(Received for publication, April 3, 1995; and in revised form, May 4, 1995)
From the
The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans is composed of at least 14 dissimilar
subunits which are designated NQO1-14 and contains one
noncovalently bound FMN and at least five EPR-visible iron-sulfur
clusters (N1a, N1b, N2, N3, and N4) as prosthetic groups. Comparison of
the deduced primary structures of the subunits with consensus sequences
for the cofactor binding sites has predicted that NQO1, NQO2, NQO3,
NQO9, and probably NQO6 subunits are cofactor binding subunits.
Previously, we have reported that the NQO2 (25 kDa) subunit was
overexpressed as a water-soluble protein in Escherichia coli and was found to ligate a single [2Fe-2S] cluster with
rhombic symmetry (g
It is generally accepted that there are two types of
NADH-quinone (Q) ( The Paracoccus
denitrificans NDH-1 is one of the well characterized enzyme
complexes of bacterial NDH-1. It has been isolated from membrane and
characterized (Yagi, 1986), and was shown to be similar to the
mitochondrial Complex I in terms of enzymatic properties and immuno
cross-reactivity. The structural genes encoding the P.
denitrificans NDH-1 have been cloned and sequenced (Xu et
al., 1991a, 1991b, 1992a, 1992b, 1993). It was found that these
genes constitute a cluster composed of 14 structural genes and 6
unidentified reading frames (URFs), which are designated NQO1-14 and URF1-6, respectively (Yagi et
al., 1992). All of the 14 subunits are highly homologous to their
counterparts in bovine heart Complex I, which is composed of at least
41 subunits (Walker, 1992). In addition, the P. denitrificans NDH-1 shows striking EPR spectral similarity to the bovine heart
Complex I. These facts make the P. denitrificans NDH-1 a
useful model system for study of the structure and mechanism of action
of mitochondrial Complex I. In order to elucidate the electron
transfer reaction of the enzyme complex, it is a prerequisite to
identify the number and location of its redox components. It has been
shown that the P. denitrificans NDH-1 contains one FMN (Yagi,
1986) and at least 5 EPR-visible iron-sulfur clusters analogous to
those in bovine heart Complex I (Meinhardt et al., 1987).
These iron-sulfur clusters are tentatively designated as N1a, N1b, N2,
N3, and N4. The N1a and N1b are [2Fe-2S] clusters, while all
others are [4Fe-4S] clusters. On the basis of sequence
comparison of the deduced primary structures of the P.
denitrificans NDH-1 subunits with their homologues regarding
iron-sulfur cluster binding sites (Matsubara and Saeki, 1992), and of
the resolution data of the bovine Complex I (Hatefi, 1985), NQO1, NQO2,
NQO3, NQO9, and possibly NQO6 have been suggested to contain
iron-sulfur clusters (Yagi, 1993; Yagi et al., 1993).
Recently, Yano et al. (1994a) have succeeded in overexpressing
the NQO2 (25 kDa) subunit in Escherichia coli. The expressed
NQO2 subunit has been purified and characterized by UV-visible and EPR
spectroscopy, which revealed that the NQO2 subunit ligates a single
[2Fe-2S] cluster with rhombic EPR signal (g The NQO3 (66 kDa) subunit, which is
homologous to the 75-kDa subunit of the iron-sulfur (IP) subcomplex of
Complex I, is also a putative iron-sulfur cluster ligating subunit.
Therefore, we attempted to express the NQO3 subunit in E. coli in hopes that the NQO3 subunit might be natively expressed, as was
the NQO2 subunit, and might better lend itself to a study of the
properties of its iron-sulfur cluster(s). In the present paper we
report the characterization of the P. denitrificans NQO3
subunit expressed in E. coli using pET11a expression vector.
The expressed subunit was found predominantly in the cytoplasm and
contains iron-sulfur clusters. The subunit was purified and then
subjected to chemical analysis, UV-visible and EPR measurements to
characterize the iron-sulfur clusters. We found that the NQO3 subunit
contains at least one [2Fe-2S] and one [4Fe-4S]
cluster. The assignment of these iron-sulfur clusters to a specific
electron-transfer components of NDH-1 and possible functional role of
the NQO3 (66 kDa) subunit are discussed.
Figure 1:
Localization of the expressed NQO3 (66
kDa) subunit. The Coomassie Brilliant Blue-stained SDS-polyacrylamide
gel (panel A) and immunoblotting (Panel B). The lanes
contains cell lysate of E. coli pET(NQO3) (12 µg of
protein for lane A1 and 1.2 µg for lane B1),
soluble fraction (7.5 µg of protein for lane A2 and 0.75
µg for lane B2), and membrane fraction (2.1 µg of
protein for lane A3 and 0.21 µg for lane B3).
Immunoblotting was carried out using affinity-purified antibody against
the bovine 75-kDa subunit and alkaline phosphate-conjugated anti-rabbit
IgG antibody as described (Han et al., 1988, 1989; Hekman et al., 1991). The numbers at the left indicate the molecular mass in kilodalton and the arrows at the right indicate the expressed NQO3
subunit.
The expressed
NQO3 subunit is found mainly in the soluble fraction, whereas the yield
of the product in the membrane fraction is extremely low and could be
detected only immunologically (Fig. 1). The NQO3 subunit was
purified from the cytoplasmic fraction to nearly homogeneous form (see
``Experimental Procedures''), as revealed by
SDS-polyacrylamide gel electrophoresis and by immunoblotting with
antibody against bovine 75-kDa subunit (Fig. 2). Neither
detergents nor chaotropic agents were required for the isolation. The
yields of the purification steps are summarized in Table 1.
Approximately 80 mg of purified protein was obtained from 100 g (wet
weight) of E. coli cells. The N-terminal amino acid sequence
of the purified expressed subunit is equivalent to that of the NQO3
subunit isolated from the P. denitrificans NDH-1 (A-D-L-R-K).
The expressed subunit lacks the initial methionine which appears to be
removed post-translationally in E. coli as well as in P.
denitrificans cells (Xu et al., 1992a). The amino acid
composition of the expressed NQO3 subunit (data not shown) is in
reasonably good agreement with the composition deduced from the NQO3 gene sequence and also as determined by the amino acid
analysis of the NQO3 subunit isolated from the P. denitrificans NDH-1 enzyme (Xu et al., 1992b). In addition, the
identity of NQO3 gene product expressed in E. coli and the
66-kDa subunit isolated from P. denitrificans NDH-1 was
confirmed by SDS-polyacrylamide gel electrophoresis.
Figure 2:
SDS-PAGE and immunological analyses of
samples from different stages of purification of the NQO3 (66 kDa)
subunit. Panel A shows a Coomassie Brilliant Blue-stained
SDS-polyacrylamide gel and panel B shows an immunoblotting
using affinity-purified antibody against the bovine 75-kDa subunit.
Lanes contain cell lysate of E. coli pET(NQO3) (10.6 µg of
protein, lane A1; 1.1 µg of protein, lane B1),
soluble fraction (8.2 µg of protein, lane A2; 0.8 µg
of protein, lane B2), (NH
In order to
examine whether the soluble NQO3 subunit exists as a monomer or an
oligomer in solution without detergents, we estimated the apparent
molecular size of the purified NQO3 subunit by gel filtration
chromatography. The apparent molecular size of the NQO3 subunit was
determined as approximately 140 ± 10 kDa, indicating that the
soluble NQO3 subunits form dimers under the conditions used. No
discernible amounts of more aggregated structures could be detected
from the elution profile (data not shown). The cytoplasmic fraction
and (NH Fig. 3shows the absorption spectra of the
oxidized and reduced form of the expressed NQO3 subunit purified from
the cytoplasmic fraction. The purified subunit shows broad absorption
peaks at 320 and 417 nm in the oxidized form. In addition, it shows
small shoulders around 460 and 550 nm. When the subunit is reduced by
adding neutralized dithionite solution (5 mM final
concentration), the color is bleached in the entire visible region. In
the reduced form, a small broad peak is detected around at 550 nm.
These results indicated that the expressed NQO3 subunit contains
reducible iron-sulfur clusters in the molecule.
Figure 3:
Optical absorption spectra of the purified
NQO3 (66 kDa) subunit. The purified NQO3 subunit was diluted in 20
mM Tris-HCl (pH 8.5) containing 2.0 mM DTT and 1.0
mM EDTA and 0.1 mM PMSF to 1.25 mg/ml. The spectra
were recorded at room temperature in the oxidized form (line
A) and in the reduced form (line B). The sample was
reduced by 5 mM dithionite (pH 7.5). The difference spectrum
(oxidized form minus reduced form) is shown in the inset.
Figure 4:
EPR spectra of the iron-sulfur clusters of
the expressed NQO3 (66 kDa) subunit of P. denitrificans NDH-1 (solid line) and their computer simulation (dashed
line). The iron-sulfur clusters in the sample were
potentiometrically poised to the E
To further characterize these two iron-sulfur clusters,
a potentiometric redox titration was conducted. The E
Figure 5:
Potentiometric redox titration of the
iron-sulfur clusters in the expressed NQO3 (66 kDa) subunit. The
partially purified NQO3 subunit (after DEAE-Toyopearl chromatography)
at 10 mg/ml in 50 mM Tris-HCl (pH 8.6), containing 1.0 mM DTT and 0.1 mM PMSF was titrated anaerobically at 15
°C in the presence of the following redox mediator dyes:
2-hydroxy-1,4-naphthoquinone, 1,2-naphthoquinone-4-sulfonate,
1,4-naphthoquinone-2-sulfonate, indigo disulfonate, indigo
trisulfonate, indigo tetrasulfonate, safranin T, phenosafranin, neutral
red, methyl viologen, and benzyl viologen (50 µM each).
The reduction of the clusters was monitored by the signal amplitude at g = 1.93 at 35 K and 10-mW microwave power for the
[2Fe-2S] cluster (▴) and at g = 1.89
signal at 10 K and 5-mW microwave power for the [4Fe-4S]
cluster (▪). Other EPR conditions were as described in the legend
to Fig. 4.
In the
ferricyanide-oxidized NQO3 subunit, a characteristic EPR spectrum of an
oxidized [3Fe-4S] iron-sulfur cluster was detected. Relative
concentration of this cluster varied depending on preparation,
ferricyanide concentration, and other experimental conditions. In order
to clarify whether this cluster is an intrinsic component of the
protein or it represents a product of oxidative deterioration of the
tetranuclear cluster, we have studied the EPR spectra of the
[3Fe-4S] and the [4Fe-4S] clusters after different
incubation periods of the NQO3 subunit with 0.1 mM ferricyanide (Fig. 6, A and B). As shown
in Fig. 6, the g = 2.01 signal of the
[3Fe-4S] cluster increased with incubation time.
Concomitantly, the content of the [4Fe-4S] cluster decreased,
which was measured after reduction of the samples with dithionite (in
the presence of redox mediators). These results strongly indicate that
the [3Fe-4S] cluster observed in the preparation is not
another component of the NQO3 subunit but rather originates from the
oxidant-induced conversion of the native [4Fe-4S] cluster
into a [3Fe-4S] cluster in a process similar to that
described for a number of bacterial ferredoxins (Bell et al.,
1982; Guigliarelli et al., 1985; Morgan et al., 1984;
Thomson et al., 1981).
Figure 6:
Ferricyanide-induced conversion of
[4Fe-4S] to [3Fe-4S] cluster in the expressed NQO3
(66 kDa) subunit. EPR spectra of oxidized (A) and
dithionite-reduced (B) purified NQO3 subunit before, 1 and 10
min after the addition of ferricyanide. C, changes in the
concentration of [3Fe-4S] and [4Fe-4S] cluster
during the time course of the incubation of NQO3 subunit with
ferricyanide. Ferricyanide (0.5 mM) was added to the anaerobic
solution of the purified NQO3 subunit (5 mg/ml) (50 mM Tris-HCl, pH 8.0, 0.2 mM EDTA, 0.1 mM PMSF, 40
µM methyl viologen, and 4 µM phenazine
methosulfate), incubated in the EPR tube for the indicated time periods
at +5 °C and frozen immediately before or after reduction with
10 mM dithionite. Spectra were recorded at 10 K. EPR
conditions: microwave power, 5 mW; modulation amplitude, 8 G; microwave
frequency, 9.46 GHz. The [3Fe-4S] and [4Fe-4S]
clusters were monitored as g = 2.01 signal amplitude in
the spectra of oxidized protein, and g = 1.89 signal
amplitude of the dithionite-reduced samples, respectively. The small
difference in the EPR line shape of [4Fe-4S] cluster is due
to difference in the reduction level of the [2Fe-2S] cluster
which is contributing to g
Iron-sulfur cluster contents
determined by EPR spin quantitation are approximately 0.8 mol of each
cluster per subunit. Therefore, although the expressed NQO3 subunit is
present as a homodimer, these results exclude the possibility that the
iron-sulfur clusters are coordinated between two NQO3 subunit as
exemplified by photosystem I (Golbeck, 1992) and nitrogenase iron
protein (Georgiadis et al., 1992). Furthermore, these results
also exclude the possibility that the binuclear and the tetranuclear
clusters of the expressed NQO3 subunit share the ligand residues.
Therefore, it is likely that the NQO3 (66 kDa) subunit houses at least
one [2Fe-2S] and one [4Fe-4S] clusters in the
molecule. Based on the deduced primary structure of the P.
denitrificans NQO3 subunit and its homologues, we anticipated that
the NQO3 subunit carries plural iron-sulfur clusters (Xu et
al., 1992a). Early EPR studies of the IP fragment as a whole,
isolated from the bovine Complex I, containing the 75-kDa subunit (the
homologue of the NQO3 subunit) together with six other water soluble
polypeptides (Masui et al., 1991), showed two discernible
axial-type EPR signals with largely different spin relaxation behavior,
arising from at least one binuclear and one tetranuclear iron-sulfur
clusters with g The NQO3 subunit contains 12 cysteine
(Cys-26, -37, -48, -51, -66, -110, -113, -119, -158, -161, -164, and
-208) and 2 histidine residues (His-39 and -106) (Xu et al.,
1992a) that are conserved in complex I from various sources. Four
cysteines (Cys-158, -161, -164, and -208) constitute a consensus
sequence motif for the ligation of a [4Fe-4S] cluster
(Cx Generally speaking, 12 cysteines and 2 histidines in NQO3 provide
enough room for the ligation of three iron-sulfur clusters. In
addition, the amount of non-heme iron and acid-labile sulfide of the
NQO3 subunit (8 mol of Fe/mol and 9 mol of S Since the N3 cluster is most likely
accommodated in the NQO1 (50 kDa) subunit (Ohnishi et al.,
1981; Fecke et al., 1994; Sled' et al., 1994),
the [4Fe-4S] cluster in the NQO3 subunit may be assigned to
the N4 cluster. Two [2Fe-2S] clusters referred to as N1a and
N1b are present in the P. denitrificans NDH-1 (Meinhardt et al., 1987). The N1a cluster exhibits an EPR spectrum with
rhombic symmetry whereas the EPR spectrum of N1b cluster has an axial
symmetry. Based on the EPR properties of the [2Fe-2S]
clusters in the expressed NQO2 and NQO3 subunits, we could tentatively
assign the N1a and N1b clusters to the NQO2 and NQO3 subunits,
respectively. However, the midpoint redox potentials of the iron-sulfur
clusters in the overexpressed subunits are considerably lowered in
comparison with those in the complex. Since the subunits are solely
expressed in E. coli in the absence of neighboring subunits,
the environment of the iron-sulfur clusters might be different from
that in the native enzyme complex. The midpoint redox potential of
iron-sulfur clusters is especially sensitive to slight changes in the
vicinity of the clusters (e.g. accessibility of external
solvent). Substantial lowering of the midpoint redox potentials of
iron-sulfur clusters has also been reported for the resolved FP
subcomplex of bovine Complex I (Ohnishi et al., 1985). As
described above, all iron-sulfur clusters seem to be located in the
N-terminal region (residues 1-265) of the NQO3 subunit. It has
been reported that the N-terminal region of the NQO3 subunit is
structurally similar to the The iron-sulfur cluster
domain of the NQO3 subunit seems to interact with FP, most likely with
the NQO1 subunit. The evidence has been provided by cross-linking
experiments of bovine heart Complex I (Patel et al., 1988a,
1988b; Yamaguchi and Hatefi, 1993), which indicates that the 75-kDa
subunit is cross-linked with the 51-kDa subunit of FP (NQO1 subunit
homologue). In addition, presuming the topological similarity between
the P. denitrificans NDH-1 and the bovine heart Complex I, it
can be speculated that the NQO3 subunit plays a structural role in
connecting FP and HP with other IP subunits. Price and Gomer(1989) have
purified the 75-kDa subunit of Complex I from bovine ventricular
myocardium using a method for the isolation of the cytoskeletal
component desmin. The isolated 75-kDa subunit was found to polymerize
into the 10-nm filamentous structure in vitro. On the basis of
these results, these authors proposed that the 75-kDa subunit functions
as a membrane-associated filamentous skeleton. It is probable that this
characteristic results from the relatively hydrophobic C-terminal
region (residues 266-673) of the NQO3 subunit which may be
involved in the interaction of its hydrophilic section of the Complex I
or NDH-1 with its hydrophobic section (HP subunits). Taken together, it
is suggested that the NQO3 subunit acts as the linking protein among
FP, IP, and HP. This hypothesis should be experimentally examined in
the future.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
= 1.92, 1.95, and
2.00) (Yano, T., Sled', V. D., Ohnishi, T., and Yagi, T.(1994) Biochemistry 33, 494-499). In the present study, the
NQO3 (66 kDa) subunit, which is equivalent to the 75-kDa subunit of
bovine heart Complex I, was overexpressed in E. coli. The
expressed NQO3 subunit was found predominantly in the cytoplasmic phase
and was purified by ammonium sulfate fractionation and anion-exchange
chromatography. The chemical analyses and UV-visible and EPR
spectroscopic studies showed that the expressed NQO3 subunit contains
at least two distinct iron-sulfur clusters: a [2Fe-2S]
cluster with axial EPR signals (g
= 1.934 and 2.026, and L
= 1.8 and 3.0 millitesla) and a [4Fe-4S] cluster
with rhombic symmetry (g
= 1.892,
1.928, and 2.063, and L
= 2.40, 1.55,
and 1.75 millitesla). The midpoint redox potentials of
[2Fe-2S] and [4Fe-4S] clusters at pH 8.6 are
-472 and -391 mV, respectively. The tetranuclear cluster in
the isolated NQO3 subunit is sensitive toward oxidants and converts
into [3Fe-4S] form. The assignment of these iron-sulfur
clusters to those identified in the P. denitrificans NDH-1
enzyme complex and the possible functional role of the NQO3 subunit is
discussed.
)oxidoreductase in bacterial respiratory
chains (see Yagi(1993)). One is not an energy-coupling enzyme and is
designated NDH-2. The NDH-2 enzyme contains only a noncovalently bound
FAD as a cofactor, and is composed of a single polypeptide (Jaworowski et al., 1981). The enzymes capable of energy transformation
can be further divided into two types. One type, designated as
Na
-NDH, has been found in marine alkalophilic microbes
and is capable of translocating sodium ion as a coupling ion. It is
composed of three different subunits and contains one FMN and one FAD
as prosthetic groups (see Unemoto and Hayashi (1993)). The other type
represents the multisubunit enzyme complex which can function as a
proton pump and generates an electrochemical proton gradient across the
plasma membrane, and is designated NDH-1. The NDH-1 contains a
noncovalently bound FMN and several iron-sulfur clusters as cofactors
and is composed of at least 14 dissimilar subunits (Yagi et
al., 1992, 1993; Weidner et al., 1993; Leif et
al., 1995). In terms of polypeptide composition, cofactors, and
enzymatic properties, the bacterial NDH-1 enzyme complex appears to be
the counterpart of the mitochondrial proton-translocating
NADH-ubiquinone oxidoreductase (Complex I).
= 1.92, 1.95, and 2.00). Further
spectroscopic studies including CD, MCD, and resonance Raman
spectroscopy have confirmed that the [2Fe-2S] cluster in the
NQO2 subunit is coordinated by cysteine residues (Crouse et
al., 1994). More recently, site-directed mutagenesis studies have
indicated that four conserved cysteine residues (Cys-96, -101, -137,
and -141) coordinate the [2Fe-2S] cluster with a novel
binding motif (Cx
Cx
Cx
C) (Yano et al., 1994b).
Construction of Expression Vector
A full-length
NQO3 gene was constructed from two plasmids, pXT-1 and pXT-2, as
follows: a PstI/SphI fragment encoding the C-terminal
region of NQO3 subunit was taken from pXT-2 and ligated into PstI/SphI cleaved pTZ18U. The plasmid thus obtained
was designated pTZ18(NQO3c). In addition, a PstI fragment
encoding the remaining N-terminal region of NQO3 subunit was cleaved
from pXT-1 and ligated into PstI cleaved pTZ18(NQO3c). The
proper construct was confirmed by cleavage with several restriction
enzymes and finally by sequencing around the PstI ligation
site (Sanger et al., 1977). The resulting construct was
designated pTZ18U(NQO3). An oligonucleotide
5`-GATCTGCCATATGGTCTTCCCTT-3` (the italic and
underlined bases were altered from the P. denitrificans DNA
for the mutation) was designed in order to generate an NdeI
site at the protein initiation codon. The mutagenesis was performed
using the Bio-Rad in vitro mutagenesis kit based on the method
of Kunkel et al.(1987). The mutation was confirmed by DNA
sequence analysis of the region surrounding the initiation site. In
order to avoid sequencing of the entire region, the BglII/BamHI fragment of the mutated plasmid was
replaced with the BglII/BamHI fragment from
pTZ18U(NQO3) plasmid and the resulting plasmid was designated
pTZ18U(NQO3-NdeI). The NdeI/BamHI fragment
from pTZ18U(NQO3-NdeI) was ligated in pET11a expression vector
cleaved with NdeI/BamHI. The final construct was
designated pET11a(NQO3) and was used for expression of the NQO3 subunit
in E. coli.Expression of NQO3 (66 kDa) Subunit
Expression of
intact NQO3 subunit is highly dependent on the following conditions:
bacterial strain, medium content, growth temperature, and induction
time. Temperature is one of the key factors in obtaining the soluble
intact products. In addition, the iron-sulfur cluster content of the
expressed subunits is significantly affected by timing of induction
with IPTG. The optimal expression procedure is as follows. Competent E. coli strain BL21(DE3)pLysS was transformed with
pET11a(NQO3) and spread onto a 2xYT agar plate containing 50 µg/ml
carbenicillin. A well isolated colony was picked and used to inoculate
10 ml of 2xYT medium containing 100 µg/ml ampicillin and cultivated
at 37 °C to the stationary phase. The culture was used to inoculate
500 ml of TB medium containing 100 µg/ml ampicillin, 100 µg/ml
ammonium Fe(II) citrate, and 100 µM sodium sulfide. Cells
were grown at 25 °C until absorbance (at 600 nm) of the culture
reached approximately 0.1. IPTG was then added to 0.4 mM final
concentration and the cells were further grown for 18 h at 25 °C.
The cells were harvested by centrifugation at 6,000 rpm for 10 min in a
GSA rotor. The cell precipitates were suspended in 50 mM Tris-HCl buffer (pH 8.5) containing 1.0 mM EDTA, 0.1
mM PMSF, 1 mM DTT, and 150 mM NaCl to 20%
(w/v). The cell suspensions were rapidly frozen in liquid nitrogen, and
stored at -20 °C until use.Purification of NQO3 (66 kDa) Subunit
The cell
suspension was freeze-thawed twice using liquid nitrogen and a water
bath at 30 °C and then sonicated for a few minutes on ice until its
viscosity was reduced. After that, all procedures were performed at 4
°C. The suspension was treated twice with Parr cell disruption bomb
at >1,000 kg/cm. The suspension was centrifuged at 6,000
rpm for 10 min in SS34 rotor in a Sorvall centrifuge to remove unbroken
cells. The resulting supernatant was further centrifuged at 50,000 rpm
for 30 min in a 60Ti rotor to separate soluble and membrane fractions.
The supernatant was transferred into a beaker and the protein
concentration was adjusted to approximately 5 mg/ml with buffer A (10
mM Tris-HCl, pH 8.5, 1 mM EDTA, 0.1 mM PMSF,
and 1 mM DTT) plus 150 mM NaCl. The suspension was
fractionated with (NH
)
SO
, and the
fraction precipitating between 30 and 38%
(NH
)
SO
saturation was collected at
10,000 rpm for 15 min in SS34 rotor. The dark brown precipitate was
suspended in 10 ml of buffer A and dialyzed against 1 liter of buffer A
for 3 h. The dialyzed solution was applied on a DEAE-Toyopearl column
(3.0
15 cm) equilibrated with buffer A. The column was washed
with 10 column volumes of buffer A. The protein was eluted with a
linear gradient of 0-0.5 M NaCl in buffer A. The
fractions containing the NQO3 subunit were collected and dialyzed
against 1 liter of buffer A for 3 h. The solution was applied on a
QAE-Toyopearl column (3.0
10 cm) equilibrated with buffer A.
After washing the column with 10 column volumes of buffer A followed by
10 column volumes of buffer A containing 0.05 M NaCl, the
adsorbed proteins were eluted with a linear 400-ml gradient of
0.05-0.7 M NaCl in buffer A. The fractions containing
the NQO3 subunit were combined and concentrated by Amicon
Centriprep-30.
Gel Filtration Analysis
The purified NQO3 subunit
(0.5 mg) was applied to a gel filtration column (Bio-Rad A-5m, 1.0
45 cm) equilibrated with 10 mM Tris-HCl buffer (pH
8.5), containing 1 mM DTT and 300 mM NaCl and eluted
at a flow rate of 0.5 ml/min. The eluant was monitored at 280 nm with
Pharmacia LKB Uvicord SII monitor. Molecular size standards used were
-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum
albumin (68 kDa), and ovalbumin (43 kDa).
Sequence Analysis
GCG software programs were used
to analyze the amino acid sequence (Devereux et al., 1984).
Sequence comparison of the polypeptides were conducted with BESTFIT and
PILEUP programs. The FASTA and PROFILESEARCH programs were used to
search for homology with the GenBank/EMBL sequence data bases. Homology
search was also carried out by using the BLAST program running at the
National Center for Biotechnology Information (Altschul et
al., 1990).EPR Spectroscopy
EPR measurements were conducted
with a Bruker ESP300 E spectrometer operating at X-band (9.2 GHz). The
sample temperature was controlled with an Oxford instrument ESR-9
helium flow cryostat. The magnetic field was calibrated using a strong
or a weak pitch standard. Spin quantitations were conducted under
non-power saturated conditions using 0.5 mM Cu-EDTA as a
standard. The transition-probability corrections were made according to
Aasa and Vnng(1975). EPR spectral
simulations were performed as described by Blum and Ohnishi(1980)
assuming Gaussian line shapes and no hyperfine interactions.
Potentiometric redox titrations were done according to Dutton(1978).Other Analytical Procedures
UV-visible absorption
spectra were recorded on an SLM-Aminco DW-2000 spectrophotometer.
Protein was estimated by the methods of Lowry et al.(1951) and
Biuret in the presence of sodium deoxycholate (1 mg/ml) (Gornall et
al., 1949). SDS-polyacrylamide gel electrophoresis was carried out
by the modified method of Laemmli(1970). Immunoblotting was conducted
as described (Han et al., 1988, 1989; Hekman and Hatefi, 1991;
Hekman et al., 1991). Non-heme iron and acid labile sulfide
were determined according to Doeg and Ziegler(1962) and Forgo and
Popowski(1949), respectively. Amino acid composition (Yagi and Dinh,
1990), amino acid sequence (Matsudaira, 1987), and DNA sequences
(Sanger et al., 1977) were determined according to the
references cited. Any variations from the procedures and other details
are described in the figure legends.Materials
Acrylamide, N,N`-methylenebis(acrylamide), SDS, prestained low-range
marker proteins, and Coomassie Brilliant Blue R-250 were from Bio-Rad;
DNA sequencing kit was from U. S. Biochemical Corp;
[-
S]thio-dATP was from Amersham; alkaline
phosphatase-conjugating affinity purified antibodies to rabbit IgG were
from Calbiochem; expression vectors and E. coli strain were
from Novagen; carbenicillin was from ICN. The antiserum against the
bovine 75-kDa subunit was a kind gift from Dr. Maureen G. Price (Rice
University, Houston).
Expression of the NQO3 (66 kDa) Subunit in E.
coli
We expressed the P. denitrificans NQO3 subunit as
a non-fused protein in E. coli cells with pET11a vector (see
``Experimental Procedures''). The expression of intact NQO3
subunit is highly dependent on the growth conditions. When the
expression is induced by the addition of IPTG at 37 °C, the NQO3
subunit is overexpressed as inclusion bodies. However, when the growth
temperature was decreased to 25 °C, the subunit remained in the
cytoplasmic phase. A similar effect was also observed previously in the
case of the P. denitrificans NQO2 subunit (Yano et
al., 1994a). These phenomena could be explained by the fact that
bacterial cells grow slower at lower temperatures so that the expressed
product may be given enough time for proper folding and cofactor
incorporation. We have tried to express the NQO3 subunit in several
different forms: fused-protein with His Tag sequence in the N terminus
or C terminus, and in truncated forms with different polypeptide length
(data not shown). However, in all of these attempts we failed to obtain
comparable amounts of soluble products, and inclusion bodies were
formed even at lower culture temperatures. Moreover, the iron-sulfur
cluster content of the products in the soluble fraction was very low
(data not shown). Probably, a full-length and non-fused form of the
NQO3 subunit is essential for the proper polypeptide folding and
cofactor incorporation. Fig. 1shows typical results of
expression of NQO3 subunit in E. coli under optimal growth
conditions. The overproduced NQO3 subunit constitutes up to 10% of the
total soluble proteins in E. coli (Fig. 1A).
The expressed product is readily recognized by anti-bovine 75-kDa
antibody (Fig. 1B), which has been previously shown to
cross-react with the NQO3 subunit of the P. denitrificans NDH-1 enzyme complex (Xu et al., 1992a).
)
SO
fraction (30-38% saturation) (3.4 µg of protein, lane A3), after DEAE-Toyopearl chromatography (1.4 µg of
protein, lane A4), and after QAE-Toyopearl chromatography (1.0
µg of protein, lane A5; 0.1 µg of protein, lane
B5). The numbers at the left indicate the
molecular mass in kilodalton and the arrows at the right indicate the NQO3 subunit.
)
SO
precipitate prepared
from E. coli cells with pET11a(NQO3) plasmid are dark-brown.
This brown color becomes more distinct during the subsequent
purification steps of DEAE- and QAE-Toyopearl column chromatography,
suggesting that the expressed NQO3 subunit contains iron-sulfur
clusters. We have determined the amount of non-heme iron and
acid-labile sulfide in the preparations at different purification steps
as shown in Table 1. After (NH
)
SO
fractionation, the non-heme iron and acid-labile sulfide contents
were almost equal. It should be noted that the supernatant prepared
from E. coli cells harboring pET11a expression plasmid without
the NQO3 gene contains much lower amounts of non-heme iron and
acid-labile sulfide and shows no EPR signals characteristic of
iron-sulfur clusters (data not shown). It seems likely that some part
of iron-sulfur clusters in the expressed NQO3 subunit are lost during
purification. As described below, we found that the [4Fe-4S]
cluster in the expressed NQO3 subunit is not very stable and some of
the clusters are converted to [3Fe-4S] clusters under aerobic
condition.
EPR Study of the Expressed NQO3 (66 kDa)
Subunit
In order to determine the type and number of iron-sulfur
clusters in the expressed NQO3 subunit, EPR measurements were carried
out. Because of some loss of iron-sulfur clusters in the expressed NQO3
subunit during the last purification steps, we have used for EPR
measurements the preparation after (NH)
SO
fractionation and DEAE-Toyopearl ion-exchange chromatography. In
the reduced NQO3 subunit we have detected EPR signals from two distinct
species of iron-sulfur clusters with different spin-relaxation
behaviors. One species exhibits a spectrum of axial symmetry, and is
detectable at sample temperatures as high as 77 K, indicating that it
is a binuclear [2Fe-2S]
cluster (Fig. 4A, solid line). The other
shows a rhombic EPR spectrum arising from a rapidly relaxing species,
most likely [4Fe-4S]
cluster (Fig. 4B, solid line). Table 2summarizes the half-saturation parameters of EPR signals
from these iron-sulfur clusters at various temperatures. The EPR
spectra of the individual clusters can be simulated with reasonable fit
using the following parameters: g
= 1.934, 2.026, L
= 1.8, 3.0 millitesla for the [2Fe-2S] cluster (Fig. 4A, dashed line); and g
= 1.892, 1.928, and 2.063, L
= 2.4, 1.55, and 1.75 millitesla for
the [4Fe-4S] cluster (Fig. 4B, dashed
lines).
of
-495 mV for [2Fe-2S] (A) and -464 mV for
[4Fe-4S] clusters (B). EPR spectra were recorded at
35 K and microwave power at 10 mW for the [2Fe-2S] cluster (A); and 10K, 5 mW for the [4Fe-4S] cluster (B). Computer simulation of the clusters was conducted as
described by Blum and Ohnishi(1980); parameters used were: g
= 1.934, 1.934, and 2.026 and L
= 17.9, 18.0, and 30.0 millitesla for
the [2Fe-2S] cluster and g
=
1.892, 1.928, and 2.063 and L
= 24.0,
15.5, and 17.5 millitesla for the [4Fe-4S] cluster. Other EPR
conditions: microwave frequency, 9.2506 GHz; modulation amplitude, 10
G; time constant, 64 ms.
values of the two iron-sulfur clusters
at pH 8.6 were determined to be -472 mV for the
[2Fe-2S] cluster and -391 mV for the [4Fe-4S]
cluster (Fig. 5). No additional EPR signals were detected down
to E
-600 mV. The relative
stoichiometry of [2Fe-2S]:[4Fe-4S] clusters has
been estimated as 1:0.84 on the basis of spin quantitation.
of the spectra. The
relative large g = 2.00 free radical signal originates
from the redox mediators.
values (2.02 and 2.06,
respectively) fairly close to those described in the present paper.
However, further chaotropic resolution of the IP fraction into smaller
fractions resulted in considerable broadening of the spectra and to a
loss of cluster-characteristic EPR properties (Ohnishi et al.,
1985). In contrast, the present work revealed that the expressed single
subunit of P. denitrificans NQO3 retained both iron-sulfur
clusters. This supports the single subunit expression strategy for the
assignment of the prosthetic groups to the NDH-1 subunits, as discussed
in a series of previous papers (Yano et al., 1994a, 1994b;
Crouse et al., 1994).
Cx
Cx
CP). Therefore, it
seems likely that these cysteine residues coordinate the
[4Fe-4S] cluster of the NQO3 subunit. The deduced primary
sequence of the NQO3 subunit does not contain any cysteine sequence
motifs for the [2Fe-2S] cluster ligation: the plant-type
ferredoxins (Cx
Cx
Cx
C),
hydroxylase-type ferredoxins
(Cx
Cx
Cx
C), the NQO2
subunit (Cx
Cx
Cx
C), or the Rieske
iron-sulfur proteins (CxHx
Cx
H)
(Matsubara and Saeki, 1992; Yano et al., 1994b; Gray and
Daldal, 1995). To date, cysteine and histidine residues are the only
residues which have been experimentally shown to coordinate
[2Fe-2S] clusters in vivo. The ligand residues are
most likely conserved in various organisms. It is thus conceivable that
four out of the remaining 10 conserved residues (Cys-26, -37, -48, -51,
-66, -110, -113, -119, His-39 and -106) are likely candidates for the
[2Fe-2S] cluster ligation. Therefore, identification of the
amino acid residues coordinating the [2Fe-2S] cluster of the
NQO3 subunit would provide not only new information about the structure
of the NQO3 subunit, but also a unique sequence motif for the
[2Fe-2S] cluster ligation. This would provide additional
support for the notion that NDH-1 and Complex I are the most elaborate
of all known iron-sulfur proteins (Cammack, 1992; Johnson, 1994). In
order to identify the ligand residues of the [2Fe-2S] and
[4Fe-4S] clusters of the NQO3 subunit, site-directed
mutagenesis experiments are in progress in our laboratories.
/mol) is
approximately 1.8-fold higher than those based on the spin
concentration by EPR measurements (4.8 mol of Fe/mol and 4.8 mol of
S
/mol). These facts suggest the possible existence
of a third iron-sulfur cluster in the expressed NQO3 subunit. Bovine
heart Complex I is also considered to contain EPR-silent iron-sulfur
clusters (Hatefi, 1985). In order to verify this issue, further
characterization of the NQO3 subunit by EPR, MCD, and resonance Raman
spectroscopy is underway in collaboration with Dr. Michael K.
Johnson's group.
-subunit of Alcaligenes eutrophus NAD
-reducing hydrogenase which contains 11 of 12
cysteine residues conserved among the NQO3 subunit and its homologues
(Xu et al., 1992a). The
-subunit is thought to bear
iron-sulfur cluster(s) and to participate in wiring electrons from the
hydrogenase part of
-heterodimer subunits to the NADH-binding
site of
-subunit (Schneider et al., 1984; Tran-Betcke et al., 1990). Taken together, it is conceivable that the
N-terminal region (iron-sulfur cluster domain) of the NQO3 subunit may
participate in electron transfer reaction.
We thank Catherine Guffey and Daniel G. Owen for their
excellent technical assistance, Drs. Saeko Takano and Akemi
Matsuno-Yagi for stimulating discussion, and Drs. Youssef Hatefi and
Cecilia Hgerhll for critical
reading of the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.