(Received for publication, September 11, 1995; and in revised form, November 15, 1995)
From the
Subunit IV of Rhodobacter sphaeroides cytochrome b-c complex was over-expressed in Escherichia
coli JM109 cells as a glutathione S-transferase fusion
protein (GST-RSIV) using the expression vector, pGEX/RSIV. Maximum
yield of soluble active recombinant fusion protein was obtained from
cells harvested 3 h after induction of growth at 37 °C in LB
medium. Subunit IV was released from the fusion protein by proteolytic
cleavage with thrombin. When subjected to SDS-polyacrylamide gel
electrophoresis, isolated recombinant subunit IV showed one protein
band corresponding to subunit IV of R. sphaeroides cytochrome b-c
complex. Although the isolated recombinant
subunit IV is soluble in aqueous solution, it is in a highly aggregated
form, with an apparent molecular mass of over 1000 kDa. The addition of
detergent deaggregates the isolated protein, suggesting that the
recombinant protein exists as a hydrophobic aggregation in aqueous
solution. When the three-subunit core cytochrome b-c
complex, purified from RS
IV-adapted chromatophores
containing a fraction of the wild-type cytochrome b-c
complex activity, was reacted with varying amounts of recombinant
subunit IV, the activity increased as the subunit IV concentration
increased. Maximum activity restoration was reached when 1 mol of
subunit IV/mol of three-subunit core complex was used. The
reconstituted cytochrome b-c
complex is similar to
the wild-type complex in molecular size, apparent K
for Q
H
, and inhibitor sensitivity,
indicating that recombinant subunit IV is properly assembled into the
active cytochrome b-c
complex. A tryptophan
residue in subunit IV was found to be involved in the interaction with
the three-subunit core complex.
The Rhodobacter sphaeroides cytochrome b-c complex, which catalyzes the electron transfer
from ubiquinol to cytochrome c
(1) , has
been purified and characterized in several
laboratories(2, 3, 4, 5, 6) .
The purified complex contains four protein subunits with molecular
masses of 43, 31, 23, and 15 kDa. The three largest subunits house
cytochrome b, cytochrome c
, and a high
potential [2Fe-2S] Reiske iron-sulfur cluster. The smallest
protein subunit (subunit IV) has been proven to be an integral part of
the complex by immunochemical studies(7) . Subunit IV and
cytochrome b have been identified as ubiquinone
(Q)(
)-binding proteins in the complex by photoaffinity
labeling techniques using azido-Q derivatives(8) . However,
subunit IV is not present in other comparable bacterial cytochrome b-c
complexes, such as Rhodospirillum
rubrum(9) , Rhodobacter capsulatus(10) ,
and Paracoccus denitrificans(11) .
The involvement
of subunit IV in Q-binding and structural integrity of the R.
sphaeroides cytochrome b-c complex has been
further established by molecular genetics
studies(12, 13) . The gene for subunit IV (fbcQ) has been cloned and sequenced(12) . The fbcQ cistron is 372 base pairs long, encodes 124 amino acid
residues, and is contained in a 4.7-kilobase pair BamHI R.
sphaeroides DNA fragment. When fbcQ is deleted from the R. sphaeroides chromosome, the resulting strain (RS
IV)
requires a period of adaptation before the start of photosynthetic
growth(13) . The cytochrome b-c
complex in
adapted chromatophores is labile to detergent treatment (75%
inactivation) and shows a 4-fold increase in the K
for Q
H
(13) . The first two
changes (adaptation time and detergent lability) indicate a structural
role of subunit IV; the third change (K
increase) indicates its Q-binding function. Introducing
wild-type fbcQ on a stable low copy number plasmid, pRK415,
into RS
IV restores photosynthetic growth behavior, the apparent K
for Q
H
, and
tolerance to detergent treatment to the level of wild-type cells.
The Q-binding domain in subunit IV is located at residues
77-124, as determined by isolation and sequencing of an
[H]azido-Q-labeled, V8-digested
peptide(12) . The most likely Q-binding region is at residues
77-86, which lies on the cytoplasmic side of the chromatophore
membrane. By using site-directed mutagenesis techniques coupled with in vivo complementation, tryptophan-79 has been identified to
be responsible for Q-binding, and amino acid residues 6-11 are
responsible for the structural role of subunit IV(14) .
Although mutagenesis coupled with in vivo complementation
has generated useful information in structure-function studies of
subunit IV, this approach is often complicated by mutational effects on
the complex assembly and stability of the mutated protein. An approach
using expressed mutated recombinant protein to reconstitute, in
vitro, a subunit IV-lacking complex (three-subunit core complex)
alleviates the problem of assembly and stability of mutated protein and
thus complements the in vivo complementation approach. In
order to employ this in vitro reconstitution approach, a
reconstitutively active, three-subunit core cytochrome b-c complex and an over-expressed, functionally
active subunit IV are needed.
The three-subunit core complex is
available in our laboratory. This complex is prepared from adapted
chromatophores of RSIV by a method involving dodecylmaltoside
solubilization and DEAE-Biogel A and DEAE-Sepharose 6 B column
chromatography(13) . Recently, we have over-expressed subunit
IV in Escherichia coli as a glutathione S-transferase
(GST) fusion protein by using the pGEX expression vector
system(15) . The pGEX expression system allows one-step
affinity purification of the recombinant fusion protein with
glutathione-agarose gel(16) . The recombinant protein is then
released from the fusion protein by thrombin cleavage(17) .
Herein we report construction of a subunit IV expression vector,
pGEX/RSIV, conditions for high expression of an active soluble form of
the GST-RSIV fusion protein in E. coli JM109, and isolation
and characterization of pure recombinant subunit IV. Reconstitution of
the R. sphaeroides cytochrome b-c
complex
from the three-subunit core complex and recombinant subunit IV is
described, and properties of the reconstituted complex are examined.
The amino acid residues essential for reconstitutive activity of
subunit IV are also identified.
SDS-PAGE(21) , nondenaturing blue gel
electrophoresis(22) , Western blots(23) , and protein (24) and cytochromes b and c contents (13) were determined according to methods
previously described.
Titration of recombinant subunit IV by N-bromosuccimide was performed according to the method
described by Spande and Witkop(25) . The amount of tryptophan
being oxidized by N-bromosuccimide was determined by measuring
the decrease in absorbance at 280 nm. A molar extinction coefficient of
585 M cm
for tryptophan
in 50 mM Tris-Cl, pH 8.0, containing 300 mM NaCl and
0.01% dodecylmaltoside was used for calculations.
Figure 1: Construction of the expression vector for R. sphaeroides subunit IV, pGEX/RSIV.
Figure 2: Recovery of purified GST-RSIV fusion protein from cells after various IPTG induction growth times. A, lane 1 is molecular weight references. Lane 2 represents cells grown without IPTG. Lanes 3-6 represent cells grown for 1, 2, 3, and 5 h, respectively, after the addition of IPTG in LB media containing 50 µg/ml ampicillin. The isolation of GST-RSIV fusion protein by affinity glutathione-agarose gel is as described under ``Experimental Procedures.'' 30 µl of glutathione-agarose eluates were applied to SDS-PAGE. B, the proteins on the gel of A were electrophoretically transferred to a nitrocellulose membrane without staining and then reacted with anti-subunit IV antibodies. Protein A-horseradish peroxidase conjugate was used as a second antibody.
The susceptibility of recombinant subunit IV to the protease digestion is further evident from the presence of three smaller molecular mass protein bands, with apparent molecular sizes of 31, 28, and 26 kDa, following SDS-PAGE of glutathione-agarose eluates (see Fig. 2A). The 31- and 28-kDa protein bands, which reacted with antibodies against subunit IV and antibodies against GST and disappeared after thrombin digestion, may derive from partial C-terminal digestion of recombinant fusion protein. The 26-kDa protein band, which reacted only with antibodies against GST, may result from the complete digestion of subunit IV from the fusion protein.
The addition of protease inhibitors, such as phenylmethylsulfonyl fluoride and leupeptin, during cell extract preparation did not prevent degradation of the recombinant subunit IV. Expression of the GST-RSIV fusion protein in E. coli KS1000, which is deficient in the Tsp protease, a periplasmic protease, also did not prevent the degradation of recombinant subunit IV. The Tsp protease (26) was reported to degrade cytoplasmically expressed proteins in crude cell extracts, and presumably it can degrade proteins expressed in the periplasm as well.
It should be noted that our routine cell extract procedure included treatment with 1% Triton X-100. If this treatment was omitted, the yield of soluble recombinant GST-RSIV fusion protein decreased 55%. This suggests that some GST-RSIV fusion protein is either in a membrane fraction or an inclusion body aggregate that can be solubilized by Triton X-100 while maintaining the GST-active site recognizable by glutathione-agarose gel. Recombinant GST-RSIV fusion protein obtained from cell extracts prepared with Triton X-100 has the same molecular size and reconstitutive activity as protein obtained without Triton X-100 treatment.
Recombinant subunit IV was released from the fusion protein by thrombin digestion. When the fusion protein was incubated with thrombin at a weight ratio of 1:500 at room temperature, about 80% of the subunit IV was recovered within 1 h. Although prolonged incubation can complete cleavage, it is often accompanied by irreversible denaturation of protein. Therefore, a 1-h digestion time was used. The released GST and uncleaved GST-RSIV fusion protein in the treated sample were removed by glutathione-agarose beads, and thrombin was removed by gel filtration.
Following SDS-PAGE, isolated
subunit IV showed a single protein band that corresponds to subunit IV (M = 14,384) of R. sphaeroides cytochrome b-c
complex (see Fig. 3).
The partial N-terminal amino acid sequence of recombinant subunit IV
was determined to be GSMFSFI-, indicating that two additional amino
acid residues, glycine and serine, are present at the N terminus of
subunit IV. These two residues result from the recombinant
manipulation. The true molecular mass of recombinant subunit IV should
be 201 daltons more than that of native subunit IV.
Figure 3:
SDS-PAGE of recombinant subunit IV. Lane 1, purified recombinant subunit IV (3 µg). Lane
2, purified wild-type cytochrome b-c complex
(20 µg). Lane 3, the molecular weight reference
markers.
Table 1shows the
protective effect of subunit IV on the RSIV cytochrome b-c
complex toward detergent treatment. When
RS
IV chromatophores were added to varying amounts of recombinant
subunit IV before being subjected to dodecylmaltoside treatment, the
cytochrome b-c
complex activity in the
detergent-solubilized chromatophore fraction increased with the amount
of recombinant subunit IV added. Maximum restoration (68%) was reached
when recombinant subunit IV and the RS
IV b-c
complex were present in a 1:1 molar ratio. The addition of
subunit IV to the wild-type or complement chromatophores had no effect
on cytochrome b-c
complex activity upon detergent
solubilization. The further addition of subunit IV to the subunit
IV-treated, detergent-solubilized chromatophores fraction did not
further increase cytochrome b-c
complex activity.
The incomplete restoration of detergent tolerance to the cytochrome b-c
complex in RS
IV chromatophores by
recombinant subunit IV may result from a decrease in binding affinity
of the three-subunit core complex to recombinant subunit IV in the
presence of high concentrations of dodecylmaltoside, as used in
solubilization.
Fig. 4shows the restoration of the
cytochrome b-c complex activity from the purified
three-subunit core complex by recombinant subunit IV. When the core
complex was incubated with varying concentrations of subunit IV,
activity increased as the concentration of subunit IV increased.
Maximum restoration was reached when 1 mol of subunit IV/mol of
three-subunit core complex was used. The activity was restored to the
same level as that of the wild-type complex, indicating that
recombinant subunit IV is fully active. Because recombinant subunit IV
can fully restore cytochrome b-c
complex activity
to the three-subunit core complex, the structural requirement for the
amino acid residues near the N terminus of subunit IV are not
stringent, as recombinant subunit IV has two amino acid residues,
serine and glycine, added to the N terminus. This is in line with the
gene deletion study showing that the first five amino acid residues
from the N terminus are not essential for subunit IV(14) .
Restoration of the cytochrome b-c
complex activity
to the three-subunit complex by subunit IV was found to be incubation
time-dependent (see Fig. 5). Maximum activity restoration was
observed after 1 h of incubation at 0 °C. The incubation time
dependence of reconstitution may result from deaggregation of
recombinant subunit IV or conformational change of the reconstituted
complex.
Figure 4:
Effect of subunit IV concentration on
restoration of ubiquinol-cytochrome c reductase activity from
the three-subunit core complex. Aliquots (40 µl) of the wild-type
() and the three-subunit core cytochrome b-c
complexes (
), 0.45 mg/ml in 50 mM Tris-HCl, pH 8.0,
containing 300 mM NaCl and 0.01% dodecyl maltoside were added
to the indicated amounts of purified subunit IV in 50 mM Tris-Cl, pH 8.0. After 1 h of incubation at 0 °C, aliquots
were withdrawn for ubiquinol-cytochrome c reductase activity
assay. Each data point represents the average of duplicate
assays.
Figure 5:
Effect of incubation time on activity
restoration of three-subunit core cytochrome b-c complex by subunit IV. 26 µg of three-subunit cytochrome b-c
complex in 50 µl of 50 mM Tris-Cl, pH 8.0, containing 0.01% dodecylmaltoside and 300 mM NaCl was added to 4 µg of isolated recombinant subunit IV in
50 µl of 50 mM Tris-Cl buffer, pH 8.0, and incubated at 0
°C. At indicated time intervals, 5-µl aliquots were withdrawn
and assayed for ubiquinol-cytochrome c reductase
activity.
The addition of recombinant subunit IV to the three-subunit
core complex not only restored the enzymatic activity but also the
Q-binding environment. Fig. 6shows the
QH
-dependent activity titration curves for the
wild-type, reconstituted, and RS
IV cytochrome b-c
complexes. The apparent K
for
Q
H
of wild-type, reconstituted, and
three-subunit core cytochrome b-c
complexes were
2.2, 2.5, and 10.8, respectively. The restoration of the K
in the reconstituted complex further confirms
the involvement of subunit IV in Q binding of this complex.
Figure 6:
Titration of ubiquinol-cytochrome c reductase activity in wild-type, three-subunit core, and
reconstituted cytochrome b-c complexes with
various concentrations of Q
H
. Aliquots of
wild-type (
), RS
IV (
), and reconstituted (
)
cytochrome b-c
complexes in 50 mM Tris-HCl, pH 8.0, containing 0.01% dodecylmaltoside and 300 mM NaCl were added to a 1-ml assay mixture containing the indicated
concentrations of Q
H
. Reconstituted cytochrome b-c
complex was prepared by adding 10 µl of
purified subunit IV (1.0 mg/ml) to 140 µl (0.45 mg/ml) of RS
IV
complex and incubated at 0 °C for 1 h.
The
ubiquinol-cytochrome c reductase activity in the reconstituted
four-subunit complex is fully sensitive to antimycin treatment. A 50%
inhibition was found with 1 mol of antimycin/mol cytochrome c, a level identical to that observed for the
wild-type cytochrome b-c
complex.
The
reconstituted cytochrome b-c complex has the same
molecular size as the wild-type complex as revealed by the same
electrophoretic mobility of these two complexes in a nondenaturing blue
gel electrophoresis (data not shown). The apparent molecular mass of
these two complexes was estimated to be around 240 kDa, indicating that
they exist in dimer form. The isolated three-subunit core complex also
occurs in dimer form with a slightly higher electrophoretic mobility
than the wild-type complex in nondenaturing blue gel. Because the
isolated recombinant subunit IV in aqueous solution has an apparent
molecular mass of over one million, whereas the reconstituted
cytochrome b-c
complex has only 240 kDa,
deaggregation of subunit IV must occur during the reconstitution
process. This correlates with the observation that incubation time is
required for maximum reconstitution.
Because there are five tryptophans in subunit IV, the number of
tryptophans involved was unclear. To address this question, the
correlation between loss of reconstitutive activity of recombinant
subunit IV and tryptophan residues in subunit IV reacting with N-bromosuccimide was established. When recombinant subunit IV
was incubated with various concentrations of N-bromosuccimide
at room temperature for 10 min, the reaction of N-bromosuccimide with tryptophan residues was directly
proportional to the loss of reconstitutive activity of subunit IV, up
to a 6 molar excess of N-bromosuccimide (Fig. 7). About
70% of the reconstitutive activity of subunit IV was abolished when one
tryptophan residue was modified. The direct correlation between
activity loss and the tryptophan modification suggests that the first
tryptophan residue modified in subunit IV is required for interaction
with the three-subunit core complex. This tryptophan residue is more
reactive toward N-bromosuccimide than other tryptophan
residues in subunit IV. N-Bromosuccimide reacted with maximum
of three tryptophans in subunit IV even though there are five
tryptophans present. Because isolated recombinant subunit IV is in
decamer form under the modification conditions, the N-bromosuccimide inactive tryptophans must be buried inside
the aggregate. When wild-type four-subunit and three-subunit core
complexes were incubated with a 10 molar excess of N-bromosuccimide at room temperature for 10 min, no loss of
activity was observed. This indicates that the tryptophan residue in
subunit IV responsible for interaction with other core subunits is
shielded by their interacting subunit in the cytochrome b-c complex. Identification of the subunit IV
tryptophan residue responsible for this interaction is currently in
progress in our laboratory.
Figure 7:
Correlation between tryptophan
modification and inactivation of recombinant subunit IV. 1-ml aliquots
of recombinant subunit IV, 1 mg/ml, in 50 mM Tris-Cl, pH 8.0,
containing 300 mM NaCl and 0.01% dodecylmaltoside were added
to 25 µl of water containing indicated amounts of N-bromosuccimide (NBS) at room temperature. After
incubation at room temperature for 10 min, the amount of tryptophan
oxidized by N-bromosuccimide in each sample () was
determined as described under ``Experimental Procedures.''
Immediately after the oxidized tryptophan was quantitized, 3-µl
aliquots were withdrawn from each tube, added to 2-µl aliquots of
tryptophan (50 mM), incubated for another 5 min, and
reconstituted with 20 µl of the three-subunit core complex, 1
mg/ml, in 50 mM Tris-HCl, pH 8.0, containing 300 mM NaCl and 0.01% dodecylmaltoside. Ubiquinol-cytochrome c reductase activity (
) was assayed after incubation at 0
°C for 1 h.