(Received for publication, October 12, 1994; and in revised form, December 27, 1994)
From the
A flavoprotein from Amphibacillus xylanus catalyzes the
reduction of oxygen to hydrogen peroxide. Each polypeptide chain in the
tetrameric enzyme contains 5 cysteine residues. The complete reduction
of enzyme by dithionite requires 6 electrons. Such behavior indicates
the presence of redox centers in addition to the FAD, and these could
be disulfides. In order to assess the catalytic role of disulfide in
the enzyme, 2 of the cysteines (Cys-337 and Cys-340), which show a high
degree of homology with alkyl hydroperoxide reductase F52a protein and
thioredoxin reductase, have been changed to serines by site-directed
mutagenesis of the cloned flavoprotein gene (individually and in a
double mutant). Titration of the three mutant enzymes, lacking Cys-337,
Cys-340, or both cysteines, requires only 2 electron eq to reach the
reduced flavin state. These results indicate the absence of a
redox-active disulfide and demonstrate the involvement of Cys-337 and
Cys-340 in the redox-active disulfide. The catalytic activity of the
three enzymes was examined by steady-state analysis. The K for NADH and oxygen and the k
value of these mutant enzymes were essentially
the same as those of wild type. The NADH oxidase activities were also
accelerated markedly in the presence of free FAD, which is the case for
wild-type enzyme. The NADH:5,5`-dithiobis(2-nitrobenzoic acid) (DTNB)
oxidoreductase activities of all mutant enzymes were less than 3% of
the activity of wild-type enzyme. The weak DTNB reductase activities in
the mutant enzymes lacking Cys-337 or Cys-340 may occur through direct
reduction of the mixed disulfide Cys-337-thiol or Cys-340-thiol and
nitrothiobenzoate by FADH
. However, the weak DTNB reductase
activity in the mutant enzyme lacking both cysteines indicates that
FADH
can reduce either DTNB or another disulfide directly,
albeit inefficiently. These results suggest intramolecular
dithiol-disulfide interchange reactions in the flavoprotein.
A new group of facultatively anaerobic bacteria has been
isolated from an alkaline compost and named Amphibacillus
xylanus(1, 2) . Because A. xylanus metabolizing via either anaerobic or aerobic pathways produces
similar amounts of ATP, the bacteria grow well and have the same growth
rate and cell yield under both conditions in spite of lacking a
respiratory system(2, 5) . We have proposed that
NAD is regenerated by NADH oxidase from NADH produced
from glycolysis and pyruvate oxidation in the aerobic
pathway(5) .
A flavoprotein that is functional as NADH
oxidase is purified from aerobically grown A.
xylanus(3) . The flavoprotein is a homotetramer composed
of a subunit (M = 56,000) containing 1 mol
of FAD and catalyzes the reduction of oxygen to hydrogen peroxide with
-NADH as the preferred electron donor(3) . The enzyme is
also observed to catalyze a thiol-disulfide interchange reaction,
NADH:DTNB (
)oxidoreductase(4) .
The NADH oxidase
activity of the flavoprotein has a ping-pong type mechanism, and the K for NADH and the K
for oxygen are 33.3 µM and 1.7 mM,
respectively. The K
value for oxygen is
too high to catalyze the efficient reoxidization of NADH by oxygen in
the cell of A. xylanus. In the presence of free FAD, however,
the K
value for oxygen becomes much lower
so that the accurate determination of the value by the usual assay
method is not possible. NADH oxidase activity is accelerated markedly
in the presence of free FAD. The intracellular FAD concentration of A. xylanus is calculated to be about 13 µM, and
the NADH oxidase activity is accelerated sufficiently with this FAD
concentration. These results indicate that the flavoprotein acts as an
effective NADH oxidase in the cell of A. xylanus(4) .
Dithionite and NADH titration of the flavoprotein require 3 eq of reductant/FAD for full reduction. During the addition of the first 4 electrons in the dithionite titration, the two bands of the oxidized flavin spectrum decrease gradually, and a new absorbance band at 585 nm forms. The long wavelength absorbance is ascribed to a neutral (blue) flavin semiquinone. The second phase of the reduction results in a further 2-electron reduction of the enzyme and spectral changes associated with full reduction of the flavin component. These results indicated that the flavoprotein has non-flavin redox center(s) in addition to the FAD(4) . Two disulfide bonds have been demonstrated in the flavoprotein. In order to explain the observed 6-electron reduction from titration, we proposed that the two disulfides act as the non-flavin redox centers(4) .
The amino acid sequence of A. xylanus flavoprotein is highly homologous with the alkyl hydroperoxide reductase F52a pro- tein component from Salmonella typhimurium(11) and the NADH dehydrogenase from an alkalophilic Bacillus sp. YN-1 (12) in all domains, showing identity of 51.2 and 72.5%, respectively(3) . Further, the short segment of the flavoprotein, which contained Cys-337 and Cys-340, shows a high degree of homology with Escherichia coli thioredoxin reductase(4) . The homologous region of thioredoxin reductase contains the redox-active disulfide, Cys-135 and Cys-138, which is active in catalysis(19, 20, 24) . Alkyl hydroperoxide reductase F52a protein is related to thioredoxin reductase, but there is an additional domain at the N terminus of alkyl hydroperoxide reductase F52a protein. Both enzymes contain FAD and a redox-active disulfide, in each monomer, in homologous positions. The N-terminal extension of alkyl hydroperoxide reductase F52a protein contains an additional disulfide that appears to be able to interchange with the redox-active disulfide(11, 13) . Therefore, we hypothesized that Cys-337 and Cys-340 of the flavoprotein might create a disulfide and be involved in the flow of electrons. To confirm our hypothesis, we have created three site-directed mutations of Cys-337 and Cys-340 of A. xylanus flavoprotein in an attempt to assign specific catalytic roles to these thiols. In this report, we describe the spectral and steady-state kinetic analysis of these altered enzymes.
Figure 1: Construction of mutant expression plasmid. Construction of the wild-type expression plasmid has been described(4) . The filled boxes represent the A. xylanus genes, and the open boxes represent the 1.9-kilobase HindIII fragments containing the sequence of the flavoprotein gene. The hatched boxes represent the tac promoters, and the cross-hatched boxes represent the Amp gene. Asterisks indicate the mutated codons. The position of the flavoprotein and the mutant flavoprotein gene giving rise to translated protein is indicated by the labeled arrow. kb, kilobase.
The
mutagenic oligonucleotides used were 5`-TAGCATATTCTACACAC-3` (C337S),
5`-GTACACACTCCGATGCC-3` (C340S), and
5`TGTAGCATATTCTACACACTCCGATGCCCC-3` (C337S/C340S). Mutagenesis
reactions were carried out as described by Kunkel (7) employing the
site-directed mutagenesis system Mutan-K. Single-stranded pNOSS906
served as the template (Fig. 1). Double-stranded circular DNA
resulting from the mutagenesis reactions was transformed into E.
coli MV1184. The resulting DNA prepared from individual colonies
was sequenced to verify the presence of the desired mutations. Dideoxy
sequencing was performed using the BcaBEST dideoxy sequencing
kit (Takara Shuzo Co.) according to the manufacturer's
instructions with [-
P]dCTP (Amersham).
Replicative form DNA of phagemids carrying the mutations for C337S, C340S, and C337S/C340S was digested with BglII, and the resulting 216-base pair fragment was recovered from 1.5% agarose gel. The fragment was inserted into BglII-cleaved pNOH1850 to construct the mutant expression plasmids, and the resulting plasmids were transformed into E. coli JM109. Plasmid DNA was isolated by using standard alkaline lysis methods, and manipulation was performed according to standard methods(8) .
Titration of C337S, C340S, and C337S/C340S mutant enzymes with
dithionite requires only 2 electron eq to reach the reduced flavin
state (Fig. 2). These results show the absence of the
redox-active disulfide and demonstrate the involvement of Cys-337 and
Cys-340 in the redox-active disulfide. In the 1-electron-reduced state,
the flavin absorbance of the oxidized C337S, C340S, and C337S/C340S
mutant enzymes diminishes and a new band appears at around 580 nm. The
new band, having a shoulder at 600 nm, is typical of the neutral (blue)
flavin semiquinone(16, 18) , which is also observed in
the 4-electron reduction of wild-type enzyme(4) . In the C340S
and C337S/C340S mutant enzymes, the maximal formation of semiquinone is
45% of the total enzyme flavin. The absorbance values at 580 and 450 nm
of 100% semiquinone were extrapolated from a plot of A versus A
(18) , revealing that the
extinction coefficients of flavin semiquinone are 4,800-4,900 M
cm
at 580 nm and
4,400-4,600 M
cm
at 450 nm in both mutant enzymes. These extinction coefficients
are higher than that of wild-type enzyme (4,400 M
cm
and 4100 M
cm
, respectively). Because the absorption band
of FAD at 450 nm did not change shape or peak position and because no
absorption band around 750 nm was observed, this indicates there was no
stable thiolate-flavin charge-transfer species(33) . In
contrast, the maximal formation of semiquinone is 17% of the total
enzyme in C337S mutant enzyme, and the extinction coefficient of this
band is 3,700 M
cm
and
3,200 M
cm
at 580 and
450 nm, respectively. These values are lower than those of the
wild-type enzyme. Further addition of dithionite reduces the remaining
FAD and FADH
to FADH
.
Figure 2: Spectral titration of flavoprotein with dithionite. The NADH oxidase, 30.0-37.5 µM in 50 mM sodium phosphate buffer, pH 6.6, containing 0.5 mM EDTA was titrated at 20 °C with dithionite. Spectra were recorded after each addition when no further absorbance changes occurred. The spectra are of oxidized enzyme (top lines) and enzyme after reduction by dithionite. The insets show the absorbance at 450 nm versus added dithionite. A, C337S mutant enzyme; B, C340S mutant enzyme; C, C337S/C340S mutant enzyme.
Figure 3: Spectral titration of flavoprotein with NADH. The NADH oxidase, 28.2-32.0 µM flavoprotein in 50 mM sodium phosphate buffer, pH 6.6, containing 0.5 mM EDTA was titrated at 20 °C with NADH. The spectra are of oxidized enzyme (top lines) and enzyme after reduction by NADH. The insets show the absorbance at 450 nm versus added NADH. A, C337S mutant enzyme; B, C340S mutant enzyme; C, C337S/C340S mutant enzyme.
Reduction of each of the mutant enzymes with NADH shows that only the FAD prosthetic group is involved. A comparison of these results with NADH titration of wild-type enzyme confirms that Cys-337 and Cys-340 constitute the active-site disulfide in direct communication with flavin. This redox pair in turn is presumably responsible for reduction of the remaining disulfide.
NADH oxidase activity of wild-type enzyme was accelerated markedly in the presence of additional free FAD, but FMN and riboflavin did not affect the NADH oxidase activity(4) . The NADH oxidase activity of all mutant enzymes was accelerated markedly in the presence of free FAD (Fig. 4), as with the wild-type enzyme(4) . The optimum pH value for the activity of all mutant enzymes was not significantly altered relative to that of the wild type (data not shown). These results indicated that Cys-337 and Cys-340 of the flavoprotein are not involved in the NADH oxidase activity.
Figure 4: Effect of FAD on NADH oxidase activity. Assay conditions were 50 mM sodium phosphate buffer, pH 6.6, containing 0.5 mM EDTA and 100 µM NADH at 25 °C. Open circles, wild-type enzyme; filled circles, C337S; open squares, C340S; filled triangles, C337S/C340S.
The A. xylanus flavoprotein that functions as NADH oxidase has unique functional properties that are the difference from known NADH oxidases(25, 26, 27, 28, 29, 30, 31) . Several enzymes referred to as NADH oxidase are known to be able to catalyze electron transfer from NADH to various electron acceptors such as methylene blue, cytochrome c, 2,6-dichloroindophenol, and potassium ferricyanide(26, 27, 30, 31) . However, none of these enzymes have been reported to be able to catalyze thiol-disulfide interchange reactions. A. xylanus flavoprotein was obtained to catalyze electron transfer between NADH and DTNB(4) . We have hypothesized that Cys-337 and Cys-340 of the flavoprotein are involved in the flow of electrons from NADH as the non-redox center(4) .
Pseudomonas aeruginosa mercuric reductase, which is a key component of an organomercurial
detoxification system, has a redox-active disulfide (Cys-135,Cys-140)
and a FAD(41) . Mercuric reductase partially reduced with NADPH
is an EH species whose spectral features have been
attributed to a charge-transfer interaction between the thiolate of
Cys-140 and FAD(41) . Further, the enzyme reduced by 0.8 eq of
dithionite shows fluorescence intensity of the enzyme flavin diminished
by 44% but only shows a 16% thiolate-FAD charge transfer, indicating
that the second disulfide is in close proximity to the
FAD(22) . Miller et al.(22) designated the
Cys-558,Cys-559 disulfide as the auxiliary disulfide and suggested that
reduction of the auxiliary disulfide by NADPH occurs via
dithiol-disulfide interchange with the redox-active cysteine pair
(Cys-135, Cys-140).
Reduction of the enzyme FAD by the strong reductant dithionite occurred during the total uptake of 6 electrons in the A. xylanus wild-type flavoprotein. Because the FAD can accept only 2 electrons, the 6-electron uptake indicates that other redox-active acceptors are present. The amino acid sequence suggests that these are disulfides (4) . Titration of C337S, C340S, and C337S/C340S mutant enzymes with NADH required only 2 electrons for full reduction. These results clearly indicate the absence of redox-active disulfide and demonstrate the involvement of Cys-337 and Cys-340 in the redox-active disulfide. This agrees with the results of thiol quantitation in that the number of thiols is the same in oxidized and reduced mutant enzymes. These results show that the redox-active disulfide, Cys-337 and Cys-340, is reduced directly via the 2-electron-reduced FAD coenzyme.
The NADH oxidase activity of
wild-type enzyme is accelerated markedly in the presence of additional
free FAD, and the K value for oxygen decreases
dramatically(4) . The K
values for oxygen
and NADH and the k
values in the three mutant
enzymes were not significantly changed. The acceleration of NADH
oxidase activity by excess FAD is also observed in all three mutant
enzymes. The NADH oxidase activity of the mutant enzymes that lack the
redox-active disulfide shows that the activity does not depend on the
other disulfide. Thus, it is clear that neither disulfide bond in the
wild-type enzyme is involved in the reduction of oxygen to hydrogen
peroxide. This suggests that the reaction of 2-electron-reduced FAD
coenzyme with oxygen may form a flavin-C-4a-hydroperoxide
adduct(34) , followed by the elimination of hydrogen peroxide.
Ahmed and Claiborne (35, 36) proposed that the
peroxidatic reaction in Streptococcus faecalis NADH oxidase,
which catalyzes the reduction of oxygen to water, in contrast, would
involve the redox-active cysteinyl derivative.
The pyridine nucleotide-disulfide oxidoreductases form a family of homodimeric flavoproteins, having a redox-active disulfide and FAD in each monomer. Interaction of the 2electron-reduced enzymes involves sequential thiol-disulfide interchange reactions. Each nascent thiol of 2-electron-reduced enzyme has a distinct function; the interchange thiol reacts with the disulfide substrate, and the electron-transfer thiol reacts with the FAD(20) . Studies of two active site mutations of E. coli thioredoxin reductase, Ser-135,Cys-138 and Cys-135,Ser-138, have shown that Cys-138 interacts more closely with the FAD than does Cys-135(37, 38, 39) . This was confirmed by the study of x-ray crystal structure; Cys-138 is indeed close to the flavin, 3.0 Å from the C-4a position of the isoalloxazine ring, whereas Cys-135 is 4.4 Å from the C-5a position of the isoalloxazine ring(40) . It is suggested that Cys-135 might be the interchange thiol, because Cys-138 interacts with the FAD(20, 38, 39) .
The active site
mutant of mercuric reductase from P. aeruginosa,
Cys-135,Ser-140, shows 3% of the rate of DTNB reduction of the
wild-type enzyme. Schultz et al. (42) suggested that
the mixed disulfide between Cys-135-thiol and nitrothiobenzoate is
reduced directly by FADH. Further, they suggest that the
Ser-135,Cys-140 mutant enzyme lacks DTNB reductase activity due to
steric hindrance in the formation of the mixed disulfide(42) .
All three mutant enzymes showed weak NADH:DTNB oxidoreductase
activities, less than 3% of the rate of the wild-type enzyme. Thus, it
is clear that Cys-337 and Cys-340 are involved with the NADH:DTNB
oxidoreductase activity in the wild-type enzyme. These activities were
the same in both mutant enzymes, suggesting that the thiol-disulfide
interchange reactions differ from that of thioredoxin reductase, in
spite of the high degree of conservation around the active site
cysteines. The weak DTNB reduction in the C337S and C340S mutant
enzymes may occur slowly through direct reduction by FADH of the mixed disulfide between Cys-337-thiol or Cys-340-thiol and
nitrothiobenzoate. Surprisingly, the C337S/C340S mutant enzyme, lacking
both thiols, also showed the same 3% DTNB reductase activity. This
result indicates that the other disulfide bond in the C337S/C340S
mutant enzyme may also have thiol-disulfide interchange activity. We
suggest that the thiol-disulfide interchange reaction in the wild-type
flavoprotein may involve an intramolecular dithiol-disulfide
interchange between the second disulfide and the Cys-337,Cys-340
thiols, reduced via FADH
. This would differ from the known
members of the pyridine nucleotide-disulfide oxidoreductase family of
flavoprotein(20) , except for mercuric reductase(22) .