From the Department of Microbiology and Immunology,
Temple University School of Medicine, Philadelphia, Pennsylvania 19140 and the § Department of Microbiology, Lund University,
S-223 62 Lund, Sweden
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The menaquinone:cytochrome c reductase, or bc complex, of Bacillus subtilis belongs to a third class of bc-type complex, distinct from the bc1 and b6f classes. Using a mutagenesis approach, we demonstrate that the cytochrome b (QcrB) and c (QcrC) subunits of the complex give rise to bands at 22 and 29 kDa, respectively, after denaturing electrophoresis; that both subunits are required for proper complex assembly and/or stability; and that both subunits retain one heme molecule under denaturing conditions. This unusual property of a b-type cytochrome was investigated further. We present evidence for the existence of a covalent linkage between the polypeptide and heme bH and of an important role for Cys43 in binding of heme bH. It is proposed that heme is also covalently attached to the cytochrome b subunit of b6f complexes of chloroplasts and cyanobacteria.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The cytochrome bc1 complex (quinol:cytochrome c oxidoreductase) is an integral membrane protein complex that functions as part of an electron transfer chain by passing electrons from quinol in the membrane to a c-type cytochrome. Coupled to electron transfer is the transport of protons across the membrane, and consequently, the enzyme contributes to the proton motive force. The complex has been isolated from mitochondria and several bacteria, and a similar complex, called the b6f complex, has been isolated from plant chloroplasts and from cyanobacteria. Recently, crystal structures of the soluble part of the Rieske protein (1) and of the intact complex (2) from bovine heart mitochondria have been solved.
There is considerable variation, depending on the source, in the number
of subunits making up the complex, but three subunits are always
present: an iron-sulfur (Rieske) protein containing a high potential
[2Fe-2S] cluster with cysteine and histidine ligation; a cytochrome
b containing two low spin b-type hemes, bL and bH, both with
bis-histidine ligation but with different electrochemical properties;
and a cytochrome c containing one c-type heme
with histidine/methionine axial ligation in the case of
c1 and histidine/tyrosine (-amino group) in
the case of f (3). The cytochrome b of
bc1 complexes consists of approximately 400 amino acid residues, arranged as eight transmembrane helices, while the
cytochrome b of b6f
complexes may be regarded as being split into two subunits: one of
~220 residues, containing four transmembrane helices, which house the
two b-type hemes; and the other, called subunit IV, of
~160 residues, which contains three transmembrane helices.
Relatively little is known about the bc-type complexes of Gram-positive bacteria. Only one complex, that from the thermophilic bacterium Bacillus sp. PS3, has been purified to date (4). Genes (called qcrABC) encoding bc-type complexes have recently been identified in Bacillus subtilis (5) and Bacillus stearothermophilus (6). Their predicted protein products appear to constitute a distinct, third class of complex, which bears greater similarity to b6f than to bc1 complexes. The predicted 224-amino acid residue cytochrome b subunit (QcrB) is similar to that of b6f complexes. However, the third subunit of the complex, QcrC, can be regarded as a fusion protein consisting of subunit IV and a cytochrome c. The latter bears little similarity to either cytochrome c1 or cytochrome f but, rather, resembles small Bacillus c-type cytochromes.
Cytochromes of c-type are distinct from other cytochromes in that the heme molecule is covalently attached, generally via two thioether linkages, to the polypeptide. SDS-PAGE1 of Bacillus subtilis membrane preparations from cells grown in the presence of the heme biosynthetic precursor, 5-aminolevulinic acid, 14C-labeled, has previously been used to identify membrane-bound c-type cytochromes because noncovalently attached heme (presumably, all heme except heme c) is lost from its protein ligands under the denaturing conditions of the gel (7). Such an analysis of wild-type B. subtilis reveals four major radioactive bands at 36, 29, 22, and 16 kDa, respectively. Two of the four bands have been identified unambiguously; the 36-kDa band is due to subunit II of the cytochrome c oxidase, caa3, and the 16-kDa band is due to cytochrome c550 (8). The 16-kDa c550 band masks an additional small cytochrome c, which has been recently identified.2 The two remaining bands, of 29 and 22 kDa, are associated with the qcr operon, since deletion of the promoter region results in the loss of both bands (5). However, it is unclear why the qcr operon, which encodes one c-type cytochrome, should give rise to two bands. It was proposed that the 29-kDa band may be QcrC (predicted mass = 28 kDa) and that the 22-kDa band might be a degradation product of QcrC. Studies using radioactively labeled heme in place of aminolevulinic acid confirmed that both the 22 and 29 kDa bands contain heme and not an alternative prosthetic group for which aminolevulinic acid is a precursor (9). Here we describe an investigation of the identities of the bands, using a combination of qcr gene deletion, insertion, and site-directed mutagenesis in B. subtilis, which shows that the 29- and 22-kDa bands correspond to the cytochrome c (QcrC) and cytochrome b (QcrB) subunits of the bc complex, respectively. Normally, b-type heme dissociates from its ligating polypeptide under denaturing conditions, but the case of QcrB is clearly an exception. Resistance to extraction by acidified acetone suggests that one heme of QcrB is covalently linked to the polypeptide. We present evidence from site-directed mutagenesis studies of QcrB that indicates that Cys43 is involved in binding heme bH; and we propose that heme may also be covalently bound to cytochrome b subunits of b6f-type complexes.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Strains and Growth Media-- The bacterial strains and plasmids used are presented in Table I. The B. subtilis 168 strain used in this study was found to be oligosporogenic. We have observed that c-type cytochromes in sporulating strains undergo much greater proteolytic degradation than those of strains defective in sporulation. Escherichia coli strains were grown at 37 °C in LB broth (10) or on LA plates consisting of LB with 0.9% (w/v) agar, or on TBAB (Difco) plates. B. subtilis strains were grown at 37 °C in NSMP (11) or on TBAB plates. TBAB plates containing 1.5% (w/v) starch were used to test strains for amylase production. Antibiotics, where appropriate, were added to liquid media and plates at the following concentrations: chloramphenicol, 4 mg/liter (B. subtilis) or 12.5 mg/liter (E. coli); erythromycin, 1 mg/liter; ampicillin, 100 mg/liter; neomycin, 4 mg/liter; tetracycline, 12.5 mg/liter.
|
Genetic Techniques--
General molecular genetics techniques
were used as described in Sambrook et al. (10). Plasmid DNA
was isolated using Bio-Rad or Promega Wizard miniprep kits. Chromosomal
DNA from B. subtilis strains was isolated as described by
Marmur (12). E. coli strains were transformed by
electroporation (13) using a Bio-Rad Gene pulser. B. subtilis-competent cells were prepared and transformed essentially
as described previously (14). DNA sequence analysis was carried out
using the dideoxynucleotide termination method (15), with Sequenase II
(Stratagene/Amersham Pharmacia Biotech) and [-35S]ATP
(Amersham Pharmacia Biotech).
Construction of Plasmids pPP503, pPP502, and pPP491-- Plasmid pPP503 (Fig. 1) was constructed as follows. The 2.8-kb XbaI-ClaI fragment of plasmid pPP499 containing qcrABC was ligated into similarly digested pDH88. A 63-base pair XbaI-NotI fragment of the resulting plasmid was removed, and the plasmid was recircularized by ligation after the ends had been made blunt by Klenow enzyme treatment. A 4.4-kb EcoRI-BamHI fragment of the plasmid containing qcrABC under Pspac was then ligated into similarly digested pVK47, a derivative of pDH32 lacking a 2.6-kb PvuII fragment of lacZ, resulting in pPP503.
|
Site-directed Mutagenesis-- The Altered Sites II in vitro Site-specific mutagenesis system (Promega) was used to introduce mutations into qcrB. Six mutagenic oligonucleotides were synthesized (The Great American Gene Company, Ransom Hill Bioscience Inc.). The substituted nucleotides are underlined: C43S, 5'-GCGTTTGTGTATTCTTTTGGGGGAC-3'; G45D, 5'-GTGTATTGCTTTGACGGACTGACGT-3'; R91Q, 5'-GGCCAGATTGTCCAGGGGATGCA-3'; H94D, 5'-GGGGGATGGACCACTGGGG-3'; R111Q, 5'-TACATACGCTGCAGGTCTTTTTCC-3'; W126A, 5'-GCGAGCTGAACGCGATTGTCGG-3'.
Mutagenesis was carried out on the pALTER-1 derivative pPP511. The desired mutations were confirmed by DNA sequence analysis using custom primers. Plasmids for insertion of mutant qcrB variants under the Pspac promoter at the amyE locus on the B. subtilis chromosome were obtained by digesting the mutant pPP511 plasmids with HindIII and ligating the 1.6-kb qcrAB fragment into HindIII-digested pPP502.Heme-specific Labeling-- Growth of strains, heme-specific labeling, and preparation of membranes were carried out as described previously (5). Briefly, strains were grown overnight at 37 °C in 25 ml of NSMP supplemented with 2 µM 5-[4-14C]aminolevulinic acid (51 mCi/mmol). Cells were collected by centrifugation and treated with lysosyme, and the particulate fraction was collected, washed, and suspended in 20 mM MOPS buffer, pH 7.4. The particulate fraction was incubated at 40 °C for 30 min or at 100 °C for 5 min in the presence of SDS. SDS-PAGE and subsequent treatment of the gel was carried out as described previously except that the gel was not treated with salicylic acid prior to drying (5). The dried gel was exposed to a storage phosphor screen for 1 week, and the screen was scanned using a PhosphorImager SI (Molecular Dynamics). The resulting digitized autoradiogram was analyzed using the program ImageQuant (Molecular Dynamics).
Heme Extraction-- Isolated B. subtilis membranes (75 µl, protein concentration approximately 14 mg/ml) were precipitated by the addition of 1 ml of ice cold acetone and mixing. The suspension was centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was removed. The pellet was twice extracted with 1 ml of ice-cold acidified acetone (24 mM in HCl) and subsequently twice more with ice-cold acetone, also at 4 °C. Finally, the pellet was resuspended in 20 µl of 40 mM Tris borate buffer containing 3.5 mM SDS, pH 8.64.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Analysis of qcrB and qcrC Mutants-- Mutants of strain 168 containing an insertion in qcrB (LUH51) and a deletion of qcrC (LUH52), respectively, were constructed and grown in the presence of 5-[4-14C]aminolevulinic acid. SDS-PAGE of resulting membrane preparations followed by autoradiography revealed that both mutants lacked the 29-kDa band. LUH51 also lacked the 22-kDa band, while LUH52 contained a faint band at 22 kDa (autoradiogram not shown). The latter result is consistent with the assignment of the 29-kDa band as QcrC, while the absence of the 29-kDa band from the qcrB mutant, LUH51, might arise from a polar effect on transcription of qcrC resulting from the insertion of the neo gene cassette into qcrB. To investigate this further, two additional mutants were constructed. These strains, LUH58 and LUH59, contained the same qcrB and qcrC mutations as LUH51 and LUH52, respectively, and in addition an intact copy of qcrC under the control of the inducible Pspac promoter, inserted elsewhere on the chromosome, at the amyE locus. The strains were grown in the presence of 5-[4-14C]aminolevulinic acid and in the presence and absence of the inducer, IPTG. An autoradiogram of a SDS gel of the membrane preparations is shown in Fig. 2. Strain LUH59, grown in the presence of inducer (lane 3), gives rise to both 29- and 22-kDa bands, while in the absence of inducer (lane 4) the 29-kDa band was not observed and only a faint band at 22-kDa was detected. Thus, the deletion of qcrC from the qcr operon can be complemented in trans by qcrC. Lanes 1 and 2, which result from strain LUH58 grown in the presence and absence of the inducer, respectively, contain neither the 29- nor the 22-kDa band. These data show that both QcrB and QcrC subunits are required in order for either the 29- or 22-kDa band to be observed at normal intensity. This may be due to the instability of the complex in the absence of either cytochrome subunit or may result from the requirement of both subunits for proper folding and cofactor insertion into the subunits. The presence of a faint 22-kDa band in strains LUH52 and LUH59 (grown in the absence of IPTG) suggested that the 22-kDa band is encoded by qcrB.
|
Mutagenesis of QcrB--
The above data failed to distinguish
unequivocally the identities of the 29- and 22-kDa bands. To identify
the bands, we sought to alter the assembly and/or the heme binding
properties of QcrB, and accordingly, site-directed variants of QcrB
were generated. Few previous mutagenesis studies of a
b6f-type cytochrome b
protein have been reported (17), so we relied upon data available from studies of bc1-type cytochrome b
proteins but considered also the possibility that the replacement of
residues important for heme binding and subunit folding in the
bc1-type protein might affect the
b6f-type protein differently. The
mutations selected were as follows: Gly45 Asp,
Arg91
Gln, His94
Asp,
Arg111
Gln, and Trp126
Ala. Three of
the residues, Gly45, His94, and
Trp126, are invariant, and the remaining two are highly
conserved, among cytochrome b sequences (18).
Gly45 is believed to form part of the heme
bH pocket (19) but is also proposed to be
important for packing of heme bL, while
Arg111 and Trp126 are predicted to be close to
heme bH at the cytoplasmic boundary (20).
His94 serves as a ligand to heme bL
(19), and Arg91 is predicted to be lying close to it
(18).
|
|
Heme Attached to QcrB Survives Extraction with Acidified Acetone-- In b-type cytochromes, heme is bound to the protein via noncovalent interactions, which are usually easily disrupted when the protein is denatured. That heme remains associated with QcrB under denaturing conditions indicates that heme may be covalently attached to QcrB. To investigate this possibility further, membranes from B. subtilis strain 168 with 14C-labeled heme were treated with acidified acetone, according to the procedure described by Rieske (21) for the extraction of noncovalently bound heme from cytochromes. The autoradiogram resulting from SDS-PAGE analysis of extracted membranes is shown in Fig. 4. Lanes 2 and 3 correspond to treatment with acetone and acidified acetone, respectively. The lanes differ only at the gel front, where the band due to dissociated heme is significantly reduced in intensity after acidified acetone extraction. Clearly, the intensity of the band due to QcrB is unaffected relative to QcrC, consistent with the covalent attachment of heme to QcrB.
Investigation of the Role of Cys43 in Heme Binding in
QcrB--
Tight binding of heme to QcrB is also found in some other
bc-type complexes. Studies of cytochrome
b6f complex from pea and spinach
chloroplasts indicated, by use of peroxidase activity as a stain for
heme, that heme remains associated with the b6 subunit, even under denaturing conditions (22, 23). A similar observation was made from SDS-PAGE analysis of purified bc
complex from Bacillus sp. PS3 (which is similar to B. stearothermophilus) (4). However, early studies of a bacterial
bc1 complex by SDS-PAGE analysis showed, also
through peroxidase activity staining, that the heme groups of
cytochrome b are very easily released from the protein in
the presence of -mercaptoethanol (24).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The work described here completes the assignment of all
heme-containing protein bands observed after SDS-PAGE analysis of membrane preparations from B. subtilis. Data from deletion
studies and the site-directed QcrB variants Arg111 Gln
and Cys43
Ser show clearly that the bands at 29 and 22 kDa belong to the QcrC and QcrB subunits of the bc complex,
respectively.
Relatively little is known about the synthesis and assembly of bc1/b6f complexes. A tentative model for maturation of bacterial bc1 complexes was recently proposed, in which cytochromes b and c1 form a protease-resistant primary complex to which the Rieske iron-sulfur protein then associates (27). In B. subtilis, both QcrB and QcrC are required in order that either subunit be observed at normal intensity, indicating that the presence of both subunits is essential for the correct folding and assembly of the complex. Lane 4 of Fig. 2, arising from LUH59 (a qcrC deletion mutant), contains a faint band at 22 kDa, indicating a small amount of holo-QcrB. In the absence of QcrB, however, no trace of QcrC is detected (Fig. 2, lanes 1 and 2). These findings suggest that QcrB can be inserted into the membrane and bind heme to some degree in the absence of QcrC but that in the absence of QcrB no holo-QcrC is formed.
The substitutions Gly45 Asp, Arg91
Gln,
His94
Asp, and Trp126
Ala did not
result in a detectable complex, and we conclude that these
substitutions result in a QcrB protein that is unable to fold
correctly. Gly45 is one of several invariant glycine
residues that are believed to be important for the structure of
cytochrome b and particularly for the packing of the two
hemes. Replacement of the equivalent glycine residue of R. sphaeroides (Gly48) and S. cerevisiae
(Gly33) with aspartate also resulted in the failure of the
complex to assemble (26, 28). His94 is one of the four
invariant histidine residues that serve as ligands to the two
b-type hemes. Mutagenesis studies of R. sphaeroides cytochrome b showed that this residue
(His97 in R. sphaeroides) ligates heme
bL (19), a conclusion that was recently
confirmed by the x-ray structure of the bc1
complex from bovine heart (2). Mutagenesis studies also indicated that assembly and stability of the Rhodobacter protein is more
sensitive to changes in the heme ligands of bL
than to those of bH. An assembled complex
deficient in heme bH could be obtained, but one
deficient in heme bL could not (19). Data
presented here indicate that the structure/assembly of B. subtilis QcrB is similarly sensitive to changes in the
bL heme pocket, since neither Arg91
Gln nor His94
Asp gave a detectable complex.
Arg111 and Trp126 are predicted to be near the
cytoplasmic surface of the QcrB subunit, close to heme
bH (20). The equivalent residues
(Arg114 and Trp129) of R. sphaeroides are not absolutely essential for assembly/stability of
the complex or for binding of heme bH, although
Arg114 could be replaced only with another positively
charged residue, i.e. lysine. The data reported here
indicate that the bH site in the cytochrome
b subunit of B. subtilis must be somewhat
different from that of R. sphaeroides. The location of a
positive charge at position 111 was not found to be essential for
assembly/stability of the B. subtilis bc complex, since the
replacement Arg111
Gln resulted in an assembled
complex, albeit with altered bH binding
properties. It is not known whether heme bH is
bound to this variant enzyme complex in vivo and
subsequently lost during electrophoresis or whether it fails to insert
at all. The Trp126
Ala substitution did not lead to
assembled complex, and we conclude that this residue is more critical
for assembly/stability of the bc complex from B. subtilis than it is for the bc1 complex from R. sphaeroides.
Although it appears that proper assembly of QcrB will not survive
substitutions in the region of heme bL, previous
studies of bc1-type complexes indicate that this
heme may be more easily lost than heme bH during
purification (29, 30). However, under denaturing conditions both hemes
are readily lost from the cytochrome b subunit of
bc1-type complexes. Here, we have demonstrated
that QcrB from B. subtilis retains heme under these
conditions. Acidified acetone extraction of noncovalently bound heme
from membrane preparations of B. subtilis, prior to SDS-PAGE
analysis, failed to affect the intensity of the QcrB band, consistent
with the covalent attachment of heme to QcrB. Studies of the purified
bc complex from Bacillus PS3 indicate that this
is likely to be a common property of Bacillus QcrB subunits
(4). In addition, studies indicate that Bacillus firmus OF4
has the same pattern of heme-containing bands as B. subtilis
after electrophoresis of membrane preparations, although with some
variation in apparent size (31). The ability to heme-stain cytochrome
b of the b6f complex of
chloroplasts after SDS-PAGE suggests that this may be a property of all
b6f-type cytochrome b
proteins (22, 23). Mutagenesis studies reported here indicate that it
is likely that heme bH, rather than
bL, remains bound, and sequence alignment
studies show further that there is a cysteine residue
(Cys43) lying close to the bH site,
which is invariant among b6f-type QcrB proteins but not among bc1-type proteins.
The substitution Cys43 Ser abolished the ability of
QcrB to retain heme under denaturing conditions, although the complex
was still assembled. This could simply be due to an alteration of the
bH heme binding pocket that prevents heme
insertion. However, the replacement residue, serine, is of similar size
to cysteine, has similar electrostatic properties, and would not be
expected to cause a significant structural rearrangement. The
equivalent residue in other cytochrome b proteins varies
considerably, from asparagine in most mitochondrial proteins to
isoleucine in Rhodobacter to threonine in
Bradyrhizobium (18), suggesting that it is not essential for
protein folding. Indeed, substitution of the equivalent residue of
S. cerevisiae cytochrome b, Asn31
(Fig. 5A) with lysine did not affect the heme binding
properties of the cytochrome b subunit but affected the
sensitivity of the complex to inhibitors of electron transport that
bind at the quinone reduction (Qi or QN) site
(32). Thus, the data are consistent with a direct role for
Cys43 in heme bH binding to B. subtilis QcrB.
Recently, several examples of unusual covalent bonds within metalloproteins have been reported, including the covalent attachment of heme to tyrosine (33) and to lysine residues (34). Therefore, although we have shown that Cys43 is important for heme bH binding, it is possible that a residue other than Cys43 is the site of covalent attachment. If covalent attachment between Cys43 and heme bH does occur, it is not clear whether such a bond is of functional importance or what the nature of the attachment is. Cysteine residues of c-type cytochromes form thioether bonds with the heme vinyl side chains. However, these cysteines occur in the conserved motif Cys-X-X-Cys-His, which is not present in QcrB. There are examples of c-type hemes that are attached by only one cysteine instead of the usual two (e.g. cytochrome c1 of Euglena gracilis (35)), but in these cases a shorter Cys-His motif is found. What is certain, however, is that the covalent attachment is not formed by the general cellular machinery for cytochrome c biosynthesis; holo-QcrB is formed in a B. subtilis strain deleted for ccdA, a gene essential for the synthesis of c-type cytochromes (36).
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. Lars Hederstedt and Patrick J. Piggot for their help and support. We thank Dr. Thomas A. Link for helpful discussions and Dr. Edward A. Berry for providing structural information on the chicken bc1 complex.
![]() |
FOOTNOTES |
---|
* This work was supported by Swedish Natural Science Research Council Grant BU 01637-321/323 (to Dr. Lars Hederstedt); by a European Molecular Biology long term fellowship (to NLB); and by Public Service Grant GM43577 (to Dr. Patrick J. Piggot).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Microbiology, Lund University, Sölvegatan 12, S-223 62 Lund, Sweden.
1
The abbreviations used are: PAGE, polyacrylamide
gel electrophoresis; TBAB, tryptose blood agar base; NSMP, nutrient
sporulation medium phosphate; IPTG, isopropyl
-D-thiogalactoside; kb, kilobase pair; MOPS,
4-morpholinepropanesulfonic acid.
2 J. Bengtsson, C. Rivolta, L. Hederstedt, and D. Karamata, unpublished data.
3 E. A. Berry, personal communication.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|