(Received for publication, September 6, 1995; and in revised form, October 24, 1995)
From the
Complex II (succinate-ubiquinone oxidoreductase) from Escherichia coli is composed of four nonidentical subunits
encoded by the sdhCDAB operon. Gene products of sdhC and sdhD are small hydrophobic subunits that anchor the
hydrophilic catalytic subunits (flavoprotein and iron-sulfur protein)
to the cytoplasmic membrane and are believed to be the components of
cytochrome b in E. coli complex II. In
the present study, to elucidate the role of two hydrophobic subunits in
the heme b ligation and functional assembly of complex II, plasmids
carrying portions of the sdh gene were constructed and
introduced into E. coli MK3, which lacks succinate
dehydrogenase and fumarate reductase activities. The expression of
polypeptides with molecular masses of about 19 and 17 kDa was observed
when sdhC and sdhD were introduced into MK3,
respectively, indicating that sdhC encodes the large subunit
(cybL) and sdhD the small subunit (cybS) of cytochrome b
. An increase in cytochrome b content
was found in the membrane when sdhD was introduced, while the
cytochrome b content did not change when sdhC was
introduced. However, the cytochrome b expressed by the plasmid
carrying sdhD differed from cytochrome b
in its CO reactivity and red shift of the
absorption peak
to 557.5 nm at 77 K. Neither hydrophobic subunit was able to bind the
catalytic portion to the membrane, and only succinate dehydrogenase
activity, not succinate-ubiquinone oxidoreductase activity, was found
in the cytoplasmic fractions of the cells. In contrast, significantly
higher amounts of cytochrome b
were expressed in
the membrane when sdhC and sdhD genes were both
present, and the catalytic portion was found to be localized in the
membrane with succinate-ubiquinone oxidoreductase and succinate oxidase
activities. These results strongly suggest that both hydrophobic
subunits are required for heme insertion into cytochrome b
and are essential for the functional assembly
of E. coli complex II in the membrane. Accumulation of the
catalytic portion in the cytoplasm was found when sdhCDAB was
introduced into a heme synthesis mutant, suggesting the importance of
heme in the assembly of E. coli complex II.
Complex II (succinate-ubiquinone oxidoreductase) is a
tricarboxylic acid cycle enzyme associated with the inner membrane of
mitochondria or the cytoplasmic membrane of
bacteria(1, 2) . Complex II in Escherichia coli is encoded by the sdhCDAB operon (3, 4) and contains four nonidentical subunits and
five prosthetic groups(5, 6) . The largest hydrophilic
subunit (flavoprotein; Fp), ()encoded by sdhA, has
covalently bound FAD, and the second largest subunit (iron-sulfur
protein; Ip), encoded by sdhB, contains three different types
of iron-sulfur clusters termed S-1[2Fe-2S],
S-2[4Fe-4S], and S-3[3Fe-4S]. Two hydrophobic heme
b-containing subunits are encoded by sdhC and sdhD,
and at least the sdhC product is a component of cytochrome b
(7) .
Generally, the Fp and Ip subunits comprise the catalytic portion of complex II and catalyze electron transfer from succinate to artificial electron acceptors such as phenazine methosulfate or 2,6-dichlorophenol-indophenol (DCIP) (succinate dehydrogenase; SDH). The amino acid sequences of Fp and Ip are highly conserved among species. For example, the amino acid sequences of Fp and Ip from human liver (8, 9) show 50% similarity to their counterparts in E. coli complex II. The Fp and Ip subunits of succinate-ubiquinone oxidoreductase are also closely related to those of bacterial and mitochondrial fumarate reductases (FRDs), which catalyze the reverse reaction of SDH activity(10, 11, 12) . In E. coli, FRD encoded by the frdABCD operon is synthesized during anaerobic growth, whereas complex II encoded by the sdh operon is induced in aerobic culture. Studies on the primary structures of different Fps and Ips and the effect of site-directed mutagenesis reveal structural and functional relationships in the binding of prosthetic groups and substrate recognition(1, 2) .
It is commonly accepted that the intact four-subunit complex II
contains a cytochrome b component composed of hydrophobic
large (cybL) and small (cybS) subunits. Exceptions are the enzyme from Bacillus subtilis, which contains two cytochrome b components bound to a single large hydrophobic subunit (13) , and FRD from E. coli which does not contain
heme b. In contrast to the well characterized Fp and Ip subunits,
numerous questions concerning the cytochrome b in complex II
remain to be solved due to limited information about the primary
structures of the subunits. The nucleotide sequences of both
hydrophobic subunits in complex II have only been determined for E.
coli(3) and Saccharomyces cerevisiae(14, 15, 16) , while that of cybL has
been reported in bovine complex II (17, 18) . Amino
acid sequences of these hydrophobic subunits in complex II from
different species show little conservation. The contents and redox
properties of heme b in complex II from different organisms vary
greatly. B. subtilis complex II contains two hemes b, and the
heme with the higher potential (E` =
+65 mV) is reduced by succinate, while the heme with the lower
potential (E
` = -95 mV) is
not(19, 20) . E. coli and Ascaris suum complex II have one heme b in complex II, and the heme b in E.
coli complex II (E
` = +36 mV) is
fully reducible by succinate(6) , while the heme b in A.
suum complex II (E
` = -34 mV) is
partially reduced by succinate(21, 22) . Bovine
cytochrome b
(E
` =
-185 mV) is not readily reducible by succinate, and low
substoichiometric amounts of heme b are found in this enzyme
complex(23) . Furthermore, purified complex II from yeast has
no heme b(24) . Extensive studies on the role of cytochrome b in complex II have been carried out using the B.
subtilis enzyme. In B. subtilis complex II, insertion of
heme into the apocytochrome b
subunit is
critical for the assembly of the membrane-bound
enzyme(25, 26) , suggesting a structural role for the
cytochrome. Electron paramagnetic resonance (EPR) and near-infrared
magnetic circular dichroism (MCD) studies showed that each of the two
hemes has bis-histidine ligation(26, 27) .
Furthermore, site-directed mutagenesis studies have identified the
histidine residues that are ligated to heme
b(25, 26) . However, it is difficult to define
accurately the structure and function of the two-subunit cytochrome b in complex II, because the B. subtilis complex II
has a single large hydrophobic subunit, although this subunit seems to
have arisen due to protein fusion between cybL and cybS of the
two-subunit cytochrome b(28) .
Another example of well characterized hydrophobic subunits in complex II is the frdC and frdD gene products in E. coli FRD(29, 30, 31) . The role of the two hydrophobic subunits in assembly and the amino acid residues essential for quinone binding have been analyzed(32, 33, 34) . However, this enzyme complex does not contain heme b, so the function of heme b in the two-subunit cytochrome b found in most bacterial and mitochondrial complex II has not been analyzed.
Cytochrome b from E. coli complex II has been
purified and characterized by the present authors during the systematic
analysis of cytochromes, including cytochrome bo and bd oxidase, in the membranes of aerobically grown E.
coli(35, 36, 37) . The first 24 residues
from the N-terminal amino acid sequence of the cytochrome are identical
to residues 4-27 deduced from E. coli sdhC(7) .
We also have established a one-step protocol to purify complex II by
chromatography using an E. coli strain that overproduces the
enzyme by 10-fold due to the presence of a multicopy plasmid containing
the cloned sdh operon(6) . Using this preparation,
bis-histidine ligation of the heme in cytochrome b
was demonstrated by EPR and MCD(38) . However, it is not
known whether cybL alone or cybL plus cybS provide coordination to the
heme.
In the present study, the role of the two hydrophobic subunits in heme ligation and the functional assembly of complex II in the membrane was investigated using plasmids carrying portions of the E. coli sdh operon.
Figure 1: The sdh operon in E. coli. Restriction endonuclease sites are indicated. B, BamHI; K, KpnI; Hi, HindIII; Ha, HaeIII, N, NaeI; S, SacI. The lines below the sdh operon show the fragments in various plasmids listed in Table 1.
Figure 2:
Western blotting of homogenates of various E. coli strains with antibody against bovine Fp. Ten µg of
protein was loaded onto each lane of a 12.5% (w/v)
SDS-polyacrylamide gel. Lane 1, NM522; lane 2, GVK124 (sdh); lane 3, MI1443 (frd
); lane 4, MK3 (sdh
, frd
). The closed arrowhead indicates Fp of fumarate reductase, and the open arrowhead indicates that of complex
II.
Figure 3: Absorption spectra of E. coli membranes. The dithionite-reduced minus air-oxidized difference spectra of crude membranes were recorded at 77 K. The cuvette light path length was 1 mm. The right panel shows the second derivatives of the spectra in the left panel. a, NM522 membranes (protein concentration, 4.7 mg/ml); b, MK3 membranes (6.5 mg/ml); c, MK3/pSDHCD membranes (2.1 mg/ml); d, MK3/pSDHD membranes (4.5 mg/ml) in 50 mM Tris-HCl (pH 7.5). The bar indicates absorbance of 0.1 (a, b, and d) or 0.2 (c).
Figure 4:
Elution profiles from HPLC gel filtration
chromatography. Sarkosyl-solubilized membranes (final protein
concentration, 4 mg/ml; 100 µl each) were applied to the column,
and the elution of cytochrome b was monitored at 412 nm. The
flow rate was 1 ml/min. a, NM522; b, MK3; c,
MK3/pSDHCD; d, MK3/pSDHD. Cytochrome b eluted at 17.8 min. The bar indicates an absorbance of
0.01 (a and b) or 0.02 (c and d).
The content of cytochrome b in
MK3/pSDHD membranes was 4-fold higher than in MK3 membranes, while
MK3/pSDHC membranes (Table 2) contained amounts of cytochrome b similar to those in MK3 membranes. However, the properties
of the cytochrome b expressed in pSDHD membranes were
different from those of cytochrome b, although
the elution profiles of both cytochromes were similar. The cytochrome b from MK3/pSDHD membranes showed an
absorption peak at
557.5 nm in the reduced minus oxidized difference spectrum at 77 K. In
addition, these membranes contained more CO-reactive cytochrome b than membranes from other transformants. Fig. 5shows the
SDS-polyacrylamide gel electrophoresis profile of the fractions eluted
from the gel filtration column at 17.8 min (peak III corresponding to
cytochrome b
in Fig. 4) visualized by
silver staining. The expression of polypeptides with molecular masses
of about 19 and 17 kDa was observed when sdhC and sdhD were introduced into MK3, respectively, and these two peptides
were highly expressed in MK3/pSDHCDAB and MK3/pSDHCD. These results
indicate that sdhC encodes the large subunit (cybL) and sdhD encodes the small subunit (cybS) of cytochrome b
and that both subunits are essential
components of cytochrome b
in E. coli complex II.
Figure 5: Silver-stained SDS-polyacrylamide gel of the peak eluted at 17.8 min on HPLC. The proteins in 120 µl of the fractions eluted between 17.3 and 18.3 min (1 ml) were concentrated by treatment with 10% (w/v) trichloroacetic acid. All of the proteins were then dissolved in sample buffer (without boiling) and separated on a 15-25% (w/v) gradient SDS-polyacrylamide gel. Lane 1, purified E. coli complex II (protein concentration, 0.3 µg); lane 2, MK3; lane 3, MK3/pSDHCDAB; lane 4, MK3/pSDHCD; lane 5, MK3/pSDHD; lane 6, MK3/pSDHC. The closed arrowhead indicates the cybL subunit, and the open arrowhead indicates the cybS subunit.
Table 4shows the effect of deficiency in heme
synthesis on the assembly of complex II. No SDH activity was found in
the cytoplasmic or membrane fractions of the hemA mutant, H500, grown on buffered LB medium with glucose. When H500
was transformed with pSDHCDAB, SDH activity was found in the cell
homogenates, although the specific activity (80.2 nmol/min/mg) was
lower than that from wild-type strain NM522. More than 98% of this
activity was localized in the cytoplasmic fraction, suggesting the
importance of heme b in the assembly of E. coli complex II.
This was confirmed by the association of succinate-ubiquinone
oxidoreductase activities in the membrane fractions when H500 and H500
transformed with pSDHCDAB were grown in the presence of
5-aminolevulinic acid. The localization of the Fp peptide, analyzed by
Western blotting (data not shown), correlated well with the
distribution of enzyme activity.
The data presented here clearly demonstrate that two
hydrophobic subunits, cybL and cybS, encoded, respectively, by sdhC and sdhD in the E. coli sdh operon, are required
for heme ligation into cytochrome b and are
essential for the functional assembly of E. coli complex II in
the membrane. The strategy for this study was in vivo complementation by a combination of sdh genes on
different vectors, a method that was used successfully to investigate
the assembly of E. coli FRD(33) . Our recent analysis
by EPR and MCD showed that the heme b
component of E.
coli complex II is ligated to the protein by two histidine
residues(38) . Furthermore, His
in cybL and
His
in cybS were determined to be possible heme axial
ligands in cytochrome b
as shown using
site-directed mutants. (
)The data from the present study are
consistent with these observations, because cytochrome b
was expressed only when the sdhC and sdhD genes were both present. Bis-histidine ligation of the
heme b between histidine residues located in the hydrophobic segments
was also found for cytochrome b in B. subtilis complex II, although it contains two cytochromes b in a
single hydrophobic peptide(26, 27) . The cytochrome b expressed by pSDHD localized in the membrane and eluted from
the gel filtration column with the same retention time (17.8 min) as
cytochrome b
. However, this cytochrome b differs from cytochrome b
in that it binds
CO and shows a 1-nm red shift of the
absorption peak in the low
temperature difference spectrum. The main fraction of the sdhC product (cybL) eluted at 18.5 min from the column when membranes
from the strain MK3/pSDHC were solubilized and analyzed by HPLC,
suggesting that the cytochrome b from pSDHD eluting at 17.8
min is a dimer of the cybS peptide. It is likely that heme b bridges
two histidine residues in the cybS dimer. His
is a
candidate for one of these residues because the increase in cytochrome b was not observed when His
in pSDHD was
substituted by glutamine; on the other hand, substitution of His
did not affect the increase in cytochrome b. (
)The cytochrome b content of membranes from
MK3/pSDHCDAB was about 5-fold higher than that from wild type strain
NM522, whereas the SDH activity of the strains differed only slightly.
The reason for this difference remains unclear. However, it is
speculated that the amount of cytochrome b
synthesized from the plasmid is higher than that of the catalytic
portion, Fp and Ip, for some reason (e.g. polar effects). The
higher ratio of succinate-ubiquinone oxidoreductase to SDH in membranes
from pSDHCDAB as compared with other membranes (Table 3) may be
related to the high content of cytochrome b
. The
association of heme b in mitochondrial complex II is still unclear
except for A. suum complex II(21, 45) , even
though the genes for SDH3 and SDH4 from S. cerevisiae(14, 15, 16) and the cDNA for bovine
cybL (17, 18) have been cloned and sequenced. Our
present report is the first indication that both hydrophobic subunits
are indispensable for heme b ligation into the two-subunit cytochrome b in complex II.
The presence of the two hydrophobic
subunits, cybL and cybS, is essential for the functional assembly of E. coli complex II and its localization in membranes as well
as for heme b ligation. Neither hydrophobic subunit alone is able to
bind the catalytic Fp and Ip subunits to the membrane, and the
catalytic portion in the cytoplasmic fraction does not transfer
reducing equivalents to ubiquinone. This is quite similar to the
situation in E. coli FRD(33) , including the effects
of deficiency on the growth of the strains. In contrast, gene
disruption of SDH4 in S. cerevisiae, which
corresponds to E. coli sdhD, does not lead to any growth
defect on glycerol-rich medium(16) . Membrane attachment of the
catalytic portion of this mutant is impaired but not abolished. A
tighter association between the catalytic portion and cytochrome b in mitochondrial complex II compared with the bacterial enzyme may
explain this difference. Active mitochondrial complex II is eluted from
the gel filtration column in the presence of an anionic detergent,
Sarkosyl(41, 45) , while cytochrome b in E. coli complex II is dissociated from the complex
and can be purified by the same column (35) ; this supports the
above idea. A difference in the biogenesis of the enzyme complex was
also found between complex II and FRD of E. coli. The
introduction of the frdABC and frdD genes on separate
plasmids into E. coli MI1443, which lacks a chromosomal frd operon, fails to restore anaerobic growth on glycerol and
fumarate(33) . Moreover, this strain is unable to interact with
quinone analogues, although the enzyme complex is completely functional
in catalyzing fumarate reduction by reduced benzyl viologen and is
localized in the membrane. In this study it was claimed that
co-translational association of frdC and frdD products is required for the functional assembly of E. coli FRD(33) . In contrast to FRD, we observed active complex
II in membranes when both pSDHC and pSDHDAB were transformed into MK3,
and the enzyme complex supported aerobic growth on succinate,
indicating differences in the assembly processes of the two enzyme
complexes.
An accumulation of the unassociated catalytic portion of
complex II in the cytoplasm was observed in heme synthesis mutants, and
normal complex II was assembled in the membrane when 5-aminolevulinic
acid was present in the culture. This result implies that the heme is
important for the functional assembly of E. coli complex II.
Interestingly, our recent analysis of site-directed mutants showed that
complex II in cybL His and cybS His
mutants,
which lack heme b in their cytochrome b
, is
associated with the membrane and catalyzes succinate-ubiquinone
oxidoreductase activity.
The presence of heme b appears to
be essential for the initial step in the assembly of complex II into
the membrane and to be unnecessary for electron transfer to ubiquinone
once the enzyme complex is assembled.