(Received for publication, October 6, 1995; and in revised form, December 24, 1995)
From the
This study reports the expression of the flavoprotein (FP)
subcomplex of the proton-translocating NADH-quinone oxidoreductase
(NDH-1) from Paracoccus denitrificans, which is composed of
the NQO1 (50 kDa) and the NQO2 (25 kDa) subunits. The two subunits are
co-expressed in Escherichia coli using a double expression
plasmid system. The expressed subunits form a water-soluble heterodimer
complex with 1:1 stoichiometry. The expressed complex contained one
[2Fe-2S] cluster but almost no FMN or
[4Fe-4S] cluster. The two latter prosthetic groups
could be partially reconstituted with FMN, NaS, and
(NH
)
Fe(SO
)
in vitro under anaerobic conditions. The reconstituted FP subcomplex showed
EPR signals from two distinct species of iron-sulfur cluster. One
resonance transition originates from a [2Fe-2S] cluster
with g values of g
= 1.92, 1.95, and
2.00 and slow spin relaxation, which was tentatively assigned to the
cluster N1a. These EPR properties are very similar to those reported
for the NQO2 subunit expressed alone (Yano, T., Sled', V. D.,
Ohnishi, T., and Yagi, T. (1994) Biochemistry 33,
494-499). The other originates from a [4Fe-4S]
cluster with g values of g
= 1.87,
1.94, and 2.04 and fast relaxing behavior, which are reminiscent of the
cluster N3 in the membrane bound enzyme complex. After reconstitution
with FMN, the FP subcomplex catalyzed electron transfer from NADH and
from deamino-NADH to a variety of electron acceptors. The enzymatic
properties of the FP subcomplex, reconstituted with FMN and
iron-sulfur, correspond to those of the isolated P. denitrificans NADH-dehydrogenase complex.
The respiratory chain of aerobically grown Paracoccus
denitrificans consists of NADH-quinone oxidoreductase (designated
as Complex I or NDH-1), ()succinate-quinone oxidoreductase,
quinol-cytochrome c oxidoreductase, and cytochrome c oxidase(1, 2) , and thus resembles the
mitochondrial respiratory chain. Complex I from mammalian mitochondria
is composed of at least 41 unlike subunits(3) . The simplest
bacterial NDH-1 described thus far consists of 14 subunits and can be
regarded as a minimal functional unit required to drive the
oxidation-reduction reaction coupled to the proton-translocating
activity of coupling site 1(4, 5) . As has been
revealed from the EPR analysis of membrane preparations, the P.
denitrificans NDH-1 contains at least 5 EPR-detectable iron-sulfur
clusters (6) with EPR and redox properties very similar to
those of the bovine enzyme(7, 8) . The five EPR
visible iron-sulfur clusters are designated as N1a, N1b, N2, N3, and
N4. The clusters N1a and N1b are binuclear and the clusters N2, N3, and
N4 are tetranuclear.
The NADH-quinone oxidoreductase has been isolated and characterized (9) . The gene cluster encoding the P. denitrificans NDH-1 enzyme has been cloned and sequenced(10, 11, 12, 13, 14) . This gene cluster is composed of 14 structural genes and 6 unidentified reading frames which are designated as NQO1-14 and URF1-6, respectively(15) . It was demonstrated that the P. denitrificans genes encoding NDH-1 show striking sequence identities to their mammalian counterparts. The P. denitrificans and the bovine Complex I polypeptides show immunocrossreactivity(9) . Furthermore, enzyme activity and sensitivity to specific inhibitors are very similar in the two complexes. Therefore, P. denitrificans NDH-1 is a suitable model for further understanding of the mitochondrial Complex I.
To understand the electron transfer mechanism of the NDH-1 enzyme complex, it is important to locate and identify all redox components at the molecular level. Sequence analysis allows us to predict that NQO1, NQO2, NQO3, NQO9, and possibly NQO6, are iron-sulfur cluster ligating subunits and that the NQO1 subunit contains a FMN-binding site(16, 17) . In addition, it has been shown that the NQO1 subunit bears the NADH-binding site(18) . These subunits seem to be all located in the peripheral part of the enzyme(5, 19) .
In previous work we have expressed
single P. denitrificans NDH-1 subunits in Escherichia coli and characterized their iron-sulfur cluster(s). These studies
showed that the NQO2 subunit contains a [2Fe-2S]
cluster which is most likely N1a(20, 21) .
Furthermore, by expressing and characterizing NQO2 altered by
site-directed mutagenesis, we have shown that the
[2Fe-2S] cluster is coordinated by four conserved
cysteine residues (C, C
, C
,
and C
)(22) . Recently, the expression and
characterization of the NQO3 (66 kDa) subunit (equivalent to the bovine
75-kDa subunit) was carried out. The expressed NQO3 subunit contained a
[2Fe-2S] cluster (N1b), a [4Fe-4S]
cluster(N4), and possibly an additional iron-sulfur
cluster(23) . These experiments have demonstrated that the
strategy of expressing individual subunits is useful for investigating
the location of individual redox components and refines previous
studies in which mitochondrial Complex I or bacterial NDH-1 were split
into different
subfractions(5, 19, 24, 25) . We
have attempted to express the NQO1 subunit in E. coli to
characterize its properties. When the NQO1 subunit was solely expressed
in E. coli, the subunit was produced as aggregated form
(inclusion body) under any conditions used. Any attempts to refold the
NQO1 subunit have not been successful so far. Therefore, we attempted
to co-express the NQO1 and NQO2 subunits in E. coli. The
co-expression strategy may be an alternative way to express intact
subunits on the basis of the following facts. 1) Bovine flavoprotein
(FP) subcomplex is composed of the 51-kDa subunit (homologue of the
NQO1 subunit), 24-kDa subunit (homologue of the NQO2 subunit), and
9-kDa subunit (the P. denitrificans NDH-1 lacks a 9-kDa
subunit counterpart), and is water-soluble(26, 27) . (2) The FP subcomplex is homologous to the
-subunit of
NAD
-reducing hydrogenase of Alcaligenes
eutrophus(10, 28, 29, 30) .
In this study the two FP subunits, NQO1 and NQO2, were co-expressed as soluble proteins in E. coli, forming a complex with 1:1 stoichiometry in situ. The isolated subcomplex did not contain FMN or any [4Fe-4S] cluster, but did contain a [2Fe-2S] cluster (N1a) which had EPR properties almost identical to those previously reported for the NQO2 subunit expressed alone. The FMN and the [4Fe-4S] cluster could be successfully reconstituted in vitro. EPR studies of the reconstituted FP subcomplex revealed that the subcomplex exhibits two distinct EPR signals: one arises from the [2Fe-2S] cluster (N1a), whereas the other, rapidly relaxing, signal originates from a [4Fe-4S] cluster. Furthermore, the reconstitution of FMN and [4Fe-4S] cluster into the subcomplex restored NADH-oxidizing activity with a variety of soluble electron acceptors to the level of that of the isolated P. denitrificans NADH dehydrogenase complex. The molecular and enzymatic properties of the expressed and reconstituted FP subcomplex as well as the location and assignment of the cofactors are discussed.
To examine whether the NQO1 and NQO2 subunits expressed in trans from different plasmids could form a FP subcomplex in situ, we employed a fusion protein expression strategy where a His-Tag sequence was attached to the N-terminal region of either the NQO1 or the NQO2 subunit. His-fusion proteins have the advantage of easy purification of target proteins using nickel chelation column chromatography. If the two subunits formed a complex in situ, it should be possible to purify the NQO1 + NQO2 subcomplex using this affinity chromatography. We found that when the His-Tag was fused to the N terminus of the NQO1 subunit, the nonfused NQO2 subunit was co-purified with the NQO1 subunit (Fig. 1, lane 3) apparently in 1:1 stoichiometry. As shown in Table 1, the amino acid analysis of the purified complex is in good agreement with that deduced from the DNA sequence for the NQO1 and NQO2 subunits together. Furthermore, we determined the apparent molecular mass of the expressed subcomplex to be 65 kDa by gel filtration analysis, suggesting that the complex exists as a heterodimer in solution. The purified subcomplex was well recognized by both affinity-purified anti-NQO1 antibodies and anti-NQO2 antibodies (Fig. 1, panel B). Thus, it can be concluded that the NQO1 and NQO2 subunits were properly expressed and form a complex with 1:1 stoichiometry in situ.
Figure 1:
Localization of the
expressed FP subcomplex of P. denitrificans NDH-1. The
Coomassie Blue-stained SDS-polyacrylamide gel (Panel A) and
immunoblotting (Panel B). The lanes contain cell lysate of E. coli harboring pKT16b(NQO1) and pET11a(NQO2) (12 µg of
protein for Panel A, lane 1), soluble fraction (7.5
µg of protein for Panel A, lane 2), and purified
FP subcomplex (2.0 µg of protein for Panel A, lane
3; 0.2 µg of protein each for Panel B, lanes 1 and 2). Immunoblotting was carried out using
affinity-purified antibodies against the P. denitrificans NQO1 (Panel B, lane 1) and NQO2 subunits (Panel
B, lane 2) and alkaline phosphate-conjugated anti-rabbit
IgG antibody as described previously (39, 40, 41) The arrows at the right indicate the expressed NQO1 (50 kDa) (upper)
and NQO2 (25 kDa) subunits (lower). The molecular marker (Panel A, lane M) includes myosin (200 kDa),
-galactosidase (116 kDa), phosphorylase B (97 kDa), bovine serum
albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa),
trypsin inhibitor (21.5 kDa), lysozyme (14.4 kDa), and aprotinin (6.5
kDa).
As described in the Introduction, we attempted to express the NQO1 subunit alone in E. coli. However, this subunit could not be expressed as an intact product under any of the conditions we used, always resulting in the formation of inclusion body. We tried to solubilize the NQO1 inclusion bodies with strong chaotropic agents such as 6 M guanidine HCl and subsequently refold the denatured polypeptides by lowering the chaotrope concentration in a stepwise manner by dialysis. However, the solubilized NQO1 subunit began to reaggregate whenever the guanidine HCl concentration was lowered below 4 M (data not shown). Thus, the NQO1 subunit could be expressed in the cytoplasm only when it was co-expressed with the NQO2 subunit (Fig. 1, lane 2). These results suggest that the NQO2 subunit plays an important structural role in stabilizing the NQO1 subunit and for the formation of FP subcomplex in situ.
Figure 2: Absorption spectra of the expressed subcomplex of P. denitrificans NDH-1 reconstituted with iron and sulfide, with FMN, or with FMN, iron, and sulfide. The subcomplex preparations were diluted in 50 mM Tris-HCl (pH 8.5) containing 5.0 mM DTT and 0.1 mM PMSF to 1.0 mg/ml. The spectra were recorded at room temperature. The subcomplex as isolated (line A), only FeS cluster-reconstituted subcomplex (line B), only FMN-reconstituted subcomplex (line C), and FMN and FeS cluster reconstituted FP subcomplex (line D).
Therefore, we attempted
to reconstitute FMN and iron-sulfur clusters in vitro. The
standard reconstitution procedures were described under
``Experimental Procedures.'' When iron and S but no FMN were added in the reaction mixture, both non-heme iron
and acid-labile sulfide contents increased to 4.1-4.2 mol/mol (Table 2). The reconstituted subcomplex showed broad absorption
peaks in the visible region which are due to the iron-sulfur clusters (Fig. 2, spectrum B). EPR analysis suggested that the
reconstituted iron-sulfur cluster was a [4Fe-4S]
cluster (data not shown). This demonstrates that an iron-sulfur cluster
was reconstituted in vitro. The [4Fe-4S]
cluster-reconstituted subcomplex was unable to oxidize NADH (Table 2). When only FMN was added to the reconstitution mixture,
FMN was incorporated into the subcomplex up to 0.25 mol/mol whereas the
iron-sulfur cluster contents were unaltered (Table 2). The
subcomplex reconstituted with only FMN showed an absorption spectrum in
which the absorbance characteristic for the FMN moiety could be seen (Fig. 2, spectrum C). Incorporation of FMN into the
subcomplex partially restored the enzymatic activity (Table 2).
These results clearly indicated that the [4Fe-4S]
cluster and the FMN, to some extent can be incorporated independently
and that the FMN molecule is an essential component for the
NADH-oxidizing activity of the subcomplex. When the reconstitution was
performed in the presence of FMN, iron, and S
, both
FMN and iron-sulfur clusters were incorporated into the subcomplex (Table 2). This FMN + FeS cluster-reconstituted subcomplex
(referred to as the FP subcomplex) showed an absorption spectrum in
which the absorption peaks attributable to FMN and iron-sulfur clusters
could be seen (Fig. 2, spectrum D). Fig. 3shows
the absorption spectra of the oxidized, NADH-reduced, and
dithionite-reduced forms of the reconstituted FP subcomplex. Upon
addition of NADH, the absorbance was slightly diminished, similar to
that in the isolated NDH-1 complex. The difference spectra, oxidized
form minus NADH-reduced form, showed that the spectral changes
could be attributed to the reduction of FMN (data not shown). As
described below, none of the iron-sulfur clusters were reduced by NADH.
Upon addition of dithionite, the absorbance was bleached in the entire
visible region, indicating that all redox components in the
reconstituted FP subcomplex, FMN, [2Fe-2S], and
[4Fe-4S] clusters were reduced. In the reconstituted FP
subcomplex, the NADH-oxidizing activity with several artificial
electron acceptors was restored. Turnover number (FMN basis) of the
reconstituted FP subcomplex was approximately 2-fold higher than that
of the subcomplex reconstituted with only FMN (Table 2). This
suggests that the presence of the [4Fe-4S] cluster is
an important factor for effective flavination and for the complete
restoration of NADH-oxidizing activity. It may be suggested that the
[4Fe-4S] cluster affects the structure or interacts
with the FMN- and/or the NADH-binding site. It should also be noted
that flavination of the subcomplex seems to be FMN specific. FAD was
not incorporated under any conditions we tested (data not shown).
Moreover, incorporation of FMN and the iron-sulfur cluster into the
expressed subcomplex required a prolonged incubation time. In case of
other flavoproteins, FMN incorporation into apoproteins has been
reported to take place within a few minutes of incubation(47) .
Figure 3: Absorption spectra of the expressed-reconstituted FP subcomplex of P. denitrificans NDH-1. The expressed-reconstituted FP subcomplex was diluted in 50 mM Tris-HCl (pH 8.5) containing 5.0 mM DTT and 0.1 mM PMSF to 0.7 mg/ml. The spectra were recorded at room temperature in the oxidized form (as prepared) (line A), in the NADH-reduced form (line B), and in the dithionite-reduced form (line C). The final concentrations of NADH and dithionite were 5 mM and 10 mM, respectively.
Figure 4: Resolved EPR spectra of the iron-sulfur clusters of the expressed-reconstituted FP subcomplex of P. denitrificans NDH-1. Spectrum of [2Fe-2S] cluster was recorded at microwave power 25.3 µW (1); in order to resolve the spectrum of [4Fe-4S] cluster, the contribution of saturated binuclear cluster was eliminated by recording the weighted difference between spectra at 100 mW and 12.7 mW (2). EPR condition: sample temperature, 13 K; microwave field frequency, 9.449 GHz; modulation amplitude, 1 mT; modulation frequency, 100 kHz; time constant, 0.16 s; scan rate, 0.596 mT/s. Principal g values for the clusters are indicated by arrows.
Figure 5:
Power saturation behavior of the
iron-sulfur clusters in the reconstituted FP subcomplex and the
[2Fe-2S] cluster in the expressed NQO2 subunit. Panel A, power saturation of the g = 2.00 signal
(g component of [2Fe-2S] cluster) in
isolated NQO2 subunit (1) and in reconstituted FP subcomplex (2). Sample temperature was 57 K. Panel B, power
saturation profiles of the g = 2.04 signal (g
component of [4Fe-4S] cluster) in reconstituted
FP subcomplex (1), and of the g = 2.00 signals (g
component of [2Fe-2S] cluster) in isolated NQO2
subunit (2) and in reconstituted FP subcomplex (3).
Sample temperature was 13 K. Other EPR conditions were as in Fig. 4. Half-saturation parameters were determined according to
Blum and Ohnishi (53) and P
values
obtained are indicated in the figures.
The present expression study demonstrated that the NQO1 and NQO2 subunits, produced in trans from different plasmids, can form a subcomplex in E. coli. Although the two subunits form a subcomplex in 1:1 stoichiometry, with seemingly native conformation, the incorporation of FMN and [4Fe-4S] cluster does not take place in situ. These data imply that either the P. denitrificans-specific machinery or interaction with other neighboring subunits, such as the NQO3 subunit, may be required for the cofactor incorporation and to form a more stable subcomplex. These issues will be addressed in future studies.
The reconstitution
experiments described herein provided some insight into the structural
location and function of the cofactors in the FP subcomplex. The
subcomplex as isolated contains almost equimolar amounts of the
[2Fe-2S] cluster and its EPR properties are identical
to those of the previously studied NQO2 subunit by itself(20) ,
which was assigned as cluster N1a(23) . The NQO2 subunit does
not contain any additional conserved cysteine residues except for the 4
cysteines that were shown to ligate the [2Fe-2S]
cluster in our previous study (22) . Thus, the reconstituted
[4Fe-4S] cluster most likely resides in the NQO1
subunit. Since the NQO1 subunit contains a conserved sequence motif for
[4Fe-4S] cluster ligation,
CXXC
XXC
-C
(P. denitrificans numbering), the
[4Fe-4S] cluster is probably coordinated by these
cysteine residues. Considering the EPR properties of the reconstituted
[4Fe-4S] cluster, it seems to correspond to cluster N3
in P. denitrificans NDH-1(6) . Our results from the
present study provided experimental evidence that the FMN molecule is
an indispensable component for NADH-oxidizing activity, suggesting that
the FMN molecule is the primary electron acceptor from NADH.
Nevertheless, we have no experimental evidence for the exact location
of the FMN-binding site. Either FMN is embedded within the NQO1 subunit
or both the NQO1 and the NQO2 subunits contribute to the FMN-binding
site. Since flavin could be incorporated into the expressed subcomplex in vitro, we will attempt to employ direct labeling techniques
with FMN analogues as has been done with other
flavoproteins(47) . Identification of amino acid residues in
the binding pocket by labeling experiments should be followed by
site-directed mutagenesis in order to probe the specific functional
role of the targeted residues. These experiments will help us to
further understand the molecular mechanism of the NADH dehydrogenase
section of NDH-1 enzyme complex.
Mitochondrial and bacterial
NADH-ubiquinone oxidoreductases consist of two distinct parts, one
peripheral arm protruding into the matrix or cytoplasm, and one
hydrophobic arm spanning the membrane. Our recent immunological studies
on subunit location within the P. denitrificans NDH-1 enzyme
complex using specific antibodies against each subunit have suggested
that the NQO1-6 and 9 subunits constitute the peripheral part of
the enzyme complex. None of these subunits seem to penetrate through
the cytoplasmic membrane. ()(
)A series of
expression studies of the putative cofactor ligating subunits of the P. denitrificans NDH-1 have shown that the NQO2 subunit
contains cluster N1a (20) and that the NQO3 subunit bears
cluster N1b and N4, and possibly an additional iron-sulfur
cluster(23) . The present study suggests that the NQO1 subunit
contains cluster N3 and at least contributes to the FMN-binding site,
which agrees with data on the disruption of NUO51 gene
encoding the 51-kDa subunit (NQO1 homologue) of Neurospora crassa Complex I(52) . Sequence analysis regarding iron-sulfur
cluster ligation sites predicts that only the NQO9 and NQO6 subunits
may contain additional iron-sulfur clusters. Thus, all prosthetic
groups seem to be located in the peripheral part of the enzyme. A major
future challenge is to elucidate how electron transfer through the
peripheral part of NDH-1 is coupled to proton translocation across the
membrane.