(Received for publication, June 21, 1995; and in revised form, August 8, 1995)
From the
The flavoprotein NADH oxidase from Amphibacillus xylanus consumes oxygen to produce hydrogen peroxide. The amino acid
sequence of this flavoprotein shows 51.2% identity to the F-52a
component, denoted AhpF, of the alkyl-hydroperoxide reductase from Salmonella typhimurium. AhpF also catalyzes NADH-dependent
hydrogen peroxide formation under aerobic conditions, albeit at a
somewhat slower rate than the Amphibacillus protein. In the
presence of the 22-kDa colorless component (AhpC) of the Salmonella alkyl-hydroperoxide reductase, both proteins catalyze the
4-electron reduction of oxygen to water. Both flavoproteins are active
as AhpC reductases and mediate electron transfer, resulting in the
NADH-dependent reduction of hydrogen peroxide and cumene hydroperoxide.
Both enzymes' K values for hydrogen
peroxide, cumene hydroperoxide, and NADH are so low that they could not
be determined accurately. V
values for hydrogen
peroxide or cumene hydroperoxide reduction are >10,000
min
at 25 °C. These values are almost the same
as the reduction rate of the flavoprotein component by NADH. The
involvement in catalysis of a redox-active disulfide of the A.
xylanus flavoprotein was shown by construction of three mutant
enzymes, C337S, C340S, and C337S/C340S. Very little activity for
hydrogen peroxide or cumene hydroperoxide was found with the single
mutants (C337S and C340S), and none with the double mutant
(C337S/C340S).
Analysis of the DNA sequence upstream of the Amphibacillus flavoprotein structural gene indicated the presence of a partial open reading frame homologous to the Salmonella ahpC structural gene (64.3% identical at the amino acid sequence level), suggesting that the NADH oxidase protein of A. xylanus is also part of a functional alkyl-hydroperoxide reductase system within these catalase-lacking bacteria.
We recently have isolated a new group of facultatively anaerobic bacteria from alkaline compost(1) . The bacteria have unique phenotypic and chemotaxonomic characteristics (2) as well as bioenergetic properties (3) and were named Amphibacillus xylanus(2) . A. xylanus, lacking a respiratory system and hemeproteins, catalase, and peroxidase, grows well and has the same growth rate and cell yield under strictly anaerobic and aerobic conditions(2) . This growth characteristic of A. xylanus is due to the presence of anaerobic and aerobic pathways producing similar amounts of ATP(4) . Under aerobic conditions, NADH is thought to be responsible for maintenance of the intracellular redox balance(4) .
A flavoprotein functional as NADH oxidase
was purified from aerobically grown A. xylanus(5) .
The flavoprotein is a homotetramer composed of subunits (M = 56,000) containing 1 mol of FAD and
also catalyzes a thiol-disulfide interchange reaction, NADH:DTNB (
)oxidoreductase(6) . The complete reduction of
enzyme by dithionite requires 6 electrons/subunit(6) . Such
behavior indicates the presence of redox centers in addition to the
FAD, and these were postulated to be disulfides(6) . To assess
the catalytic role of disulfide in the enzyme, two of the cysteines
(Cys-337 and Cys-340), which show a high degree of homology to
thioredoxin reductase, had been changed to serines by site-directed
mutagenesis of the cloned flavoprotein gene (individually and in a
double mutant)(7) . Titration of the three mutant enzymes,
lacking Cys-337, Cys-340, or both cysteines, required only 2 electron
eq to reach the reduced flavin state(7) . The NADH:DTNB
oxidoreductase activities of all mutant enzymes were <3% of the
activity of the wild-type enzyme(7) . These results indicate
that Cys-337 and Cys-340 participate in the NADH:DTNB oxidoreductase
activity in the wild-type enzyme and demonstrate the involvement of
Cys-337 and Cys-340 as the redox-active disulfide(7) .
The
amino acid sequence of A. xylanus NADH oxidase exhibits 51.2%
identity in comparison with the alkyl-hydroperoxide reductase F-52a
component (AhpF) from Salmonella typhimurium(5) , a
flavoprotein that has also been shown to possess NADH oxidase activity.
Together with the 22-kDa protein component (AhpC) of the S.
typhimurium alkyl-hydroperoxide reductase, AhpF was reported to
catalyze the NADH-dependent reduction of alkyl hydroperoxides, but not
HO
(5) . We have now found that both S. typhimurium AhpF and A. xylanus NADH oxidase
scavenge not only alkyl hydroperoxides, but also hydrogen peroxide in
the presence of S. typhimurium AhpC. We describe the
properties of such enzyme activities in this report.
Turnover studies of hydrogen peroxide or alkyl-hydroperoxide reductase activities were carried out in a temperature-controlled stopped-flow spectrophotometer (Hi-tech SF-61) interfaced with a Dell 325D computer. The activity was measured under anaerobic conditions at 25 °C. The flavoprotein/AhpC mixture (10 ml in volume), containing 50 mM sodium phosphate buffer, pH 7.0, 0.5 mM EDTA, 300 mM ammonium sulfate, 1.12 µM flavoprotein (A. xylanus NADH oxidase or S. typhimurium AhpF), and 70.4 µM AhpC, was loaded into a tonometer. After establishing anaerobiosis by repeated evacuation and equilibration with oxygen-free argon and equilibration at 25 °C, the reaction was started by mixing the protein solution with varying mixtures of NADH and peroxide substrates, and the reaction was monitored at 340 nm. The NADH/peroxide mixture, containing 50 mM sodium phosphate buffer, pH 7.0, 0.5 mM EDTA, 0.06-2 mM hydrogen peroxide or cumene hydroperoxide, and 150-600 µM NADH (concentrations were halved after mixing in the stopped-flow spectrophotometer), was bubbled with oxygen-free argon at 25 °C. Although most of these determinations were carried out under anaerobic conditions, to avoid possible complications from reaction with oxygen, we found that in the presence of AhpC, the peroxide reductase activities were in fact the same in air-saturated solution as those determined anaerobically. We did not bubble argon into NADH/hydrogen peroxide mixtures in the experiments in which the effect of changing salt concentrations was evaluated.
Figure 1:
Time course for the oxidation of NADH
by hydrogen peroxide in the presence of A. xylanus NADH
oxidase or S. typhimurium AhpF. The activities were measured
under anaerobic conditions at 25 °C. 0.56 µMA.
xylanus NADH oxidase (solid line) or S. typhimurium AhpF (dotted line) and 35.2 µM AhpC were
mixed with 0.5 mM hydrogen peroxide and 150 µM NADH and monitored at 340 nm. Conditions are described under
``Experimental Procedures.'' The hydrogen-peroxide reductase
activities of A. xylanus NADH oxidase and S. typhimurium AhpF were 118 and 145 s,
respectively.
Studies employing an oxygen electrode to directly monitor oxygen consumption also confirmed hydrogen peroxide-producing oxidase activities for both the NADH oxidase and AhpF flavoproteins in the absence of the AhpC component, although considerably more AhpF was required in these studies to achieve rates similar to the oxidase (2.3 nmol of AhpF gave rates comparable to those with 0.22 nmol of NADH oxidase). On addition of catalase following full reaction with limiting oxygen, excess NADH (600 µM), and NADH oxidase (Fig. 2) or AhpF (data not shown), oxygen was produced, indicating the formation of hydrogen peroxide during turnover with oxygen. When the same experiments were repeated in the presence of 8.8 nmol of AhpC, however, no oxygen was detected following addition of catalase (Fig. 2), indicating that no hydrogen peroxide accumulated under these conditions. That oxygen consumption was the result of 2-electron reduction in the presence of flavoprotein only or 4-electron reduction in the presence of AhpC and either flavoprotein was confirmed by determination of the stoichiometry of NADH reduction relative to oxygen consumption in the presence of limiting NADH amounts. Thus, in experiments conducted with AhpF in the absence or presence of AhpC, the ratios of NADH oxidation relative to oxygen consumed were 1.11 and 2.35, respectively. The overall reaction catalyzed by both flavoproteins (AhpF and A. xylanus NADH oxidase) in the presence of AhpC is envisaged to occur as outlined in Fig. S1(see ``Discussion'' for further details).
Figure 2: Oxygen consumption during A. xylanus NADH oxidase catalysis in the presence and absence of AhpC. In the absence of AhpC (upper panel), the air-equilibrated reaction mixture (2 ml) contained 50 mM sodium phosphate buffer, pH 7.0, including 0.5 mM EDTA, 400 µg of bovine plasma albumin, and 600 µM NADH. In the presence of AhpC (lower panel), the reaction mixture contained the same components plus 8.8 nmol of AhpC. The reaction was started at 25 °C by addition of 0.22 (upper panel) and 0.44 (lower panel) nmol of NADH oxidase, as indicated by arrow a. After oxygen consumption, 15 µg of catalase was added, as indicated by arrow b.
Figure S1: Scheme 1Proposed reaction mechanism for oxygen reduction catalyzed by A. xylanus NADH oxidase in the presence of AhpC.
Figure 3:
Time course for the oxidation of NADH by
alkyl hydroperoxide in the presence of A. xylanus NADH oxidase
or S. typhimurium AhpF. The activities were measured under
anaerobic conditions at 25 °C. 0.56 µMA. xylanus NADH oxidase (dotted line) or S. typhimurium AhpF (solid line) and 35.2 µM AhpC were
mixed with 0.5 mM cumene hydroperoxide and 150 µM NADH and monitored at 340 nm. Conditions are described under
``Experimental Procedures.'' The alkyl-hydroperoxide
reductase activities of A. xylanus NADH oxidase and S.
typhimurium AhpF under these conditions were 117 and 138
s, respectively.
Stoichiometric electron transfer by A. xylanus NADH oxidase in the presence of AhpC was indicated by the consumption of 1.68 µmol of cumene hydroperoxide on oxidation of 1.73 µmol of NADH and by the consumption of 1.39 µmol of t-butyl hydroperoxide on oxidation of 1.68 µmol of NADH (data not shown). Thus, alkyl-hydroperoxide reductase activity is also supported by the A. xylanus NADH oxidase flavoprotein in the presence of AhpC.
As a direct indication of the stoichiometric transfer of electrons from NADH to AhpC via the A. xylanus NADH oxidase, anaerobic NADH titration of AhpC in the presence of a catalytic amount of the flavoprotein was carried out, followed by DTNB assay to detect protein thiols. Anaerobic titration of AhpC in the presence of a 200-fold lower amount of the A. xylanus flavoprotein resulted in the oxidation of 1.06 eq of NADH/subunit of AhpC (data not shown). The DTNB assay for free thiols indicated the generation of 1.83 thiols/subunit of AhpC, fully consistent with results obtained earlier using the AhpF flavoprotein from S. typhimurium instead of the A. xylanus flavoprotein(8) .
Figure 4:
Time course for the oxidation of NADH by
hydrogen peroxide in the presence of AhpC plus wild-type and mutant
C340S, C337S, and C340S/C337S A. xylanus NADH oxidases. The
activities were measured under anaerobic conditions at 25 °C. 0.56
µMA. xylanus NADH oxidase (dashed
line), mutant C340S (solid line), mutant C337S (dotted line), or mutant C340S/C337S (dashed-dotted
line) and 35.2 µM AhpC were mixed with 0.5 mM hydrogen peroxide and 150 µM NADH and monitored at
340 nm. Conditions are described under ``Experimental
Procedures.'' The hydrogen-peroxide reductase activities of
wild-type and mutant C340S, C337S, and C340S/C337S A. xylanus NADH oxidases were 117, 0.4, 0.2, and 0.0 s respectively.
Figure 5:
Ionic strength dependence of the
hydrogen-peroxide reductase activities of the A. xylanus NADH
oxidase-AhpC system. The activities were measured under aerobic
conditions at 25 °C. 0.56 µMA. xylanus NADH
oxidase and 35.2 µM AhpC were mixed with 0.5 mM hydrogen peroxide and 150 µM NADH in the presence of
salts at varying ionic strength: potassium phosphate (),
ammonium sulfate (
), and potassium chloride (
). Conditions
are described under ``Experimental
Procedures.''
The A. xylanus NADH oxidase flavoprotein has unique functional properties that are different from other known NADH oxidases (14, 15, 16, 17, 18, 19, 20) . The flavoprotein was shown to catalyze electron transfer between NADH and DTNB, and the complete reduction of the enzyme by dithionite required 6 electron eq/mol of enzyme-bound flavin(6) . Such behavior indicates the presence of redox centers in addition to the FAD, and these were shown to be two disulfides(7) .
The
alkyl-hydroperoxide reductase of S. typhimurium was first
recognized through the isolation of mutant cells that were resistant to
mutagenesis by alkyl hydroperoxide(11) , and its scavenging
activity for hydrogen peroxide was not found in early
research(11) . The A. xylanus NADH oxidase is thought
to function in vivo to regenerate NAD from NADH produced in
the aerobic pathway, and hydrogen peroxide was shown to be the final
product in the isolated NADH oxidase reaction(5) . These two
enzyme systems were presumed to be unable to reduce hydrogen peroxide.
However, in the presence of saturating concentrations of AhpC, the V values for hydrogen peroxide and alkyl
hydroperoxide are almost the same as the reduction rate by NADH of the
enzyme-bound FAD in the NADH oxidase, suggesting that these values may
be limited by the reduction rate of the flavoprotein component. K
values for hydrogen peroxide, cumene
hydroperoxide, and NADH are too low to allow accurate determination of
their values in these experiments. Several enzymes that show scavenging
activity for hydrogen peroxide and alkyl hydroperoxides have been
purified and characterized from bacteria or mammalian
sources(9, 11, 21, 22, 23, 24, 25, 26) .
The V
value for the flavin-containing NADH
peroxidase from Streptococcus faecalis is 121 s
at pH 5.4(9) . The K
values for
hydrogen peroxide of this enzyme are 130 µM at pH 5.4 and
10.1 µM at pH 7.5(9) . The K
value for hydrogen peroxide of horse liver catalase is 1.1 M(21) . The K
value for cumene
hydroperoxide with the glutathione peroxidase from pig erythrocytes is
40 µM at pH 8.0(22) . None of these enzymes has
been reported to show such high turnover numbers and low K
values for both hydrogen peroxide and alkyl
hydroperoxide as the enzymes described here.
Amphibacillus sp. are Gram-positive microorganisms and taxonomically far distant
from Gram-negative microorganisms, such as Salmonella sp.
However, the amino acid sequences of Amphibacillus NADH
oxidase and Salmonella AhpF show a high identity of
51.2%(5) . In the absence of AhpC, Salmonella AhpF
also shows NADH oxidase activity that is accelerated in the presence of
additional free FAD, in a manner similar to that of A. xylanus NADH oxidase. Furthermore, in the presence of S. typhimurium AhpC, both enzymes catalyze the NADH-dependent 4-electron
reduction of oxygen to water (Fig. S1) or the 2-electron
reduction of hydrogen peroxide or alkyl hydroperoxides to water or the
respective alcohol products (Fig. S2). The two peroxidase
activities of NADH oxidase and AhpF in the presence of AhpC give
turnover numbers of similar magnitude. The efficiency of the
two-protein component systems of Schemes 1 and 2 deserves comment. The
overall rate of NADH:peroxide reductase activity, with
HO
or cumene hydroperoxide, appears to be
limited by the rate of flavin reduction by NADH. The bimolecular rate
constant for the binding of NADH in this reaction, derived from k
/K
, is at
least 1
10
M
s
at 25 °C. What is even more remarkable,
however, is the efficiency of electron transfer between the reduced
flavin and the Cys-337-Cys-340 disulfide of the flavoprotein and
the thiol-disulfide interchange with the disulfide of AhpC. All of
these events must occur considerably faster than the limiting rate of
flavin reduction, i.e. >200 s
at 25
°C. (
)
Figure S2: Scheme 2Proposed reaction mechanism for hydrogen peroxide or alkyl hydroperoxide reduction catalyzed by A. xylanus NADH oxidase in the presence of AhpC.
With A. xylanus, which lacks a respiratory chain, NADH oxidase should regenerate NAD from NADH formed in the aerobic pathway (Fig. S3), and a lot of hydrogen peroxide is presumed to be produced in aerobic growing cells. Despite lacking catalase, A. xylanus can grow well under aerobic conditions(2) . The location of a partial open reading frame upstream of the NADH oxidase locus (from positions -15 to -312; (5) ) with homology to S. typhimurium AhpC (64.3% identity of the deduced amino acid sequences; (34) and Fig. 6) leads us to suggest that the A. xylanus NADH oxidase protein also exists as one component of an alkyl-hydroperoxide reductase system in A. xylanus.
Figure S3: Scheme 3Proposed reaction mechanism for oxygen reduction catalyzed by A. xylanus NADH oxidase in the absence of AhpC.
Figure 6: Comparison of the partial amino acid sequence of the S. typhimurium AhpC component (27) and the amino acid sequence deduced from the nucleotide sequence of the region upstream (from positions -15 to -312 relative to the ATG start site) of the NADH oxidase gene from A. xylanus(5, 34) . Sequence alignments were generated using the Genetic Computer Group BESTFIT program, employing the default parameters. ORF, open reading frame; Nox, NADH oxidase.
The region of the NADH
oxidase surrounding Cys-337 and Cys-340 is highly conserved with
respect to Escherichia coli thioredoxin
reductase(6, 28) . This region of thioredoxin
reductase contains the redox-active disulfide, which is composed of
Cys-135 and Cys-138(29) . The steady-state kinetic analysis of
two active-site mutants of thioredoxin reductase, Ser-135,Cys-138 and
Cys-135,Ser-138, shows 10 and 50%, respectively, of the thioredoxin
reductase activity of the wild-type enzyme(30) . Williams and
co-workers (30) suggested that the remaining thiol can carry
out interchange with the disulfide of thioredoxin, and the resulting
mixed disulfide can be reduced by NADH via FAD. On the other hand, the
single mutants of NADH oxidase (C337S and C340S) show very little
activity for hydrogen peroxide and cumene hydroperoxide, and no
activity was observed in the double mutant (C337S/C340S). These results
indicate that neither the remaining thiol nor the second disulfide
alone has thiol-disulfide interchange activity with AhpC. It is clear
that thiol-disulfide interchange reactions also differ from that of
thioredoxin reductase, in spite of the high degree of conservation
around the active-site cysteines. We found that the mutants require
only 2 electron eq to reach the reduced flavin state (7) ,
while the complete reduction of the wild-type enzyme requires 6
electron eq(6) . Thus, electrons from FADH must
pass through Cys-337 and Cys-340 to reduce the second disulfide of the
NADH oxidase or the disulfide of AhpC.
Rhee and co-workers (31, 32, 33, 34) have purified a
25-kDa protein from yeast and rat brain that can participate in the
reduction of hydrogen peroxide or alkyl hydroperoxides and named this
protein thiol-specific antioxidant. The 25-kDa protein, whose sequence
is 40% identical to that of AhpC(33, 34) , was shown
to be a peroxidase that reduces hydrogen peroxide and alkyl
hydroperoxides with the use of reducing equivalents derived from
thioredoxin, thioredoxin reductase, and NADPH (35) . These
activities, however, were quite low, partially because of inactivation
of the 25-kDa protein by peroxides, especially by alkyl
hydroperoxides(35) . No inactivation by peroxides was observed
in the NADH oxidase-AhpC or AhpF-AhpC systems, and the peroxide
reductase activity for both substrates appears to be limited in rate by
the reduction by NADH of the flavin in the NADH oxidase
component.