RESEARCH LETTER
Catalytic properties of Na1 -translocating NADH:quinone
oxidoreductases from Vibrio harveyi , Klebsiella pneumoniae ,
and Azotobacter vinelandii
Maria S. Fadeeva1, Cinthia Núñez2, Yulia V. Bertsova1, Guadalupe Espı́n2 & Alexander V. Bogachev1
1
Department of Molecular Energetics of Microorganisms, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow,
Russia; and 2Departamento de Microbiologı́a Molecular, Instituto de Biotecnologı́a, Universidad Nacional Autónoma de México, Morelos, México
Correspondence: Alexander V. Bogachev,
Department of Molecular Energetics of
Microorganisms, A.N. Belozersky Institute
of Physico-Chemical Biology, Moscow
State University, Leninskie Gory, Moscow
119992, Russia. Tel.: 17 495 930 0086;
fax: 17 495 939 0338; e-mail:
[email protected]
Received 26 September 2007; accepted 6
November 2007.
First published online December 2007.
Abstract
The catalytic properties of sodium-translocating NADH:quinone oxidoreductases
(Na1-NQRs) from the marine bacterium Vibrio harveyi, the enterobacterium
Klebsiella pneumoniae, and the soil microorganism Azotobacter vinelandii have
been comparatively analyzed. It is shown that these enzymes drastically differ in
their affinity to sodium ions. The enzymes also possess different sensitivity to
inhibitors. Na1-NQR from A. vinelandii is not sensitive to low 2-n-heptyl-4hydroxyquinoline N-oxide (HQNO) concentrations, while Na1-NQR from
K. pneumoniae is fully resistant to either Ag1 or N-ethylmaleimide. All the Na1NQR-type enzymes are sensitive to diphenyliodonium, which is shown to modify
the noncovalently bound FAD of the enzyme.
DOI:10.1111/j.1574-6968.2007.01015.x
Editor: Jörg Simon
Keywords
Na1-translocating NADH:quinone
oxidoreductase; sodium potential; Vibrio ;
respiratory chain; diphenyliodonium.
Introduction
The Na1-translocating NADH:quinone oxidoreductase
(Na1-NQR) generates a redox-driven transmembrane electrochemical Na1 potential (Tokuda & Unemoto, 1981, 1982;
Hayashi et al., 2001b; Bogachev & Verkhovsky, 2005). The
enzyme consists of six subunits (NqrA-F) (Nakayama et al.,
1998) encoded by the six genes of the nqr operon (Hayashi
et al., 1995; Rich et al., 1995). Na1-NQR is thought to
contain the following set of prosthetic groups: one 2Fe–2S
cluster, one noncovalently bound FAD, two covalently
bound FMN residues, and possibly also one ubiquinone-8
(Bogachev & Verkhovsky, 2005). Subunit NqrF possesses
binding motifs for NADH, FAD, and the 2Fe–2S cluster
(Rich et al., 1995; Barquera et al., 2004; Turk et al., 2004),
whereas the covalently bound FMN residues are attached by
phosphoester bonds to threonine residues in the subunits
NqrB and NqrC (Zhou et al., 1999; Nakayama et al., 2000;
Hayashi et al., 2001a).
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In vivo, Na1-NQR oxidizes NADH and transfers two
electrons to ubiquinone with production of ubiquinol. This
redox reaction is coupled with a transfer of two sodium ions
across the membrane, i.e. the Na1/e ratio for Na1-NQR is
1 (Bogachev et al., 1997). Sodium ions are indispensable
components of the Na1-NQR-catalyzed reaction; therefore,
its rate depends on the concentration of Na1, and the
reaction virtually does not occur in media depleted in this
ion. All other ions studied failed to activate Na1-NQR; they
only nonspecifically stimulated it because increase in ionic
strength increases the affinity of Na1-NQRs for sodium ions
(Unemoto et al., 1977).
Only a few inhibitors of Na1-NQR are known. The
antibiotic korormicin has recently been described; it specifically inhibits Na1-NQR at the level of its interaction with
ubiquinone (Yoshikawa et al., 1999; Hayashi et al., 2001b).
Korormicin affects the enzyme without competition with
quinone, and its inhibition constant is 0.1 nM (Yoshikawa et al., 1999). The effect of 2-n-heptyl-4-hydroxyquinoline
FEMS Microbiol Lett 279 (2008) 116–123
Catalytic properties of Na1-NQR
117
N-oxide (HQNO) on Na1-NQR is similar (Tokuda &
Unemoto, 1984), but the affinity of this inhibitor to the
enzyme is significantly weaker (Yoshikawa et al., 1999;
Hayashi et al., 2001b). Na1-NQR is also sensitive to low
concentrations of silver ions (Asano et al., 1985) and some
other ions of heavy metals (Cd21, Pb21, Zn21, Cu21)
(Bourne & Rich, 1992). These ions influence the initial
reactions of the catalytic cycle of Na1-NQR and seem to
prevent its interaction with NADH (Bourne & Rich, 1992).
In addition to the quinone-reductase reaction, Na1-NQR
can catalyze in vitro the so-called NADH-dehydrogenase
reaction during interaction with soluble quinones or some
other artificial electron acceptors (Hayashi & Unemoto,
1984). The NADH-dehydrogenase activity does not depend
on Na1 concentration, is inhibited by heavy metal ions, and
is insensitive to HQNO and korormicin (Bourne & Rich,
1992; Hayashi et al., 2001b). Only the FAD binding domain
of the NqrF subunit seems to be required for this activity
(Barquera et al., 2004; Turk et al., 2004).
The Na1-NQR enzymes have been investigated only in
different marine bacteria of the genus Vibrio, such as Vibrio
alginolyticus (Tokuda & Unemoto, 1982, 1984), Vibrio
harveyi (Bogachev et al., 2001, 2002), and Vibrio cholerae
(Barquera et al., 2002, 2004), i.e. on enzymes from closely
related microorganisms. However, it has recently been
shown that the nqr operon is widely distributed among
various bacteria, including several pathogenic microorganisms (Zhou et al., 1999; Häse et al., 2001). Thus, it was
important to study, under the same experimental conditions, the catalytic properties of Na1-NQRs from different
bacteria, which inhabit diverse ecological niches, in order to
reveal the characteristics of Na1-NQRs that are common for
this family of enzymes.
grown in Luria broth (LB) medium at 37 1C. Vibrio harveyi
and Azotobacter vinelandii were grown at 32 1C in rich
medium FM (Fadeeva et al., 2007) and modified Burk’s
medium BSN (D’Mello et al., 1994) media, respectively.
Antibiotic concentrations for E. coli were ampicillin at
100 mg mL1, tetracycline at 10 mg mL1, and kanamycin at
50 mg mL1; for K. pneumoniae: rifampicin at 100 mg mL1,
tetracycline at 3.3 mg mL1, ampicillin at 100 mg mL1, and
chloramphenicol at 40 mg mL1; for V. harveyi: rifampicin at
100 mg mL1, kanamycin at 50 mg mL1, and tetracycline at
5 mg mL1; for A. vinelandii: rifampicin at 10 mg mL1,
kanamycin at 1 mg mL1, and spectinomycin at 50 mg mL1.
Materials and methods
Construction of NDH-2-deficient V. harveyi strain
Bacterial strains, growth, and medium
composition
The bacterial strains used in this study are listed in Table 1.
Escherichia coli and Klebsiella pneumoniae strains were
Genetic manipulations
Construction of Na1-NQR-deficient V. harveyi
strain
The nqrA gene of V. harveyi was amplified by PCR with Taq
polymerase and primers nqrA_dir 5 0 -CAAGTGCCCAT
GGTTACAATA and nqrA_rev 5 0 -GCATGAATTCCCCTT
CCT. The amplified 1.4-kbp fragment was cloned into
pGEM-T vector, resulting in the pHA001 plasmid. A kanamycin-resistance cassette was inserted into the Bse8I site of the
nqrA gene, and a plasmid (pHAKm4) bearing the nqrA gene
together with the unidirectionally transcribing kanamycinresistance cassette was selected. The nqrA<Km fragment from
pHAKm4 was subcloned into suicide vector pKNOCK-Tc
(Alexeyev, 1999), resulting in pNockA2. This plasmid was
transferred into the V. harveyi R3 strain via conjugation using
E. coli SM10lpir as the donor, and a TcS KmR RfR phenotype
clone (R3A10, nqrA<Km) characteristic of a double-crossover
introduced mutation was selected.
A V. harveyi DNA fragment containing the ndh gene
(EDL67931 locus) was amplified by PCR using primers
VH_ndh_dir 5 0 -GGATGTATGACCGCAGAGCAC and
VH_ndh_rev 5 0 -CCAGATAACACCGCCAGTCAG. The amplified 1.4-kbp fragment was cloned into pGEM-T vector,
Table 1. Bacterial strains used in this study
Strains
Relevant details
References or sources
V. harveyi R3
V. harveyi R3A10
V. harveyi NDKm34
K. pneumoniae KNQ197
K. pneumoniae KNU210
K. pneumoniae KND038
A. vinelandii DN165
A. vinelandii GG4
E. coli Sm10 lpir
RfR
nqrA<Km, KmR RfR
ndh<Km, KmR RfR
nqrA<Km, RfR KmR
nuoB<Cm, RfR CmR
nuoB<Cm ndh<Ap, CmR ApR RfR
ndh<OTc, RifR TcR
nqrE<Tn5, SpR
thi thr leu tonA lacY supE recA<RP4-2-Tc<Mu, KmR
Fadeeva et al. (2007)
This study
This study
Bertsova & Bogachev (2004)
Bertsova & Bogachev (2004)
This study
Bertsova et al. (2001)
Núñez et al. (unpublished data)
Miller & Mekalanos (1988)
FEMS Microbiol Lett 279 (2008) 116–123
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118
resulting in the pG_ndhVh7 plasmid. A kanamycinresistance cassette was inserted into the BlpI site of the ndh
gene, and a plasmid (pG_ndhKm7) bearing the ndh gene
together with the unidirectionally transcribing kanamycinresistance cassette was selected. The ndh<Km fragment
from pG_ndhKm7 was subcloned into suicide vector
pKNOCK-Tc, resulting in pKn_ndhVhKm1. This plasmid
was transferred into the V. harveyi R3 strain via conjugation
using E. coli SM10lpir as the donor, and a TcS KmR RfR
phenotype clone (NDKm34, ndh<Km) characteristic of a
double-crossover introduced mutation was selected. Localization of the mutations in the V. harveyi chromosome was
verified by PCR analysis.
M.S. Fadeeva et al.
Rates of NADH, NADPH, and dNADH oxidation by SBP
were measured in medium 2 at 30 1C using a Hitachi-557
spectrophotometer. The reduced pyridine dinucleotides
were used at final concentrations of 150 mM.
Na1-NQR from V. harveyi cells was purified as described
previously (Bogachev et al., 2006).
EPR spectra of purified Na1-NQR from V. harveyi were
measured as described in Bogachev et al. (2002).
Protein concentration was determined by the bicinchoninic acid method with bovine serum albumin as standard.
Sodium concentration was measured by flame photometry.
Results and discussion
Construction of double NDH-2/NDH-1-deficient
K. pneumoniae strain
The K. pneumoniae ndh fragment was amplified with
primers Kpn_ndh_dir 5 0 -GGACCGTAACCACAGCCATC
and Kpn_ndh_rev 5 0 -CAGCGGTTTGCCTTTCATCT. The
amplified 946 bp fragment was cloned into a ‘pGEM-T Easy’
vector, resulting in the pGndh7 plasmid. Further, the 964 bp
EcoRI–EcoRI fragment from pGndh7, containing a part of
ndh, was subcloned into suicide vector pKNOCK-Tc, resulting in the pKn_ndh3 plasmid. An ampicillin-resistance
cassette was inserted into the pKn_ndh3 plasmid into the
SacI site of the ndh gene, and Ap-containing plasmid
(pKndhAp-1) bearing the ndh gene fragment together with
the unidirectionally transcribing ampicillin-resistance cassette was selected. This plasmid was transferred into the
K. pneumoniae KNU210 (nuoB<Cm) strain (Bertsova &
Bogachev, 2004) via conjugation using E. coli SM10lpir as
the donor, and a TcS CmR ApR RfR phenotype clone
(KND038, nuoB<Cm ndh<Ap) characteristic of a doublecrossover introduced mutation was selected. Localization of
the mutations in the K. pneumoniae chromosome was
verified by PCR analysis.
Measurement of activities
Preparation of sub-bacterial particles (SBP) from bacterial
cells. The cells were harvested by centrifugation (10 000 g,
10 min) and washed twice with medium 1 (250 mM KCl or
NaCl, 10 mM Tris-HCl, and 5 mM MgSO4, pH 8.0). The cell
pellet was suspended in medium 2 (20 mM HEPES-Tris,
5 mM MgSO4, 100 mM KCl, pH 8.0), and the suspension
was passed through a French press (16 000 psi). Unbroken
cells and cell debris were removed by centrifugation at
22 500 g (10 min), and the supernatant was further centrifuged at 180 000 g (60 min). The membrane pellet was
suspended in medium 2 at 20–30 mg protein mL1 and used
for measurements of activities.
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It is known that the respiratory chain of marine Vibrios,
along with Na1-NQR, also contains an additional NADHdehydrogenase of NDH-2 type (Hayashi et al., 1992), while
respiratory chains of K. pneumoniae and A. vinelandii, in
addition to Na1-NQR, also possess enzymes of NDH-1 as
well as NDH-2 type (Bertsova & Bogachev, 2004; C. Núñez
et al., unpublished data). In the present work, the NDH-2deficient strain of V. harveyi (NDKm34) and the double
NDH-2/NDH-1-deficient strain of K. pneumoniae
(KND038) were constructed. SBP isolated from these strains
oxidize NADH entirely via the corresponding Na1-NQR,
which makes them a useful tool to study the catalytic
properties of these enzymes. In the case of A. vinelandii the
previously obtained DN165 strain (Bertsova et al., 2001)
with disrupted gene of NDH-2 type enzyme was used.
Because the respiratory chain of this strain contains two
different NADH-dehydrogenases (NDH-1 and Na1-NQR),
in order to determine the specific Na1-NQR activity all
measurements on SBP from this strain were performed in
the presence of 4 mM rolliniastatin, which selectively inhibits
NDH-1 type enzymes (Belevich et al., 2007; C. Núñez et al.,
unpublished data). To test the possible effects of all inhibitors (activators) used on downstream to Na1-NQR respiratory chain complexes, the influence of these compounds
on NADH-oxidase activities of SBP from the Na1-NQRdeficient strains of V. harveyi, K. pneumoniae, and
A. vinelandii (strains R3A10, KNQ197, and GG4, respectively) was also studied.
Specificity of Na1 -NQRs to different reduced
pyridine dinucleotides
The Na1-NQRs from different marine Vibrios oxidize
NADH and dNADH, but cannot utilize NADPH with
measurable rates (Bourne & Rich, 1992; Zhou et al., 1999).
As can be seen from Table 2, the same results were obtained
for these enzymes from K. pneumoniae and A. vinelandii, i.e.
all the studied Na1-NQR-type enzymes possess the same
FEMS Microbiol Lett 279 (2008) 116–123
Catalytic properties of Na1-NQR
119
Table 2. NADH-, dNADH- and NADPH-oxidase activities of Na1-NQRs in
SBP isolated from different bacteria
SBP source
NADH
dNADH
V. harveyi
K. pneumoniae
A. vinelandii
0.8
0.24
1.1
0.9
0.26
1.2
NADPH
o 0.005
o 0.003
o 0.005
Na+ -NQR activity (%)
Na1-NQR activity
100
The activities were measured in medium 2 containing 50 mM NaCl and
are given in mmol of reduced pyridine dinucleotide oxidized min1 mg
protein1.
Dependence of activity of Na1 -NQRs on sodium
ion concentration
Na1-NQR is known to be absolutely specific to Na1, and in
the absence of sodium ions this enzyme is unable to reduce
ubiquinone (Hayashi et al., 2001b; Bogachev & Verkhovsky,
2005). Under physiological conditions (at high ionic
strength), the Km value for Na1 of this enzyme is about
3 mM. However, the KnNa values were determined only for
Na1-NQRs from different marine Vibrios (Unemoto et al.,
1977; Bogachev et al., 2001) and from the pathogenic
bacterium Haemophilus influenzae (Hayashi et al., 1996),
i.e. for the enzymes from microorganisms living at high Na1
concentration. The enterobacterium K. pneumoniae and
especially the soil bacterium A. vinelandii inhabit ecological
niches where Na1 concentrations can be very low. Thus, it
Na
values for the Na1-NQRs
was of interest to determine Km
from these bacteria and to compare them with such
properties of the enzyme from V. harveyi.
NADH-oxidase activities of SBP from the V. harveyi
(NDKm34), K. pneumoniae (KND038), and A. vinelandii
DN165 strains in contrast to NADH-oxidase activities of
SBP from the Na1-NQR-deficient strains of V. harveyi,
K. pneumoniae, and A. vinelandii (strains R3A10, KNQ197,
and GG4, respectively) were found to be completely sodium
dependent. The sodium ion concentration dependence of
activities of Na1-NQRs from all bacteria tested was hyperNa
for Na1-NQR from
bolic (Fig. 1). The value of apparent Km
V. harveyi was 2.7 mM. This value for the enzyme from
K. pneumoniae was lower (0.67 mM), while the Na1-NQR
from A. vinelandii was found to possess the highest affinity
to sodium ions with apparent Km 0.1 mM. Thus, there is
a correlation between the KnNa values of the Na1-NQRs and
the environmental Na1 concentration characteristic for the
corresponding microorganism, i.e. the Na1-NQRs from the
three bacteria tested are well adapted to the appropriate
in vivo conditions.
FEMS Microbiol Lett 279 (2008) 116–123
60
40
20
0
0
2
4
6
[NaCl] (mM)
8
10
12
Fig. 1. Na1 concentration dependence of activities of Na1-NQRs from
Vibrio harveyi (circles), Klebsiella pneumoniae (squares), and Azotobacter
vinelandii (triangles). The activities were measured in medium 2 containing
different concentrations of NaCl. Values of the maximal activities (100%)
are listed in Table 2.
100
Na+ -NQR activity (%)
substrate specificity with respect to reduced pyridine
dinucleotides.
80
80
60
40
20
0
0
2
1
3
4
[HQNO] (µM)
5
6
Fig. 2. Effects of HQNO on activities of Na1-NQRs from Vibrio harveyi
(circles), Klebsiella pneumoniae (squares), and Azotobacter vinelandii
(triangles). Activities were measured in medium 2 containing 50 mM
NaCl. Values of the maximal activities (100%) are listed in Table 2.
Inhibition of Na1 -NQRs by HQNO
HQNO is the most commonly used inhibitor of Na1-NQRs.
Although this compound is able to inhibit various quinoneoxidoreductases, the Na1-NQRs from different Vibrios are
sensitive to very low HQNO concentrations, thus allowing
its use as a specific inhibitor of this type of enzyme (Tokuda
& Unemoto, 1984; Zhou et al., 1999; Barquera et al.,
2002). In accordance with this, as can be seen in Fig. 2,
submicromolar HQNO concentrations inhibited NADHoxidase activities of SBP from V. harveyi (NDKm34) and
K. pneumoniae (KND038), while NADH-oxidase activities
of SBP from the Na1-NQR-deficient strains of V. harveyi
R3A10 (data not shown) and K. pneumoniae KNQ197
(Bertsova & Bogachev, 2004) were almost completely
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120
Inhibition of Na1 -NQRs by silver ions and by
N -ethylmaleimide (NEM)
It is well known that Na1-NQR from V. alginolyticus is
sensitive to submicromolar concentrations of Ag1 (Asano
et al., 1985). As can be seen in Fig. 3a, the NADH-oxidase
activity of SBP from the NDH-2-deficient strain of
V. harveyi (NDKm34) is arrested in the presence of 1 mM
Ag1, while the NADH-oxidase activity of SBP from the
Na1-NQR deficient strain of this bacterium (R3A10) is
resistant to this concentration of silver ions. Under the
experimental conditions of the authors V. harveyi Na1NQR was inhibited by Ag1 with apparent I0.5 0.1 mM.
The activity of Na1-NQR from A. vinelandii is considerably less sensitive to silver ions (Fig. 3a). Addition of 1 mM
Ag1 caused only partial and very slow inhibition of the
NADH-oxidase activity. In the case of K. pneumoniae the
Na1-NQR appears to be fully resistant to silver ions.
The same results were obtained using modification of
thiol groups. The activity of Na1-NQR from V. harveyi was
rapidly and selectively inhibited by 5 mM NEM (Fig. 3b).
This concentration of NEM inhibited Na1-NQR from
A. vinelandii only partially and very slowly, whereas the
activity of the enzyme from K. pneumoniae was almost
completely resistant to this compound.
As there was a correlation between inhibition of different
Na1-NQRs by Ag1 and NEM, it seems likely that Na1-NQR
inactivation by heavy metals ions is caused by modification
of some of its cysteine residue. Further work is required to
identify the reactive residue.
Inhibition of Na1 -NQRs by diphenyliodonium
It is well known that diphenyliodonium inhibits various
flavin-containing oxidoreductases because of covalent
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SBP
SBP
A
SBP
(a)
SBP
D
C
B
Ag+
Ag+
∆OD340 nm = 0.1
Ag+
Ag+
2 min
SBP
SBP
A
SBP
C
B
NEM
(b)
SBP
D
NEM
NEM
∆OD340 nm = 0.15
resistant to this inhibitor. Na1-NQR from V. harveyi was
inhibited by HQNO with I0.5 0.13 mM, while the affinity
of Na1-NQR from K. pneumoniae to this inhibitor was
slightly lower (I0.5 0.55 mM).
However, in the presence of rolliniastatin NADH-oxidase
activity of SBP from A. vinelandii DN165 was inhibited only
by micromolar concentrations of HQNO (Fig. 2). The same
HQNO concentrations inhibited NADH-oxidase activity of
SBP from A. vinelandii GG4, lacking Na1-NQR (data not
shown). Taken together, these results indicate that the Na1NQR from A. vinelandii, in contrast to the corresponding
enzymes from V. harveyi and K. pneumoniae, is resistant
to low HQNO concentrations. Because of the abovementioned properties of Na1-NQR from A. vinelandii (high
affinity to sodium ions and resistance to low HQNO
concentrations), this enzyme was not detected in the
authors’ early studies of the respiratory chain of this
bacterium (Bertsova et al., 1998).
M.S. Fadeeva et al.
NEM
4 min
Fig. 3. Inhibition by Ag1 (a) or by NEM (B) of NADH-oxidase activities
of (A) SBP from Vibrio harveyi R3A10 (Dnqr); (B) SBP from V. harveyi
NDKm34 (Dndh); (C) SBP from Azotobacter vinelandii DN165 (Dndh);
(D) SBP from Klebsiella pneumoniae KND038 (Dnuo Dndh). Activities
were measured in medium 2 containing 50 mM NaCl. In the case of
SBP from A. vinelandii DN165, the reaction medium was supplemented
with 4 mM of rolliniastatin. Additions: AgNO3, 1 mM; NEM 5 mM.
Transient absorption changes upon NEM additions were subtracted
for clarity.
modification of the isoalloxazine ring of the flavin prosthetic
group. This modification occurs only with the reduced, but
not with the oxidized form of the flavin (Chakraborty &
Massey, 2002).
The effect of DPI on Na1-NQR has not been studied
previously. In the present work it was shown that the
quinone-reductase activity of Na1-NQR from V. harveyi is
rapidly inhibited in the presence of 50 mM diphenyliodonium (Fig. 4b). The Na1-NQRs from K. pneumoniae and
A. vinelandii were also inhibited by diphenyliodonium in a
similar manner (data not shown). To study the effect of
diphenyliodonium on Na1-NQR in more detail, both SBP
from V. harveyi NDKm34 strain and a purified preparation
of this protein were used.
Na1-NQR contains at least three different flavin prosthetic
groups: one noncovalently bound FAD at the NADHFEMS Microbiol Lett 279 (2008) 116–123
Inhibition rate (s−1)
Catalytic properties of Na1-NQR
121
(a)
0.022
0.020
0.018
0.016
0.014
0.012
0.010
0.008
0.006
0.004
0.002
0.000
0
50
100
[DPI] (µM)
150
DPI K3 SBP DPI SBP
1500
200
(b)
800
∆OD340 nm = 0.2
K3
96
180
1 min
Fig 4. Inhibition of Vibrio harveyi Na1-NQR activities by diphenyliodonium (DPI). (a) Dependence of the rate of inhibition by DPI of NADHoxidase (squares) and NADH-dehydrogenase (circles) activities of Na1NQR upon inhibitor concentration. (b) NADH-oxidase and NADH:K3
oxidoreductase activities of SBP from V. harveyi NDKm34. Additions:
menadione (K3), 50 mM; DPI, 50 mM. Transient absorption changes upon
K3 additions were subtracted for clarity. Numbers indicate the specific
Na1-NQR activities in nmol of NADH oxidized min1 mg1 protein.
Activities were measured in medium 2 containing 50 mM NaCl.
dehydrogenase site of the enzyme (in subunit NqrF) and two
covalently bound FMN residues (in subunits NqrB and NqrC)
(Hayashi et al., 2001b). Thus, it was important to determine
which of these flavins is modified by diphenyliodonium.
As can be seen in Fig. 4a, the rate of inhibition of NADHoxidase activity of SBP from the NDH-2-deficient strain of
V. harveyi (NDKm34) depended linearly on diphenyliodonium concentration, yielding the inhibition rate constant of
420 M1 s1. The NADH-dehydrogenase activity of these
SBP was inhibited by diphenyliodonium more slowly, with
an inhibition rate constant of 80 M1 s1. These data may
indicate that the main target for modification by diphenyliodonium is located next to the NADH-dehydrogenase
site of the enzyme. However, the ratio between NADHdehydrogenase and NADH-oxidase activities was constant
over the course of the SBP incubation with NADH and
diphenyliodonium, i.e. in this case these two activities were
inhibited in a parallel way (as an illustration, see Fig. 4b).
These data imply that DPI inhibits the enzyme at the level of
FEMS Microbiol Lett 279 (2008) 116–123
its NADH-dehydrogenase site, and that the difference in the
values of the inhibition rate constant for NADH-dehydrogenase and quinone-reductase activities is related with
different steady-state levels of Na1-NQR reduction during
determination of these activities. Thus, it is most likely that
the main target for diphenyliodonium modification in Na1NQR is its noncovalently bound FAD. This is in accordance
with the fact that this prosthetic group takes electrons
directly from NADH (Turk et al., 2004), and thus it must
be accessible to the aqueous phase.
The electron paramagnetic resonance (EPR) spectra of
Na1-NQR preparations modified by diphenyliodonium
treatment was also determined. It was found that the full
inactivation of both the Na1-NQR enzymatic activities by
diphenyliodonium failed to change the spin concentration
in radical signals in oxidized as well as in reduced protein
samples (data not shown). This means that diphenyliodonium is unable to modify the covalently bound FMN
residues in Na1-NQR, apparently due to their inaccessibility
to the aqueous phase.
Conclusion
In this study the catalytic properties of Na1-NQRs from the
marine bacterium V. harveyi, the enterobacterium K. pneumoniae, and the soil microorganism A. vinelandii were
comparatively analyzed. It has been shown that these
enzymes have different sensitivity to inhibitors. Na1-NQR
from A. vinelandii is not sensitive to low HQNO concentrations, while Na1-NQR from K. pneumoniae is fully resistant
to either Ag1 or NEM. It was shown previously that
korormicin, a strong inhibitor of Na1-NQR from Vibrio
alginolyticus, has no effect on the homologous enzyme from
H. influenzae (Hayashi et al., 2002). All tested Na1-NQR
type enzymes were sensitive to diphenyliodonium, but its
effect (and that of its close analog diphenyleneiodonium) is
not very specific. For example, diphenyleneiodonium has
been shown to inhibit enzymes of NDH-1 as well as NDH-2
types (Majander et al., 1994; Roberts et al., 1995). Thus, the
main unique characteristic that distinguishes Na1-NQR
from other NADH-dehydrogenases is its specific requirement for sodium ions. However, it is noteworthy that even
this property of Na1-NQR can be not readily detectable,
because the affinity of this enzyme to Na1 can be very high,
especially in the case of enzymes from bacteria living at low
concentrations of this ion.
Acknowledgements
This work was supported by the Russian Foundation for
Basic Research (grant 07-04-00619). The authors thank
Prof. E.K. Ruuge for assistance with the EPR experiments.
2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
122
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