INTRODUCTION

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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, b_L and b_H, 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 b_H; and we propose that heme may also be covalently bound to cytochrome b subunits of b₆f-type complexes.

	EXPERIMENTAL PROCEDURES

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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.

View this table: [in this window] [in a new window]   Table I Strains and plasmids Amp, Cm, Neo, Tet, and Em are abbreviations for ampicillin, chloramphenicol, neomycin, tetracycline, and erythromycin, respectively. Resistance or sensitivity to the relevant antibiotic is indicated by r or s superscripts, respectively. An arrow indicates transformation of the indicated strain with chromosomal or plasmid DNA. + and  indicate that the inserted antibiotic resistance marker is transcribed in the same or opposite direction to the qcr operon, respectively. Base pair (bp) numbering is according to Ref. 5.

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 P_spac was then ligated into similarly digested pVK47, a derivative of pDH32 lacking a 2.6-kb PvuII fragment of lacZ, resulting in pPP503.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Map of plasmid pPP503. Relevant restriction sites are indicated. The fragments of B. subtilis amyE gene (GenBankTM accession number V00101) present in the plasmid pDH32 and its derivatives are amy-front (positions 464-1019) and amy-back (positions 1451-2680).

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'.

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

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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 P_spac 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.

View larger version (48K): [in this window] [in a new window]   Fig. 2.   Analysis of heme-containing proteins in B. subtilis membrane preparations after SDS-PAGE. Strains were grown in NSMP medium supplemented with 5-[4-14C]aminolevulinic acid. Isolated membranes were incubated at 40 °C for 30 min in a 1:1 mixture with preparation buffer and analyzed on a 10-15% polyacrylamide gradient in the Neville buffer system (37). Approximately 150 µg of protein was loaded in each lane. After electrophoresis, the gel was incubated in methanol/acetic acid. An autoradiogram of the dried gel is shown. Lanes 1 and 2, membrane preparations from strain LUH58 grown in the presence (1 mM) and absence of IPTG, respectively. Lanes 3 and 4, membrane preparations from strain LUH59 grown in the presence (1 mM) and absence of IPTG, respectively. The radioactivity running at the gel front is due to dissociated heme.

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 b_H pocket (19) but is also proposed to be important for packing of heme b_L, while Arg¹¹¹ and Trp¹²⁶ are predicted to be close to heme b_H at the cytoplasmic boundary (20). His⁹⁴ serves as a ligand to heme b_L (19), and Arg⁹¹ is predicted to be lying close to it (18).

View larger version (115K): [in this window] [in a new window]   Fig. 3.   The effect of mutagenesis of B. subtilis QcrB on heme-containing proteins in membrane preparations after SDS-PAGE. Details of the experiment were as described in the Fig. 2 legend. An autoradiogram of the dried gel is shown. Lane 1, membrane preparation from strain 168; lanes 2 and 3, membrane preparations from strain LUH62 (wild-type qcrB under Pspac control) grown in the absence and presence (1 mM) of IPTG, respectively; lane 4, membrane preparation from LUH63 (Cys43  Ser); lane 5, membrane preparation from LUH64 (Gly45  Asp); lane 6, membrane preparation from LUH65 (Arg91  Gln); lane 7, membrane preparation from LUH66 (His94  Asp); lane 8, membrane preparation from LUH67 (Arg111  Gln); lane 9, membrane preparation from LUH68 (Trp126  Ala). Strains LUH63-LUH68 were all grown in the presence of 1 mM IPTG.

View larger version (63K): [in this window] [in a new window]   Fig. 4.   Analysis of heme-containing proteins in B. subtilis membrane preparations after extraction with acidified acetone and SDS-PAGE. Details of strain growth and membrane preparations were as described in the Fig. 2 legend. For strain 168, approximately 150 µg of protein was loaded, while for the acetone and acidified acetone-treated samples, approximately 400 µg was loaded. An autoradiogram of the dried gel is shown. Lane 1, membrane preparation from strain 168; lane 2, acetone-extracted membrane preparation from strain 168; lane 3, acidified acetone-extracted membrane preparation from strain 168; lane 4, membrane preparation from strain 168 heated to 100 °C for 5 min prior to loading.

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).

View larger version (55K): [in this window] [in a new window]   Fig. 5.   A, alignment of partial sequences of cytochrome b subunits of bc-type complexes from bovine heart mitochondria (Bos taurus) (38), S. cerevisiae (39), R. sphaeroides (40), Synechocystis PCC6803 (41), and B. subtilis (5) generated using the program PileUp. Residue numbers are indicated at the beginning and end of each line. Helix A and Helix B indicate predicted transmembrane helices (18). bL and bH indicate histidine residues that serve as axial ligands to the respective heme groups. The arrows indicate residues that have been substituted in the course of this study. B, schematic representation of the folded QcrB polypeptide. The four proposed transmembrane helices are labeled A-D. The two hemes, bL and bH, are indicated.

	DISCUSSION

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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 b_L (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 b_L than to those of b_H. An assembled complex deficient in heme b_H could be obtained, but one deficient in heme b_L could not (19). Data presented here indicate that the structure/assembly of B. subtilis QcrB is similarly sensitive to changes in the b_L 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 b_H (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 b_H, although Arg¹¹⁴ could be replaced only with another positively charged residue, i.e. lysine. The data reported here indicate that the b_H 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 b_H binding properties. It is not known whether heme b_H 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 b_L, previous studies of bc₁-type complexes indicate that this heme may be more easily lost than heme b_H 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 b_H, rather than b_L, remains bound, and sequence alignment studies show further that there is a cysteine residue (Cys⁴³) lying close to the b_H 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 b_H 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 (Q_i or Q_N) site (32). Thus, the data are consistent with a direct role for Cys⁴³ in heme b_H 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 b_H binding, it is possible that a residue other than Cys⁴³ is the site of covalent attachment. If covalent attachment between Cys⁴³ and heme b_H 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).