(Received for publication, May 1, 1995)
From the
Spectroscopic and kinetic studies comparing the behavior of the
recombinant cytochrome b reductase fragment of corn leaf
nitrate reductase and a mutant in which cysteine 242 is replaced with a
serine residue (C242S) have been carried out. The visible and circular
dichroism spectra of the wild-type and mutant protein are virtually
identical and compare well with those reported for nitrate reductases
from other sources. The reduced wild-type protein forms a
charge-transfer complex with NAD that has an
absorption envelope that extends into the near infrared, with a maximum
around 800 nm. The C242S mutant forms a similar charge-transfer complex
with NAD
but to a lesser extent than the wild-type.
The reduction potential of the flavin for the wild-type protein is
-287 mV, and that for the mutant is -279 mV. The rate of
reduction by NADH of the C242S mutant is 7-fold slower than that for
the wild-type protein, and the K
is
larger by a factor of 2. These results indicate that the cysteine 242
residue plays a role principally in facilitating electron transfer from
NADH to the flavin rather than in binding of NADH to the enzyme.
Nitrate reductase (EC 1.6.6.1-3) catalyzes pyridine
nucleotide-dependent reduction of nitrate to nitrite as the first step
in nitrogen assimilation in algae, fungi, and higher plants. The enzyme
is a multicentered redox enzyme containing FAD, a b-type
cytochrome, and a molybdenum
center(1, 2, 3) . Each prosthetic group
appears to be found in structurally independent fragments of the two
identical 100-kDa subunits of nitrate reductase. Catalytically
active fragments have been demonstrated and even purified subsequent to
mild proteolysis(2, 4) . Genes of numerous nitrate
reductases have been cloned, and it is evident that the apparent
prosthetic group binding regions of nitrate reductase are laid out in a
linear fashion in the amino acid sequence on the basis of sequence
homology to enzymes of simpler
functionality(2, 3, 5) . For example, the
N-terminal fragment of nitrate reductase has high similarity to the
molybdenum binding region of mammalian sulfite
oxidase(6, 7) . An internal fragment of nitrate
reductase has sequence similarity to the soluble domain of mammalian
and higher plant cytochrome b
, and the C-terminal
fragment of nitrate reductase is highly similar in sequence to the
oxidation-reduction partner of cytochrome b
, namely,
NADH:cytochrome b
reductase(2, 3, 5) . The similarity
between the cytochrome b reductase fragment of nitrate
reductase and cytochrome b
reductase is especially
extensive(8) .
The structural independence of the several cofactor-binding fragments of nitrate reductase is further supported by the independent expression of two of these, the FAD- and heme-containing fragments, in Escherichia coli as stable and redox-active polypeptides (8, 9, 10) . The x-ray crystallographic structure of the FAD-containing cytochrome b reductase fragment of nitrate reductase has recently been reported(11) , and a potential new role for one of the enzyme's key cysteine residues has been identified by site-directed mutagenesis of the recombinant fragment(12) . The present work is an extension of these studies of the recombinant cytochrome b reductase fragment of nitrate reductase focusing on its oxidation-reduction chemistry and rapid reaction kinetic behavior.
The three-dimensional model of the cytochrome b reductase fragment of nitrate reductase has established its
FAD-binding region as a member of the structurally related, but a
functionally diverse group of flavin reductases known as the
``ferredoxin NADP reductase
family''(11) . The structure of ferredoxin NADP
reductase was the first of this family to be
elucidated(13) . The sequence similarity of the cytochrome b reductase fragment of nitrate reductase and ferredoxin
NADP
reductase had been recognized before the
structure was derived, but only a few key residues are strictly
conserved(13, 14) . Other members of this group
include phthalate dioxygenase reductase, an FMN-containing
NADH-dependent bacterial enzyme (15, 16, 17) and cytochrome b
reductase(18) . Several of these enzymes are
spectroscopically and kinetically well
characterized(19, 20, 21, 22) .
For many years, it has been recognized that nitrate reductase has a
cysteine that can be protected by pyridine
nucleotides(1, 2, 3) , and similar active
site cysteines have been identified in other members of the ferredoxin
NADP reductase family of
flavoenzymes(16, 19, 21, 23) .
Chemical modification of this cysteine by sulfhydryl reagents
significantly reduces activity. There are only two invariant cysteine
residues among nitrate reductases from various organisms, one in the
molybdenum-containing fragment and one in the cytochrome b reductase fragment(5, 7) . Since the cysteine in
the molybdenum-containing region has been suggested to be involved in
binding the cofactor(7) , the cysteine in the FAD-binding
region is the obvious candidate as the catalytically essential one,
especially considering the similarity of this region to the ferredoxin
NADP
reductase family of
enzymes(13, 14) . To determine if the invariant
cysteine of the recombinant cytochrome b reductase fragment of
nitrate reductase is the reactive one, each of the five cysteine
residues of this fragment have been changed to another amino acid, and
it is found that only by converting the cysteine 242 to a serine
residue (C242S) is the ferricyanide reductase activity rendered
insensitive to p-hydroxymercuribenzoate (12) .
Subsequently the structural model for this fragment of nitrate
reductase has revealed that cysteine 242 extends into the active site
crevice of the recombinant protein(11) . The steady-state
kinetics of the wild-type and C242S mutant of the cytochrome b reductase are found to be quite different, with the mutant having
a k
of about one-sixth of the wild-type; K
values for NADH and ferricyanide are
essentially unchanged(12) . These results suggest that cysteine
242 is important for reduction of the protein-bound flavin. Most
recently, the structural model for the cytochrome b reductase
fragment has been extended to include an analysis of the C242S mutant,
which indicates the side chain of the substituted serine forms a
hydrogen bond with the main chain oxygen atom of glycine 147 that is
not present in the wild-type protein. This interaction causes a local
conformational change that results in a large void in the active site (24) . Here we report a more detailed comparison of the
spectroscopic and kinetic properties of the wild-type and C242S mutant
of the recombinant cytochrome b reductase fragment in order to
gain a better understanding of the role of cysteine 242 in flavin
reduction by NADH.
The recombinant wild-type and C242S mutant cytochrome b reductase fragments of corn leaf nitrate reductase were obtained
as described previously(8, 12) . All experiments were
done in 0.1 M MOPS, ()0.1 N KCl, pH 7.0,
containing 0.1 mM EDTA, unless otherwise stated.
Ultraviolet/visible spectra were recorded using a Hewlett Packard 8452A
single-beam diode array spectrophotometer; visible/near-infrared
spectra were recorded with an On-Line Instrument Systems (OLIS)
modernized Cary-14. Circular dichroism (CD) spectroscopy was carried
out using an AVIV 40DS spectrophotopolarimeter. Reductive titrations
were carried out using anaerobic glassware described
previously(25) . Samples were made anaerobic by alternately
evacuating and flushing with oxygen-free argon on an anaerobic train
and then titrated with either sodium dithionite or NADH solution in 0.1 M MOPS, 0.1 N KCl, pH 7.0, containing 0.1 mM EDTA that had previously been made anaerobic by bubbling with
oxygen-free argon. The reduction potentials of the wild-type and mutant
enzymes were determined as described by Massey(26) , using
safranine T as the standard (
E`
=
-289 mV)(27) . Rapid mixing experiments were carried out
using a Kinetic Instruments Inc. stopped-flow apparatus interfaced with
an OLIS model 3920Z data collection system. Samples were placed in a
tonometer equipped with a three-way stopcock valve with a male Luer
connector and made anaerobic by alternately evacuating and flushing
with oxygen-free argon. The tonometer was mounted on the stopped-flow
apparatus, and samples were then rapidly mixed with anaerobic NADH
solutions. The kinetic transients obtained after mixing were monitored
as transmittance voltages collected by a high speed A/D converter and
converted to absorbance changes, and the rate constants were determined
by OLIS Inc. software.
Sodium dithionite was obtained from Virginia
Chemicals Co. Safranine T was a gift from the laboratory of Dr. Vincent
Massey, University of Michigan. NADH, NAD, and
3-aminopyridine adenine dinucleotide (AAD
) were
obtained from Sigma. All other chemicals were of reagent grade and were
used without additional purification.
Figure 1:
Visible,
near-IR, and CD spectra of nitrate reductase's cytochrome b reductase fragment and C242S mutant. A, visible spectra
of the wild-type enzyme in the oxidized form(- - -),
reduced with sodium dithionite (--), and reduced in the
presence of excess NAD (-
-). The inset shows the absorbance spectrum of the
charge-transfer complex in the visible and near-IR region. B, CD spectra of the wild-type enzyme in the oxidized form(-
- -), reduced with sodium dithionite (--), and
reduced in the presence of excess NAD
(-
-). C, visible spectra of the C242S mutant
in the oxidized form, reduced with sodium dithionite, and reduced in
the presence of excess NAD
(with same designations as
in A). The inset shows the absorbance spectrum of the
charge-transfer complex in the visible and near-IR region. D, CD spectra of the C242S mutant in the oxidized form(-
- -), reduced with sodium dithionite (--), and
reduced in the presence of excess NAD
(-
-).
The CD spectra of the
wild-type cytochrome b reductase fragment and C242S mutant are
also similar in each of the three states examined (oxidized, reduced,
and reduced plus NAD) (Fig. 1, B and D). However, as noted with the absorption spectra of the
charge-transfer complexes, the CD spectra of the reduced forms of
wild-type and C242S mutant of cytochrome b reductase in the
presence of NAD
have slight differences with the
mutant form having lower intensity (Fig. 1, B and D). Both the absorption and CD spectra of the oxidized forms
of wild-type and C242S cytochrome b reductase fragment also
compare well with those reported for the proteolytic fragment
containing the FAD domain of nitrate reductase from C.
vulgaris(22, 30) . The present results
simultaneously confirm the integrity of the wild-type recombinant and
C242S proteins and the assignment of the CD of nitrate reductase to its
flavin site(30) . Fluorescence of the FAD centers in both
wild-type and mutant proteins were examined, and there are no
noticeable differences. The emission spectra have a maximum at
approximately 525 nm (
= 460 nm) (data
not shown).
NAD and the NAD
analog
AAD
also bind to the oxidized forms of the wild-type
and C242S mutant as shown by difference spectra (Fig. 2, A and B). A previous study of the interaction of the Neurospora crassa NADPH:nitrate reductase with AAD
and AAD
phosphate (an NADP
analog derived from AAD
) demonstrated that the
analogs were competitive inhibitors of NADPH(31) . In the case
of both the wild-type and mutant cytochrome b reductase
fragment of the corn enzyme, AAD
perturbs the
absorbance spectrum of oxidized enzyme to a greater extent than does
NAD
, as is seen by a comparison between the difference
spectra; the effect is particularly pronounced in the 300-400 nm
region. The contribution of free AAD
to the orginal
spectrum was subtracted prior to obtaining the difference spectrum.
Mutation of cysteine 242 to serine has little effect on the
NAD
-induced difference spectrum, while there is a
rather more substantial effect on the AAD
difference
spectra, at least below 400 nm. This presumably reflects differences in
the flavin environment between the two forms. The x-ray
crystallographic derived model of the C242S mutant shows that while the
side-chain of cysteine 242 in the wild-type is free to interact with
NAD
, the serine hydroxyl of the mutant is
hydrogen-bonded to the main chain oxygen atom of glycine 147, resulting
in a local conformational change(24) .
Figure 2:
Difference spectra of oxidized nitrate
reductase's cytochrome b reductase fragment in the
absence and the presence of NAD and
AAD
. A, difference spectra in the presence of
NAD
. The wild-type enzyme is represented by closed
circles, and the C242S mutant is represented by open
circles. B, difference spectra in the presence of
AAD
. The contribution of free AAD to the spectrum was
subtracted prior to obtaining the difference spectra. The wild-type
enzyme is represented by closed circles, and the C242S mutant
is represented by open circles.
Figure 3: Reductive titrations with NADH. A, titration of the wild-type nitrate reductase's cytochrome b reductase fragment with NADH under anaerobic conditions. B, titration of the C242S mutant with NADH under anaerobic conditions.
Potentiometric titrations have
been performed with both wild-type and mutant enzymes by the method
described by Massey(26) . The data (Fig. 4) have been
plotted as the logarithm of oxidized/reduced concentrations of the
unknown versus the logarithm of the oxidized/reduced
concentrations of the standard dye (32) , in this case,
safranine T (E`
= -289 mV). The
reduction potential determined for the FAD/FADH
couple for
the wild-type enzyme thus obtained is -287 mV. Mutation of the
cysteine 242 to a serine increases the reduction potential only very
modestly to -279 mV. In the presence of NAD
, the
potential of the wild-type enzyme increases by 22 mV (E = -265 mV). The C242S mutant also exhibits an
increase in potential in the presence of NAD
, but the
increase is only approximately half of that observed in the case of the
wild-type (E = -268 mV). Overall, these results
are consistent with the conformation of the active site in the C242S
mutant being minimally altered relative to the wild-type(24) ,
which is reflected in the rather small change in the prosthetic
group's reduction potential. On the other hand, the smaller
change in reduction potential of the FAD upon binding NAD
implies that a less extensive charge-transfer interaction occurs
in the mutant as compared with wild-type, which correlates well with
the less intense long wavelength absorbance and CD spectrum of the
mutant when complexed with NAD
as compared with
wild-type (Fig. 1).
Figure 4:
Potentiometric titrations of nitrate
reductase's cytochrome b reductase fragment. The concentration of
enzyme used ranged between 15 and 30 µM.The enzyme was
incubated under anaerobic conditions with an equal concentration of
safranine T, 1.6 µM methyl viologen and with 240
µM xanthine, and 50 nM xanthine oxidase. Enzyme
reduction was monitored at 410 nm, an isosbestic point in the reduction
of safranine T, and the dye was monitored either at 518 or 540 nm. A, data obtained for the wild-type enzyme in the absence of
NAD (closedcircles) and in the
presence of NAD
(closedsquares). B, data obtained for the C242S mutant in the absence of
NAD
(opencircles) and in the
presence of NAD
(opensquares).
Figure 5: Time course of reduction of nitrate reductase's cytochrome b reductase fragment by NADH. A, flavin reduction was monitored at 460 nm. 7.5 µM wild-type enzyme was rapidly mixed with 50 µM NADH under anaerobic conditions (opencircles); 7 µM C242S mutant was rapidly mixed with 200 µM NADH under anaerobic conditions (closedcircles). Both enzymes were in 0.1 M MOPS, 0.1 mM EDTA, 0.1 N KCl, pH 7.0. B, formation of charge-transfer complex was monitored at 800 nm. The wild-type enzyme is shown in opencircles, and the C242S mutant is shown in closedcircles. The experimental conditions were the same as in A. These reactions were carried out at 10 °C
Figure 6:
Determination of Kfor NADH. A, the double-reciprocal plot of stopped-flow
kinetic data for wild-type enzyme. B, the double-reciprocal
plot of stopped-flow kinetic data for the C242S
mutant.
Analyses of UV/visible absorbance spectra have indicated
small differences between the wild-type and C242S mutant cytochrome b reductase fragment of nitrate reductase. In the presence of
sodium dithionite-reduced enzyme, NAD forms a
charge-transfer complex that has a long wavelength absorbance with a
maximum at 800 nm. Other pyridine-nucleotide dependent flavoproteins
that are members of the ferredoxin NADP
reductase
family of flavoproteins and holonitrate reductase are known to form
similar complexes(19, 21, 28) . The mutant
enzyme forms a similar complex but to a lesser extent than that of the
wild-type recombinant fragment of nitrate reductase. NAD
also perturbs the spectrum of oxidized enzyme as is seen in the
difference spectrum of oxidized enzyme and enzyme in the presence of
NAD
(Fig. 2A). The NAD
analog AAD
also perturbs the spectrum of
oxidized enzyme (Fig. 2B). The difference spectrum
observed with the C242S mutant is somewhat different than that obtained
for the wild-type cytochrome b reductase fragment, indicating
changes in the flavin environment that affect binding of inhibitor versus product. The potential determined for the
FAD/FADH
couple in the wild-type recombinant corn leaf
nitrate reductase cytochrome b reductase fragment (E = -287 mV, Table 1) is within the range of the
values determined for the holonitrate reductases from other
sources(30, 33) . In the presence of
NAD
, the reduction potential of the FAD of the
recombinant nitrate reductase cytochrome b reductase fragment
increases by 22 mV (Table 1), a considerably smaller shift than
in the case of the proteolytically generated FAD-containing fragment of
holonitrate reductase from C. vulgaris, where the potential
was observed to increase by 60 mV(2) . It should be noted that
the midpoint potentials of holonitrate reductase are highly influenced
by pH with the FAD reduction potential having an approximate decrease
of 30 mV/pH unit in the range of pH 6-9(30) . The major
difference between the wild-type recombinant cytochrome b reductase fragment and its C242S mutant, however, is observed in
their kinetic behavior (Table 1). The rate of reduction of the
wild-type fragment by NADH is found to be about 7-fold greater than
that of the mutant and K
for NADH about half of
that for the mutant. The k
/K
for the wild-type fragment by NADH is 14-fold greater than that
of the C242S mutant (Table 1). The K
for
NADH differing only by a factor of two between the wild-type and mutant
probably means that cysteine 242 contributes no more than 0.5 kcal/mol
to the binding of NADH. This indicates that cysteine 242 plays more of
a role in reducing the activation barrier to flavin reduction than in
binding of substrate. The effect on the activation energy to flavin
reduction is about 1 kcal/mol, making the sum approximately 1.5
kcal/mol, which is low but within the range for a hydrogen bonding
interaction. This seems consistent with the fact that in the wild-type
enzyme, the cysteine is free to interact in such a manner with the
substrate but in the mutant the serine is already hydrogen bonded to
glycine 147 and is unable to interact in a similar fashion with the
substrate. The reductive half-reaction kinetic results are in agreement
with earlier steady-state kinetic work that indicated that there was
about a 6-fold decrease in k
on substituting a
serine for cysteine 242 in a NADH:ferricyanide reductase activity assay
(12, Table 1). Thus, flavin reduction by NADH appears to be the
rate-limiting step in the overall catalysis of the recombinant
cytochrome b reductase fragment of nitrate reductase when
ferricyanide is the electron acceptor. A similar conclusion that FAD
reduction by NADH limited catalysis has been reached for cytochrome b
reductase(21) . Finally, based on the
recent structural models for both the wild-type and C242S mutant of
corn leaf nitrate reductase's recombinant cytochrome b reductase fragment(11, 24) , it can be concluded
that the thiol side chain of cysteine 242 may play a role in
positioning NADH for efficient electron transfer but does not
participate in catalysis directly. Thus, while cysteine 242 promotes
catalytic efficiency of the cytochrome b reductase fragment
(and presumably of holonitrate reductase where it is an invariant
residue in over 30 examples of nitrate reductase sequences from a wide
variety of species of higher plants, algae, and fungi), it is not
essential for NADH reduction of the enzyme flavin. Hence, the
evolutionary pressure to retain the invariant cysteine of the FAD
reduction active site of nitrate reductase and most of the other
members of the ferredoxin NADP
reductase family of
flavoprotein reductases is not because a mutation results in an
inactive form but rather in a catalytically inefficient one.