 µM Y235A FNRox (bold line) reacted
with 240 µM NADPH. Reactions were carried out in 50 mM Tris/HCl, pH 8.0, at 13 °C. Final concentrations are
given. U.A., units of absorbance.
Fast kinetic parameters for the reduction of the different Anabaena PCC
7119 FNR forms by NADPH and NADH as obtained by stopped-flow
1. This behavior is similar to that reported
for the WT enzyme for which a kobs greater than
550 s
1 has been reported when analyzed under
similar conditions (17).
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Fig. 6.
Time course of the anaerobic reactions of
T155G FNRrd with NADP+. Reaction of 7.5 µM T155G FNRrd with 200 µM
NADP+ observed at 460 nm (closed circles) and
340 nm (open circles). Other conditions are as in Fig. 5.
U.A., units of absorbance.
1 (Fig.
7A, Table V). Reaction of NADH
with R224Q, R233A, and Y235F took place with
kobs values that were within a factor of 2-5
that obtained for the WT enzyme (Table V, Fig. 7B). However, although R224Q and R233A were best fit to a mono-exponential process, two processes were observed for the Y235F mutant, which shows an
important increase in the second kobs value
while maintaining the same amplitude relationships observed for the WT
reaction. On the contrary, reaction of NADH with T155G, S223D, or Y235A FNR forms required longer time scales to reach equivalent decay amplitudes of the FNR absorbance at 460 nm (Fig. 7, B and
C, Table V), and therefore, these mutants yield observed
rate constants smaller than those detected for the WT enzyme.
Interestingly, the decay observed for the reaction of NADH with T155G
FNR shows the beginning of the second process delayed with regard to
the first one (Fig. 7C). It is noteworthy that we have
already observed this behavior for some reactions of the different FNR
forms when working at lower coenzyme concentrations and that for some
of the other mutants (Y235F, S223D for example) a very small lag phase
can be also observed at high NADH concentration (Fig. 7). These
processes were also analyzed at 600 nm (Fig. 7), where all the mutants
showed an increase in absorbance followed by a decay to above the
initial base line. The first processes observed are consistent in most
of the cases with the one observed at 460 nm. However, T155G FNR shows
a different behavior, with the final decay observed for the other FNR
forms at 600 nm replaced by a second, delayed absorbance increase that
is consistent with that observed at 460 nm (Fig. 7C,
inset). Thus far, as was the case for the reactions with
NADPH at long time scales, we do not have a good explanation for all
the observed processes. Surely the observed changes in absorbance would
reflect the association and interconversion of the
NAD(P)+/H-FNR forms, and further work along these lines is
under way and will be reported elsewhere. However, comparison of the
data obtained for the different FNR forms, including the WT enzyme, provides valuable information in the present study.
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Fig. 7.
Time course of anaerobic reactions of the
different FNR forms with NADH. A, reaction of 9 µM WT FNRrd with 2.4 mM NADH
followed at 460 nm (bold line) and 600 nm (thin
line). B, reaction of 9.5 µM S223D
FNRrd with 3.5 mM NADH observed at 460 nm
(closed squares) and 600 nm (open squares), 10 µM Y235A FNRrd with 2.8 mM NADH
followed at 460 nm (closed circles), and 600 nm (open
circles), and 8.5 µM Y235F with 2.4 mM
NADH at 460 nm (single thin line). C, 10 µM T155G FNRrd reacted with 2.7 mM NADH at 460 nm. The inset shows the same
reaction observed at 600 nm. Other conditions are as in Fig. 5.
U.A., units of absorbance.
backbone of the mutant superimposed on the native FNR backbone (Fig.
8). Prominent differences are
concentrated in the loop starting at Tyr-104 and ending at Val-113 near
the region interacting with the adenine moiety of FAD, but they are not
significant due to the poor definition of the electron density map in
this region for all FNR forms. No structural changes in the C
backbone at the mutated position were observed. This is surely a
consequence of the fact that strand 1, in which residue 155 is located,
is strongly stabilized by strands 2 and 4 of the
-sheet.
Furthermore, the turn connecting the end of this first strand with the
succeeding
-helix is stabilized by one hydrogen bond between Thr-157
and the OH group of the Tyr-303 (see Fig. 1D). However,
structural differences are observed in the Thr-155 environment
(positions 261-268) (Fig. 8). This region corresponds to the turn
linking strand 4 to the beginning of
-helix D of the
NADP+ binding domain. The structural changes in the mutated
enzyme can be explained on the basis of the altered hydrogen bond
network of this region after mutation (Table
VI and Fig.
9). The Thr-155 to Gly-155 mutation
essentially eliminates two interactions with Leu-263: the bifurcated
H-bond between the threonine OH side chain and the N and CO (the latter
through a water molecule) groups of Leu-263 (Fig. 9). Although the
position of the OH (Thr-155) group is replaced by a water molecule in
the mutated enzyme, the changes in the hydrogen bonding pattern produce
two new interactions and, as a consequence, a less extended loop as
observed by x-ray crystallography.
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Fig. 8.
Stereo view of the C
backbone superposition of FNR WT and T155G FNR mutant
(bold). Significant differences are concentrated
not at the mutated position but in its environment (loop 261-268). The
FAD cofactor and Thr-155 are represented as sticks. This
figure was drawn using MOLSCRIPT (62).
Selected non-conserved interactions between FNR WT and FNR T155G
mutant
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Fig. 9.
Hydrogen bond network and structural
differences in the FNR WT (A) and in the T155G FNR
mutant (B). In the native state, OH (Thr-155) is
making a bifurcated H-bond with the Leu-263 residue. Two new
interactions are created after mutation: O (Leu-263) stabilizes a new
interaction with N (Met-266), and O 2 (Gly-267) stabilizes a new
interaction with N (Gly-265). This produces a less extended
conformation for the 261-268 loop in the mutated enzyme.
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Fig. 10.
MOLSCRIPT drawing of the superposition of
Anabaena FNR (light) and
NADH-cytochrome b5 reductase
(dark) (58) near the position of residue 155. In
the NAD+/H-dependent enzymes, a Gly residue at
this position is favored due to the presence of a hairpin-like region
(formed by a series of prolines) that will not allow the space for a
Thr to occupy position 155 of FNR. On the contrary, the absence of this
hairpin in the NADP+/H-dependent enzymes
permits the presence of residues such as Thr or Pro at this position.
Relevant residues are labeled as chain A in FNR or chain B in
cytochrome b5 reductase.
-helix D in Anabaena FNR must
be one of the regions which determines coenzyme specificity.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Prof. R. Perham, University of Cambridge and also to Dr. J. K. Hurley and Prof. G. Tollin, University of Arizona, for their collaboration in many aspects of this work.
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FOOTNOTES |
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* This work was supported by Comisión Interministerial de Ciencia y Tecnología, Spain Grant BIO97-0912C02-01 (to C. G.-M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1bqe) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ Recipient of a travel award to the University of Cambridge from the Caja de Ahorros de la Inmaculada-Consejo Superior de Investigación y Desarrollo. To whom correspondence should be addressed: Departamento de Bioquímica y Biología Molecular y Celular. Facultad de Ciencias. Universidad de Zaragoza, Pedro Cerbuna 12, 50009 Zaragoza, Spain. Tel.: 34 976 762476; Fax: 34 976 762123; E-mail: mmedina@posta.unizar.es.
¶ Recipient of a travel award to the Universidad de Zaragoza from Universidad Nacional de Rosario and a grant from the Spanish Government. Present address: Cátedra de Física Biológica, Departamento de Ciencias Fisiológicas, Facultad de Ciencias Médicas, Santa Fé 3100, 2000 Rosario, Argentina.
Published, JBC Papers in Press, January 4, 2001, DOI 10.1074/jbc.M009287200
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ABBREVIATIONS |
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The abbreviations used are: FNR, ferredoxin-NADP+ reductase; DCPIP, 2,6-dichlorophenolindophenol; 2'-P, 2'-phosphate; WT, wild type.
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