From the Department of Life Science, Graduate School of Science, Himeji Institute of Technology, Kouto 3-2-1, Kamigori, Hyogo 678-1297, Japan
Received for publication, September 25, 2002, and in revised form, November 27, 2002
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ABSTRACT |
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Site-directed mutagenesis of
Thr66 in porcine liver NADH-cytochrome
b5 reductase demonstrated that this residue
modulates the semiquinone form of FAD and the rate-limiting step in the
catalytic sequence of electron transfer. The absorption spectrum of the T66V mutant showed a typical neutral blue semiquinone intermediate during turnover in the electron transfer from NADH to ferricyanide but
showed an anionic red semiquinone form during anaerobic photoreduction. The apparent kcat values of this mutant were
~10% of that of the wild type enzyme (WT). These data suggest that
the T66V mutation stabilizes the neutral blue semiquinone and that the
conversion of the neutral blue to the anionic red semiquinone form is
the rate-limiting step. In the WT, the value of the rate constant of
FAD reduction (kred) was consistent with the
kcat values, and the oxidized enzyme-NADH
complex was observed during the turnover with ferricyanide. This
indicates that the reduction of FAD by NADH in the WT-NADH complex is
the rate-limiting step. In the T66A mutant, the
kred value was larger than the
kcat values, but the
kred value in the presence of NAD+
was consistent with the kcat values. The
spectral shape of this mutant observed during turnover was similar to
that during the reduction with NADH in the presence of
NAD+. These data suggest that the oxidized
T66A-NADH-NAD+ ternary complex is a major intermediate in
the turnover and that the release of NAD+ from this complex
is the rate-limiting step. These results substantiate the important
role of Thr66 in the one-electron transfer reaction
catalyzed by this enzyme. On the basis of these data, we present a new
kinetic scheme to explain the mechanism of electron transfer from NADH
to one-electron acceptors including cytochrome
b5.
NADH-cytochrome b5 reductase (EC 1.6.2.2)
is a member of the large family of flavin-dependent
oxidoreductases that transfer an electron from two-electron carriers of
nicotinamide dinucleotides to one-electron carriers such as heme
proteins and ferredoxins. This enzyme catalyzes the electron transfer
from NADH to cytochrome b5
(b5)1 (1-3), and
participates in fatty acid synthesis (4, 5), cholesterol synthesis (6),
and xenobiotic oxidation (7) as a member of the electron transport
chain on the endoplasmic reticulum. In erythrocytes, this enzyme
participates in the reduction of methemoglobin (8).
The outline of the catalytic cycle of the solubilized catalytic domain
of NADH-cytochrome b5 reductase (b5R) is
understood as follows (3) (Scheme I). At
first, two electrons are transferred from NADH to FAD by hydride
(H
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) transfer. Then the two-electron reduced
enzyme-NAD+ complex (E-FADH
-NAD+)
transfers two electrons to two one-electron acceptors one by one via
the anionic red semiquinone form
(E-FAD·
-NAD+), and the reduced enzyme
returns to the oxidized state. Strittmatter (9-11) suggested that the
reduction of FAD by NADH is the rate-limiting step in electron transfer
catalyzed by b5R. Iyanagi et al. (3, 12) found
that the anionic red semiquinone of FAD in Pb5R is stabilized by
binding of NAD+. Kobayashi et al. (13) analyzed
the conversion of the neutral blue to the red semiquinone in the
presence of NAD+ using a pulse radiolysis technique. Meyer
et al. (14) also demonstrated that NAD+
stabilizes the red semiquinone of the human b5R and modulates the
electron transfer to b5. These studies suggest the importance of the
anionic red semiquinone form of the b5R-NAD+ complex in the
electron transfer.
View larger version (6K):
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Scheme I.
The preliminary tertiary structure of human erythrocyte b5R (15, 16), and the detailed tertiary structures of porcine and rat liver b5Rs at 2.1 Å resolution have been determined by x-ray crystallography (17-21). These structural studies revealed that NADH-cytochrome b5 reductase belongs to the structurally related so-called "ferredoxin reductase family" (22, 23) together with other flavoenzymes such as ferredoxin-NADP+ oxidoreductase (FNR) (22), phthalate dioxygenase reductase (24), flavodoxin reductase (25), NADPH-cytochrome P-450 reductase (26), and the cytochrome b reductase domain of nitrate reductase (27). Enzymes of this family contain a flavin-binding domain and a pyridine nucleotide-binding domain. The former domain has a highly conserved flavin-binding amino acid sequence motif, RXY(T/S). In the Pb5R, Arg63, Tyr65, and Thr66 comprise this sequence motif (19). Using site-directed mutagenesis, we demonstrated that the positive charge of Arg63 is critical for the affinities of Pb5R for both NADH and NAD+, and the specific arrangement between the side chain of Tyr65 and FAD contributes to protein stability and electron transfer (28). Marohnic and Barber (29) also reported the effects of mutations of the corresponding Arg91 in rat b5R.
The Thr66 residue in Pb5R is positioned near both the N5
atom of the isoalloxazine ring of FAD and the potential binding site of
the nicotinamide ring of NADH (20, 28) (Fig.
1). This position corresponds to
threonine or serine residues in the other members of the ferredoxin
reductase family (22, 24-27). Ser96 in spinach leaf FNR is
critical to the reductive half-reaction of FAD (30), and
Ser90 in the C-terminal Tyr208 mutant of pea
leaf FNR forms a hydrogen bond with the amide moiety on the
nicotinamide ring of the pyridine nucleotide in both the enzyme-NADP+ and enzyme-NADPH complexes (31). However, b5R
and cytochrome b reductase domain of nitrate reductase do
not have an aromatic ring corresponding to that of the C-terminal
Tyr208 in pea leaf FNR, which contacts with the
re-side of the isoalloxazine ring of FAD and moves away
accompanied with the binding of nicotinamide (31). In addition, the
main physiological role of leaf FNR is the reduction of the oxidized
pyridine nucleotide, and the direction of the electron transfer between
FAD and pyridine nucleotide is different from that of b5R. Therefore,
it is considered that Thr66 in Pb5R contributes to the
reduction of FAD and/or the stabilization of the reduced FAD. Shirabe
et al. (32) reported that mutations of the corresponding
Thr94 in human b5R affect oxidation of FAD, but the effects
of the mutations on the properties of reduced FAD in the catalytic
cycle have not been clarified.
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To analyze the role of Thr66 in catalysis in Pb5R, we
replaced Thr66 in Pb5R with serine (T66S), alanine (T66A),
and valine (T66V) and analyzed the redox properties of FAD in the
catalytic cycle using a stopped flow spectrophotometer. We present here
that the conversion of the neutral blue to the red semiquinone
intermediate and the release of NAD+ from the enzyme in the
catalytic cycle were modulated by the mutations of Thr66 in
Pb5R. In addition, we present a new model of the reaction sequence of
Pb5R containing the blue neutral semiquinone and the oxidized
enzyme-NAD+-NADH ternary complex.
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EXPERIMENTAL PROCEDURES |
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Materials-- Enzymes for recombinant DNA technology were from Takara and Toyobo. NADH and NAD+ were from Oriental Yeast. Wild type recombinant Pb5R (WT) was prepared as previously described (33).
Mutagenesis, Expression, and Purification of Mutant
Pb5Rs--
Alteration of the gene encoding Pb5R was carried out by
site-directed mutagenesis using PCR by the methods described by
Higuchi (34). Briefly, for the preparation of the mutant genes encoding the mutant proteins, two primary PCR products that overlap in sequence
were first obtained from a DNA template, pU8Pb5R, which contains the
gene encoding the WT (33). One product was generated with the
forward primer 5'-TAGGAGGTCATATGTCCACCCCGGCC-3' containing a NdeI site (underlined) and the mutagenic common
reverse primer, 5'-GGGCCGAATGACCAG-3', and the other was obtained with
the forward mutagenic primer and the reverse primer
5'-CCGCCAAGCTTCTAGAAGGCGAAGCAGC-3' containing a
HindIII site (underlined). As the mutagenic forward primers,
5'-CTGGTCATTCGGCCCTACNNNCCCGTCTC-3', which have a complementary nucleotide sequence to the 5'-end of the mutagenic forward primers, were used. In these primers, NNN are the bases corresponding to the
66th amino acid residue, and GCT, TCG, and GTG were used for the
mutations to alanine, serine, and valine, respectively. The resultant
two PCR products were mixed and reamplified with the forward and
reverse primers. The resultant secondary PCR product was inserted into
the plasmid pCW20 °C
until use.
Preparation of the Solubilized Domain of Porcine Liver
Cytochrome b5--
The recombinant solubilized domain
of porcine liver cytochrome b5 (Pb5) was
prepared as follows. The cDNA encoding the full-length porcine
liver cytochrome b5 was amplified from the
previously described first strand cDNA, which was prepared from a total
RNA preparation from porcine liver (33). The forward primer was 5'-GTTAAGAAATGGCCGAGGAGTCC-3', which has an initiator methionine codon
followed by the nucleotide sequence encoding the N-terminal tetrapeptide of natural bovine liver b5 (39). The reverse primer was
5'-CTTCGGTTACCTTCTTTTCTGACG-3'. This nucleotide sequence was complementary to the nucleotide sequence located 12-35 bases
downstream after the stop codon in the cDNA of bovine liver b5
(39). The amplified DNA fragment was blunt-ended and inserted into the
HincII site of plasmid pUC118, and plasmid pU8Pb5 was
selected. Plasmid pU8Pb5 contained the nucleotide sequence encoding 133 amino acid residues from the N-terminal Ala1 to the
C-terminal Asn133 of porcine liver b5. The deduced amino
acid sequence was identical to that previously reported except for the
difference at position 3 (40-42). The deduced amino acid residue at
position 3 was not glutamine but glutamic acid. The polypeptide
containing 87 amino acid residues from Ala7 to
Lys93 was prepared as recombinant Pb5. The cDNA
encoding recombinant Pb5 was amplified from pU8Pb5 with the forward
primer 5'-AGGAGGTCATATGGCCGTGAAGTATTACACC-3', which has an
NdeI site (underlined) containing an additional initiator methionine codon, followed by the nucleotide sequence encoding the
N-terminal hexapeptide of recombinant Pb5, and the reverse primer
5'-CCGCCAAGCTTCTACTTGGCAATCTTGATC-3', which
corresponds to the C-terminal peptide, a stop codon, and a
HindIII site (underlined). The resultant fragment was
inserted into pCW-D-thiogalactopyranoside
was added to a final concentration of 0.2 mM, and the
cultivation was continued for 14 h. The cells were lysed by
sonication in 10 mM Tris-HCl (pH 7.4) containing 1 mM EDTA and 2 mM phenylmethylsulfonyl fluoride. The lysate was subjected to centrifugation at 18,000 × g for 20 min, and the supernatant was separated on a
Sephadex G-100 (Pharmacia) column equilibrated with 10 mM
potassium phosphate (pH 7.0) (buffer A). Red colored fractions
containing recombinant Pb5 were applied to an anion exchange resin
(DE52) column equilibrated with buffer A, and the proteins were eluted
with a linear gradient of potassium chloride from 0 to 0.4 M in buffer A. Fractions containing recombinant Pb5 were
concentrated and desalted with a Sephadex G-25 (fine) (Pharmacia)
column equilibrated with 100 mM potassium phosphate (pH
7.0). The purified recombinant Pb5 showed a single band of ~10 kDa on
a 15% polyacrylamide gel after SDS-PAGE. The yield of the purified
protein from 1 liter of culture fluid was 5.9 mg. The N-terminal amino
acid sequence analyzed with a Shimadzu PSQ-1 protein sequencer was
Ala-Val-Lys-Tyr-Tyr, and most of the additional N-terminal methionine
residues were cleaved. The oxidized and reduced absorption spectra at
300-700 nm and the ability to accept electrons from the Pb5R were
almost identical to those of the natural Pb5 (43).
Protein Concentrations--
The molar extinction coefficients of
the mutant proteins at 460 nm (460) were determined by a
method similar to that described by Aliverti and Zanetti (44), as
previously described (33). The molar concentration of the WT was
determined from the absorbance at 460 nm using the molar extinction
coefficient, 1.02 × 104 M
1
cm
1 (33). The protein concentration of recombinant Pb5
was determined using the molar extinction coefficient of natural Pb5,
1.13 × 105 M
1
cm
1 at 413 nm (45).
Spectral Analyses-- Absorption spectra were measured on a Hitachi U-2010 spectrophotometer. CD spectra were measured on a Jasco J-700 spectropolarimeter. Fluorescent emission spectra were measured in 10 mM potassium phosphate (pH 7.0) at 25 °C on a Hitachi F-3010 Fluorescence spectrophotometer. The excitation wavelength was 460 nm, and emission spectra at 470-650 nm were observed.
Enzymatic Activity--
Steady-state enzymatic activities were
measured as previously described (33). The apparent
Km values for NADH
(K1 cm
1. The reduction rate of
recombinant Pb5 was measured at 556 nm using a difference in molar
extinction coefficient of natural Pb5 between the oxidized and reduced
states, 1.9 × 104 M
1
cm
1 (43). The concentration of NADH was determined using
a molar extinction coefficient, 6.3 × 103
M
1 cm
1 at 340 nm.
Stopped Flow Measurements-- Rapid reaction was analyzed with a Photal RA-401 stopped flow spectrophotometer (Otsuka Electronics) equipped with a Lauda RMS thermostatically regulated circulating water bath in 10 mM potassium phosphate (pH 7.0) at 25 °C. The spectral changes of enzymes during and after the electron transfer from NADH to ferricyanide were analyzed as follows. Equal volumes of the enzyme solution containing potassium ferricyanide and the NADH solution were rapidly mixed, and the rapid scan spectra at 460-800 nm and the time courses of the absorbance changes at 460, 530, and 620 nm were measured. The initial concentrations of NADH, oxidized enzyme, and potassium ferricyanide in the reaction mixtures were 1 mM, 10 µM, and 150 µM, respectively. Under these experimental conditions, a turnover phase and a subsequent reduction phase after the consumption of ferricyanide were observed. The rate constant for the absorbance change after the turnover (k) was determined by single exponential curve fitting of the data as previously described (33).
Spectral changes of the enzymes during the reduction with NADH were
analyzed with rapid scan spectra and time courses of the absorbance
changes at 460 nm in the presence and absence of 1 mM
NAD+. The enzyme solution, which contains or does not
contain NAD+, was rapidly mixed with the NADH solution. The
rapid scan spectra in the region at 420-560 and at 560-730 nm were
measured separately and were joined at 560 nm. In the measurement of
the rapid scan spectra, the gate time was 4 ms. The rate constants of
reduction (kred) in the presence
(k
Photoreduction-- The flavin cofactor in the enzymes was photoreduced in an anaerobic cuvette containing 40 µM protein, 1 µM 5-deazariboflavin, 5 mM EDTA, 200 µM NAD+, ~0.5 µM indigodisulfonate, and 10 mM potassium phosphate (pH 7.0). The solutions were made anaerobic by successive flushing with oxygen-free argon gas with gentle agitation for more than 50 min. The absorption spectra were observed before and after illumination at 25 °C with a 300 W halogen lamp at room temperature.
Measurement of Dissociation Constants--
The dissociation
constant of the oxidized enzyme for NAD+
(K
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RESULTS |
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Purification of Mutant Proteins--
All of the purified mutant
Pb5Rs showed a single band on an SDS-PAGE gel that was located at the
same position as the WT. The yields of the purified T66S, T66A, and
T66V mutants from 1 liter of culture fluid were 15.7, 20.3, and 14.5 mg, respectively. In all mutant proteins, the only bound flavin
detected on the TLC plate was FAD. The molar extinction coefficients of
the T66S, T66A, and T66V mutants at 460 nm were similar to that of the
WT and were 1.05 × 104, 1.01 × 104, and 1.06 × 104
M1 cm
1, respectively.
Spectral Properties of Oxidized Proteins--
The absorption
spectrum of the T66S mutant was almost identical to that of the WT
(Fig. 2A, panel a).
The absorption spectra of the T66A and T66V mutants were also similar
to that of the WT, but slight spectral changes were observed (Fig.
2A, panels b and c). The absorption
spectrum of the T66V mutant was blue-shifted by ~3 nm in comparison
with that of the WT. The intensities of the peaks at 390 nm were
slightly decreased in the spectra of the T66A and T66V mutants. The CD
spectra of the T66A, T66S, and T66V mutants were also similar to that
of the WT (Fig. 2B). All of the mutant proteins showed a
significant decrease in the intensity of the fluorescence emission of
FAD. The intensities of the fluorescence emission spectra of free FAD,
the WT, and the T66S, T66A, and T66V mutants at 524 nm were 38.9, 1.3, 1.5, 1.4, and 1.3, respectively (spectra are not shown). These data
show that the mutations did not change the overall structure of the
oxidized form of the enzyme.
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Steady-state Kinetic Parameters and Dissociation Constant for
NAD+--
The apparent steady-state kinetic parameters and
the K
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The apparent K
The K
Rapid Scan Spectra during and after Turnover--
The spectra of
the WT and mutant enzymes during and after the turnover were directly
analyzed by rapid scan analysis (Fig. 3).
In this experiment, a turnover phase and a subsequent reduction phase
by NADH after the consumption of ferricyanide were observed.
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In the case of the WT and the T66S and T66A mutants, the rapid scan
spectra in the turnover phase showed significant absorption at 460-540
nm in addition to the broad absorption around 620 nm (Fig.
3A, panels a-c). Absorption at these wavelengths
decreased simultaneously (Fig. 3B, panels a-c).
The rate constant (k) values were consistent with the
kcat values (the
k
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In the turnover phase, the T66V mutant showed peaks at 530 and 620 nm but did not show significant absorption at ~460 nm (Fig. 3A, panel d). This spectral shape was obviously different from those of the WT and the other mutants and was characteristic of a neutral blue semiquinone form. Spectra similar to that of a neutral blue semiquinone, which has peaks at ~500 and 600 nm, were observed in the flavodoxins and their mutants (46-48). Accompanied with the consumption of ferricyanide, both peaks disappeared simultaneously (Fig. 3B, panel d). The k value at 530 nm was almost identical to that at 620 nm (Table II). These data indicate that in the T66V mutant, the spectrum observed in the turnover phase is due to the neutral blue semiquinone and that the conversion of the neutral blue semiquinone is the rate-limiting step.
Photoreduction--
The enzymes were anaerobically photoreduced in
the presence of NAD+ to analyze the statically stable
semiquinone form (Fig. 4). The enzymes
showed biphasic absorbance changes at 375 and ~460 nm by successive
photoreduction and resulted in the spectra of fully reduced forms. In
the case of the WT, absorbance at 460 nm was decreased, and peaks at
375 nm and near 530 nm were increased after photoreduction for 5 min
(Fig. 4a). This indicates the formation of the anionic red
semiquinone (3). The peaks near 375 nm, which were characteristic of
the anionic red semiquinone, were observed in the spectra of the T66S,
T66A, and T66V mutants after photoreduction for 3, 5, and 4 min,
respectively (Fig. 4, b-d). In these three mutants, the
stable one-electron reduced form was an anionic red
semiquinone.
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Reduction of Enzymes without Turnover--
The WT and mutant
enzymes were reduced with NADH in the presence and absence of
NAD+ to identify the intermediates observed in the rapid
scan spectra of the WT and the T66S and
T66A mutants during the turnover with ferricyanide (Figs. 5 and
6). In the presence of 1 mM
NAD+, more than ~70% of the T66V mutant and 90-95% of
the other enzymes are in the oxidized enzyme-NAD+ complex
as judged from the
K
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In both the presence and the absence of NAD+, spectra of
the WT observed during the reduction with NADH showed a large
absorption peak at 460 nm with a shoulder at 490 nm and a broad
absorption around 620 nm, all of which decreased simultaneously (Figs.
5B, panel a, and 6B, panel
a). These spectral features were identical to that observed in the
turnover phase (Fig. 3A, panel a). In the WT, the
values of the rate constant of reduction (kred)
in the presence of NAD+
(k
In the case of the T66S mutant, the spectrum observed during the
reduction with NADH in the absence of NAD+ showed an
obvious shoulder at 490 nm and a broad peak of absorption at ~620 nm
(Fig. 6B, panel b). This spectral feature was
identical to that observed in the turnover phase (Fig. 3A,
panel b). In the T66S mutant, the
k
The spectra of the T66A mutant observed during reduction with NADH in
the presence of NAD+ showed absorption peaks at 460 nm
without an obvious shoulder at 490 nm (Fig. 5B,
panel c) as seen in the spectrum in the turnover phase (Fig. 3A, panel c). In the T66A mutant, the
k
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In the case of the T66V mutant, the spectra observed during reduction
with NADH in the presence and absence of NAD+ showed a
large absorption peak at 460 nm, with a shoulder at 490 nm and a broad
region of absorption around 620 nm, all of which decreased
simultaneously (Figs. 5B, panel d, and
6B, panel d). These spectral features are similar
to those of the WT and are different from the spectrum observed in the
turnover phase (Fig. 3A, panel d). The
k
Spectral Changes Caused by Binding of NAD+,
5'-ADP- ribose, and NADH--
To analyze the contribution of the
5'-ADP-ribose moiety to the binding of pyridine nucleotide,
NAD+, 5'-ADP-ribose, and NADH were added to the oxidized
enzymes as shown in Fig. 7. The spectral changes of the T66S and T66A
mutants caused by the addition of NAD+ were large, whereas
those of the WT and T66V mutant were small (Fig. 7A). These
spectral changes are due to the formation of the oxidized
enzyme-NAD+ complexes. The features of the spectral changes
caused by the addition of NAD+ were similar, but the
degrees of the spectral changes were different. The difference spectra
of the WT and mutant enzymes showed a positive peak around 513 nm and
negative peaks around 459 and 490 nm. These spectra of the oxidized
enzyme-NAD+ complexes were changed by the addition of
5'-ADP-ribose, which lacks a nicotinamide moiety. The spectra of the WT
and mutant enzymes obtained after the addition of 5'-ADP-ribose were
similar, and their difference spectra showed negative peaks around 470 and 500 nm. These spectra were almost identical to those obtained after
the addition of 5'-ADP-ribose to the oxidized enzymes (Fig. 7B). These spectral changes caused by the additions of
NAD+ and 5'-ADP-ribose in this order indicate that
NAD+ in the oxidized enzyme-NAD+ complexes was
released by the addition of 5'-ADP-ribose, resulting in the formation
of the oxidized enzyme-5'-ADP-ribose complexes. The spectra of the
oxidized enzyme-5'-ADP-ribose complexes were hardly changed by the
addition of NAD+ (Fig. 7B), indicating that the
affinity of the oxidized enzymes for NAD+ is lower than
that for 5'-ADP-ribose. The spectra obtained after the additions of
both NAD+ and 5'-ADP-ribose were almost identical to those
of the enzyme-5'-ADP-ribose complexes (Fig. 7). The resultant
enzyme-5'-ADP-ribose complexes were reduced by the addition of NADH and
resulted in the spectra of the reduced enzyme-NAD+
complexes, which showed a broad absorption of the charge transfer complex at 500-800 nm. The 5'-ADP-ribose moiety is necessary for both
the binding of pyridine nucleotide and the release of NAD+
from the oxidized enzyme-NAD+ complexes.
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DISCUSSION |
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In this study, we replaced Thr66 in Pb5R with serine, alanine, and valine and analyzed the effects of the mutations on the electron transfer catalyzed by Pb5R. The absorption, CD, and fluorescence spectra of the mutant proteins indicate that the amino acid substitutions did not change the overall structure of Pb5R. The T66A and T66V mutants maintained catalytic activity, indicating that the hydroxyl group of Thr66 is not essential for electron transfer. However, mutations of Thr66 in Pb5R affected catalysis.
The T66V mutant exists as the neutral blue semiquinone form during the turnover (Fig. 3), and the conversion of this form is the rate-limiting step. However, the anaerobic photoreduction spectra of the T66V mutant in the presence of NAD+ indicated the presence of the anionic red semiquinone form (Fig. 4). It is considered that the neutral blue semiquinone form converts to the red semiquinone form during turnover, and this step is the rate-limiting step. In the T66V mutant, only the hydroxyl group of the Thr66 in the WT was replaced with a methyl group. The blue neutral semiquinone of FAD (FADH·) has a hydrogen atom on the N5 atom of the isoalloxazine ring, and release of a proton from the N5 position is required for the conversion of the blue neutral semiquinone to the anionic red semiquinone (49). The substitution of Thr66 by valine is considered to be unfavorable for the effective proton release from the N5 position of the neutral blue semiquinone of FAD. Kobayashi et al. (13) observed the conversion of the blue semiquinone to the red semiquinone form of the WT using pulse radiolysis below pH 6.5, and only the stable red semiquonone appeared at 200 µs after pulse above pH 7.0. These data suggest that at pH 7.0 the blue semiquinone form of the WT is unstable, and the conversion of the blue to red semiquinone is very fast.
The spectrum of the T66A mutant, which was observed during the turnover (Fig. 3A, panel c), was assigned to be the oxidized enzyme-NADH-NAD+ ternary complex. Those of the WT and the T66S mutant (Fig. 3A, panels a and b) were assigned as the oxidized enzyme-NADH complexes, but the spectra of the T66S mutant observed during the reduction with NADH in the presence of 1 mM NAD+ were assigned as the oxidized T66S-NADH-NAD+ ternary complex (Fig. 5B, panel b). These data suggest that the oxidized enzyme-NADH complex is produced via the oxidized enzyme-NADH-NAD+ complex, and these complexes could be involved in the catalytic cycle of Pb5R.
Based on these data, we present here a new model of the reaction
sequence of b5R containing the neutral blue semiquinone form and the
oxidized enzyme-NADH-NAD+ ternary complex as intermediates
(Scheme II). This model contains the
following processes: (i) formation of the oxidized enzyme-NADH complex
(E-FAD-NADH), (ii) conversion of E-FAD-NADH to a form that has the
ability to transfer H (E-FAD-NADH*), (iii)
H
transfer from NADH to FAD, (iv) the first one-electron
transfer from the two-electron reduced enzyme complex
(E-FADH
-NAD+), (v) rapid conversion of the
neutral blue semiquinone form (E-FADH·
-NAD+) to
the anionic red semiquinone form (E-FAD·
-NAD+),
(vi) the second one-electron transfer from
E-FAD·
-NAD+, (vii) formation of the oxidized
enzyme-NADH-NAD+ ternary complex
(E-FAD-NADH-NAD+) by binding of NADH, and (viii) release of
NAD+. Although there is no direct evidence for the
conversion of E-FAD-NADH to E-FAD-NADH* (process (ii)), the existence
of the E-FAD-NADH complex, which has no ability to transfer
H
, is a reasonable assumption. This is because
H
transfer itself is generally very fast, and a two-step
mechanism for pyridine nucleotide binding has been proposed for the
related family enzymes, nitrate reductase (50), phthalate dioxygenase reductase (51, 52), and FNR (31). In the WT and the T66S mutant, the
rate-limiting step is process (ii). In the T66A mutant, the
rate-limiting step is process (viii), and the rate of process (i) is
faster than that of process (viii). In the T66V mutant, the
rate-limiting step is process (v). This model reasonably interprets the
data presented here, but more investigations are required before this
model is established.
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Massey and Hemmerich (53) proposed that the neutral blue semiquinone
form is an obligatory intermediate in flavoproteins, which are involved
in one-electron transfers. Murataliev et al. (54, 55)
suggested that the so-called "air-stable" blue semiquinone form of
the housefly NADPH-cytochrome P-450 reductase, which is also a
one-electron transfer flavoenzyme, is inactive and different from the
catalytically competent semiquinone form. The neutral blue semiquinone
form of a one-electron transfer flavoenzyme may be less active in the
one-electron oxidation than the anionic red semiquinone form. It is of
interest that the rate-limiting step of the nonphysiological diaphorase
activity of leaf FNR is the reductive half-reaction and that a stable
neutral blue semiquinone form is produced by anaerobic photoreduction
in the absence of NADP+ (30, 31). It is reasonable that the
less active neutral blue semiquinone form is required for leaf FNR,
because during photosynthesis, the one-electron reduced form of leaf
FNR must be protected from unfavorable oxidation to form the
two-electron reduced FAD (FADH), which is necessary for
the two-electron reduction of NADP+. In contrast, b5R may
not require the less active neutral blue semiquinone intermediate,
because the physiological direction of the electron transfer catalyzed
by b5R is from NADH to one-electron acceptors and opposite from that of
leaf FNR.
The hydroxyl group of the corresponding Ser90 in the
C-terminal mutant of pea leaf FNR forms a hydrogen bond with the amide moiety on the nicotinamide ring of the pyridine nucleotide in the
NADP+ and NADPH complexes (31). Although the mutations of
the Thr66 in Pb5R hardly affect the
K
In contrast to the
K
In conclusion, direct evidence for the rate-limiting step of Pb5R was
provided using stopped flow spectrophotometry. The rate-limiting steps
in the catalytic cycles of the T66V mutant, the WT and the T66S mutant,
and the T66A mutant were the conversion of the neutral blue to the red
semiquinone, the reduction of FAD in the oxidized enzyme-NADH
complexes, and the release of NAD+ from the oxidized
T66A-NADH-NAD+ ternary complex, respectively. The conserved
Thr66 in Pb5R participates in the modulations of the
semiquinone forms, the release of NAD+ from the enzyme, and
the specific electron transfer to Pb5.
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ACKNOWLEDGEMENTS |
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We thank Drs. S. Ikushiro, Y. Emi, K. Kobayashi, H. Nishida, and K. Miki for helpful discussions and S. Tobimatsu for technical assistance in preparation of the recombinant Pb5.
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FOOTNOTES |
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* This work was supported in part by Grants-in-Aid for Scientific Research and by Grant COE 21 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.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.
To whom correspondence should be addressed. Tel.:
81-791-58-0207; Fax: 81-791-58-0132; E-mail:
s-kimura@sci.himeji-tech.ac.jp.
Published, JBC Papers in Press, November 28, 2002, DOI 10.1074/jbc.M209838200
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ABBREVIATIONS |
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The abbreviations used are: b5, cytochrome b5; Pb5, solubilized domain of porcine liver cytochrome b5; b5R, NADH-cytochrome b5 reductase; Pb5R, solubilized catalytic domain of the porcine liver NADH-cytochrome b5 reductase; FNR, ferredoxin-NADP+ oxidoreductase; WT, wild type recombinant Pb5R.
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