Mutations in cytochrome assembly and periplasmic redox pathways in Bordetella pertussis

Transposon mutagenesis of Bordetella pertussis was used to discover mutations in the cytochrome c biogenesis pathway called system II. Using a tetramethyl-p-phenylenediamine cytochrome c oxidase screen, 27 oxidase-negative mutants were isolated and characterized. Nine mutants were still able to synthesize c-type cytochromes and possessed insertions in the genes for cytochrome c oxidase subunits (ctaC, -D, and -E), heme a biosynthesis (ctaB), assembly of cytochrome c oxidase (sco2), or ferrochelatase (hemZ). Eighteen mutants were unable to synthesize all c-type cytochromes. Seven of these had transposons in dipZ (dsbD), encoding the transmembrane thioreduction protein, and all seven mutants were corrected for cytochrome c assembly by exogenous dithiothreitol, which was consistent with the cytochrome c cysteinyl residues of the CXXCH motif requiring periplasmic reduction. The remaining 11 insertions were located in the ccsBA operon, suggesting that with the appropriate thiol-reducing environment, the CcsB and CcsA proteins comprise the entire system II biosynthetic pathway. Antiserum to CcsB was used to show that CcsB is absent in ccsA mutants, providing evidence for a stable CcsA-CcsB complex. No mutations were found in the genes necessary for disulfide bond formation (dsbA or dsbB). To examine whether the periplasmic disulfide bond pathway is required for cytochrome c biogenesis in B. pertussis, a targeted knockout was made in dsbB. The DsbB- mutant makes holocytochromes c like the wild type does and secretes and assembles the active periplasmic alkaline phosphatase. A dipZ mutant is not corrected by a dsbB mutation. Alternative mechanisms to oxidize disulfides in B. pertussis are analyzed and discussed.     

Haem-delivery proteins in cytochrome c maturation System II

Cytochromes of the c -type function on the outer side of the cytoplasmic membrane in bacteria where they also are assembled from apo-cytochrome polypeptide and haem. Two distinctly different systems for cytochrome c maturation are found in bacteria. System I present in Escherichia coli has eight to nine different Ccm proteins. System II is found in Bacillus subtilis and comprises four proteins: CcdA, ResA, ResB and ResC. ResB and ResC are poorly understood polytopic membrane proteins required for cytochrome c synthesis. We have analysed these two B. subtilis proteins produced in E. coli and in the native organism. ResB is shown to bind protohaem IX and haem is found covalently bound to residue Cys-138. Results in B. subtilis suggest that also ResC can bind haem. Our results complement recent findings made with Helicobacte r CcsBA supporting the hypothesis that ResBC as a complex translocates haem by attaching it to ResB on the cytoplasmic side of the membrane and then transferring it to an extra-cytoplasmic location in ResC, from where it is made available to the apo-cytochromes.     

Genes required for cytochrome c synthesis in Bacillus subtilis

Cytochromes of c-type contain covalently bound haem and in bacteria are located on the periplasmic side of the cytoplasmic membrane. More than eight different gene products have been identified as being specifically required for the synthesis of cytochromes c in Gram-negative bacteria. Corresponding genes are not found in the genome sequences of Gram-positive bacteria. Using two random mutagenesis approaches, we have searched for cytochrome c biogenesis genes in the Gram-positive bacterium Bacillus subtilis. Three genes, resB, resC and ccdA, were identified. CcdA has been found previously and is required for a late step in cytochrome c synthesis and also plays a role in spore synthesis. No function has previously been assigned for ResB and ResC but these predicted membrane proteins show sequence similarity to proteins required for cytochrome c synthesis in chloroplasts. Attempts to inactivate resB and resC in B. subtilis have indicated that these genes are essential for growth. We demonstrate that various nonsense mutations in resB or resC can block synthesis of cytochromes c with no effect on other types of cytochromes and little effect on sporulation and growth. The results strongly support the recent proposal that Gram-positive bacteria, cyanobacteria, epsilon-proteobacteria, and chloroplasts have a similar type of machinery for cytochrome c synthesis (System II), which is very different from those of most Gram-negative bacteria (System I) and mitochondria (System III).     

Role of His residues in Bacillus subtilis cytochrome b558 for haem binding and assembly of succinate: quinone oxidoreductase (complex II)

Cytochrome b558 in the cytoplasmic membrane of Bacillus subtilis constitutes the anchor and electron acceptor to the flavoprotein (Fp) and iron-sulphur protein (Ip) in succinate:quinone oxidoreductase, and seemingly contains two haem groups. EPR and MCD spectroscopic data indicate bis-imidazole ligation of the haem. Apo-cytochrome was found in the membrane fraction of haem-deficient B. subtilis, suggesting that during biogenesis of the oxidoreductase the cytochrome b558 polypeptide is embedded into the membrane prior to the incorporation of haem and subsequent binding of Fp and Ip. The six His residues in cytochrome b558 were individually changed to Tyr to attempt identification of residues serving as haem axial ligands and to analyse the role of His residues for assembly and function of the oxidoreductase. From the properties of the mutants, His-47 can be excluded as a haem ligand. The remaining His residues (at positions 13, 28, 70, 113 and 155) are located in or close to four predicted transmembrane segments. The Tyr-28 and Tyr-70 mutant proteins appeared to lack one of the two haems. Only the Tyr-13 and Tyr-47 mutant cytochromes were found to function as anchors for Fp and Ip, but the Tyr-13 mutant cytochrome assembles into an enzymatically defective succinate:quinone oxidoreductase. It is concluded from a combination of the experimental findings, sequence comparisons and membrane topology data that His-28, His-70 and His-155 are probably haem axial ligands in a dihaem cytochrome b558. His-70 and His-155 may be ligands to the same haem.     

Recombinant cytochromes c biogenesis systems I and II and analysis of haem delivery pathways in Escherichia coli

Genetic analysis has indicated that the system II pathway for c-type cytochrome biogenesis in Bordetella pertussis requires at least four biogenesis proteins (CcsB, CcsA, DsbD and CcsX). In this study, the eight genes (ccmA-H) associated with the system I pathway in Escherichia coli were deleted. Using B. pertussis cytochrome c4 as a reporter for cytochromes c assembly, it is demonstrated that a single fused ccsBA polypeptide can replace the function of the eight system I genes in E. coli. Thus, the CcsB and CcsA membrane complex of system II is likely to possess the haem delivery and periplasmic cytochrome c-haem ligation functions. Using recombinant system II and system I, both under control of IPTG, we have begun to study the capabilities and characteristics of each system in the same organism (E. coli). The ferrochelatase inhibitor N-methylprotoporphyrin was used to modulate haem levels in vivo and it is shown that system I can use endogenous haem at much lower levels than system II. Additionally, while system I encodes a covalently bound haem chaperone (holo-CcmE), no covalent intermediate has been found in system II. It is shown that this allows system I to use holo-CcmE as a haem reservoir, a capability system II does not possess.     

Identification and characterization of the ccdA gene, required for cytochrome c synthesis in Bacillus subtilis.

The gram-positive, endospore-forming bacterium Bacillus subtilis contains several membrane-bound c-type cytochromes. We have isolated a mutant pleiotropically deficient in cytochromes c. The responsible mutation resides in a gene which we have named ccdA (cytochrome c defective). This gene is located at 173 degrees on the B. subtilis chromosome. The ccdA gene was found to be specifically required for synthesis of cytochromes of the c type. CcdA is a predicted 26-kDa integral membrane protein with no clear similarity to any known cytochrome c biogenesis protein but seems to be related to a part of Escherichia coli DipZ/DsbD. The ccdA gene is cotranscribed with two other genes. These genes encode a putative 13.5-kDa single-domain response regulator, similar to B. subtilis CheY and Spo0F, and a predicted 18-kDa hydrophobic protein with no similarity to any protein in databases, respectively. Inactivation of the three genes showed that only ccdA is required for cytochrome c synthesis. The results also demonstrated that cytochromes of the c type are not needed for growth of B. subtilis.     

Bacillus subtilis CtaA and CtaB function in haem A biosynthesis

Haem A, a prosthetic group of many respiratory oxidases, is probably synthesized from haem B (protohaem IX) in a pathway in which haem O is an intermediate. Possible roles of the Bacillus subtilis ctaA and CtaB gene products in haem O and haem A synthesis were studied. Escherichia coli does not contain haem A. The CtaA gene on plasmids in E. coli resulted in haem A accumulation in membranes. The presence of CtaB together with ctaA increased the amount of haem A found in E. coli. Haem O was not detected in wild-type B. subtilis strains. A previously isolated B. subtilis CtaA deletion mutant was found to contain haem B and haem O, but not haem A. B. subtilis ctaB deletion mutants were constructed and found to tack both haem A and haem O. The results with E. coli and B. subtilis strongly suggest that the B. subtilis CtaA protein functions in haem A synthesis. It is tentatively suggested that it functions in the oxygeNatlon/oxidation of the methyl side group of carbon 8 of haem O. B. subtilis CtaB, which is homologous to Saccharomyces cerevisiae COX10 and E. coli CyoE, also has a role in haem A synthesis and seems to be required for both cytochrome a and cytochrome o synthesis.     

The Bacillus subtilis cytochrome-c oxidase. Variations on a conserved protein theme.

The structural genes of cytochrome-c oxidase in Bacillus subtilis have been isolated and sequenced. Five genes, ctaB-F, are closely spaced. ctaC, ctaD, ctaE and ctaF are the genes for subunits II, I, III and IVB, respectively, ctaB, which may encode an assembly factor, is separated and upstream from the others. In comparison to its mitochondrial counterparts, subunit I has an extended C-terminus with two additional transmembrane segments, whereas subunit III has lost two such segments from its N-terminus. The C-terminal extension in subunit II is a covalent cytochrome-c domain, previously characterized only in the thermophilic oxidases. Subunit IVB, a small hydrophobic protein, is a novel subunit. These predictions suggest that the B. subtilis cytochrome-c oxidase is structurally more related to the four-subunit Escherichia coli cytochrome-bo complex than, for instance, to the Paracoccus denitrificans enzyme. Cytochrome aa3, which was previously isolated from B. subtilis [de Vrij, W., Azzi, A. & Konings, W. N. (1983) Eur. J. Biochem. 131, 97-103] is not encoded by the ctaC-F genes; thus, there seems to be two different cytochrome-aa3-type oxidases in this Gram-positive bacterium.     

Four genes are required for the system II cytochrome c biogenesis pathway in Bordetella pertussis, a unique bacterial model

Unlike other cytochromes, c-type cytochromes have two covalent bonds formed between the two vinyl groups of haem and two cysteines of the protein. This haem ligation requires specific assembly proteins in prokaryotes or eukaryotic mitochondria and chloroplasts. Here, it is shown that Bordetella pertussis is an excellent bacterial model for the widespread system II cytochrome c synthesis pathway. Mutations in four different genes (ccsA, ccsB, ccsX and dipZ) result in B. pertussis strains unable to synthesize any of at least seven c-type cytochromes. Using a cytochrome c4:alkaline phosphatase fusion protein as a bifunctional reporter, it was demonstrated that the B. pertussis wild-type and mutant strains secrete an active alkaline phosphatase fusion protein. However, unlike the wild type, all four mutants are unable to attach haem covalently, resulting in a degraded N-terminal apocytochrome c4 component. Thus, apocytochrome c secretion is normal in each of the four mutants, but all are defective in a periplasmic assembly step (or export of haem). CcsX is related to thioredoxins, which possess a conserved CysXxxXxxCys motif. Using phoA gene fusions as reporters, CcsX was proven to be a periplasmic thioredoxin-like protein. Both the B. pertussis dipZ (i. e. dsbD) and ccsX mutants are corrected for their assembly defects by the thiol-reducing compounds, dithiothreitol and 2-mercaptoethanesulphonic acid. These results indicate that DipZ and CcsX are required for the periplasmic reduction of the cysteines of apocytochromes c before ligation. In contrast, the ccsA and ccsB mutants are not corrected by exogenous reducing agents, suggesting that CcsA and CcsB are required for the haem ligation step itself in the periplasm (or export of haem to the periplasm). Related to this suggestion, the topology of CcsB was determined experimentally, demonstrating that CcsB has four transmembrane domains and a large 435-amino-acid periplasmic region.     

Bacillus subtilis expresses two kinds of haem-A-containing terminal oxidases

The expression of two different aa3-type cytochrome oxidases is demonstrated in Bacillus subtilis. One of them (denoted caa3-605), was predicted by DNA-sequencing of Bacillus cytochrome oxidase genes, but has not been found previously. It contains covalently bound haem C in subunit II and is very similar to the enzyme previously described in the thermophilic bacterium PS3. The other oxidase (denoted aa3-600) deviates from most known oxidases of aa3 type, and is probably identical with the oxidase described by de Vrij et al. [de Vrij, W., Azzi, A. & Konings, W. N. (1983) Eur. J. Biochem. 131, 97-103]. It shows no immunological cross-reactivity to the PS3 enzyme and differs from this spectroscopically; it contains no CuA and does not oxidise cytochrome c despite of its haem-A chromophores. It catalyses oxidation of quinols, which is proposed to be its physiological function.     

The nrfI gene is essential for the attachment of the active site haem group of Wolinella succinogenes cytochrome c nitrite reductase

The cytochrome c nitrite reductase complex (NrfHA) is the terminal enzyme in the electron transport chain catalysing nitrite respiration of Wolinella succinogenes. The catalytic subunit NrfA is a pentahaem cytochrome c containing an active site haem group which is covalently bound via the cysteine residues of a unique CWTCK motif. The lysine residue serves as the axial ligand of the haem iron. The other four haem groups of NrfA are bound by conventional haem-binding motifs (CXXCH). The nrfHAIJ locus was restored on the genome of the W. succinogenes DeltanrfAIJ deletion mutant by integration of a plasmid, thus enabling the expression of modified alleles of nrfA and nrfI. A mutant (K134H) was constructed which contained a nrfA gene encoding a CWTCH motif instead of CWTCK. NrfA of strain K134H was found to be synthesized with five bound haem groups, as judged by matrix-assisted laser-desorption/ionization (MALDI) mass spectrometry. Its nitrite reduction activity with reduced benzyl viologen was 40% of the wild-type activity. Ammonia was formed as the only product of nitrite reduction. The mutant did not grow by nitrite respiration and its electron transport activity from formate to nitrite was 5% of that of the wild-type strain. The predicted nrfI gene product is similar to proteins involved in system II cytochrome c biogenesis. A mutant of W. succinogenes (stopI) was constructed that contained a nrfHAIJ gene cluster with the nrfI codons 47 and 48 altered to stop codons. The NrfA protein of this mutant did not catalyse nitrite reduction and lacked the active site haem group, whereas the other four haem groups were present. Mutant (K134H/stopI) which contained the K134H modification in NrfA in addition to the inactivated nrfI gene had essentially the same properties as strain K134H. NrfA from strain K134H/stopI contained five haem groups. It is concluded that NrfI is involved in haem attachment to the CWTCK motif in NrfA but not to any of the CXXCH motifs. The nrfI gene is obviously dispensable for maturation of a modified NrfA protein containing a CWTCH motif instead of CWTCK. Therefore, NrfI might function as a specific haem lyase that recognizes the active site lysine residue of NrfA. A similar role has been proposed for NrfE, F and G of Escherichia coli, although these proteins share no overall sequence similarity to NrfI and belong to system I cytochrome c biogenesis, which differs fundamentally from system II.     

Deletion of the COX7 gene in Saccharomyces cerevisiae reveals a role for cytochrome c oxidase subunit VII In assembly of remaining subunits

Cytochrome c oxidase from Saccharomyces cerevisiae is composed of nine subunits. Subunits I, II and III are products of mitochondrial genes, while subunits IV, V, VI, VII, VIIa and VIII are products of nuclear genes. To investigate the role of cytochrome c oxidase subunit VII in biogenesis or functioning of the active enzyme complex, a null mutation in the COX7 gene, which encodes subunit VII, was generated, and the resulting cox7 mutant strain was characterized. The strain lacked cytochrome c oxidase activity and haem a/a3 spectra. The strain also lacked subunit VII, which should not be synthesized owing to the nature of the cox7 mutation generated in this strain. The amounts of remaining cytochrome c oxidase subunits in the cox7 mutant were examined. Accumulation of subunit I, which is the product of the mitochondrial COX1 gene, was found to be decreased relative to other mitochondrial translation products. Results of pulse-chase analysis of mitochondrial translation products are consistent with either a decreased rate of translation of COX1 mRNA or a very rapid rate of degradation of nascent subunit I. The synthesis, stability or mitochondrial localization of the remaining nuclear-encoded cytochrome c oxidase subunits were not substantially affected by the absence of subunit VII. To investigate whether assembly of any of the remaining cytochrome c oxidase subunits is impaired in the mutant strain, the association of the mitochondrial-encoded subunits I, II and III with the nuclear-encoded subunit IV was investigated.(ABSTRACT TRUNCATED AT 250 WORDS)     

A dedicated haem lyase is required for the maturation of a novel bacterial cytochrome c with unconventional covalent haem binding

In bacterial c-type cytochromes, the haem cofactor is covalently attached via two cysteine residues organized in a haem c-binding motif. Here, a novel octa-haem c protein, MccA, is described that contains only seven conventional haem c-binding motifs (CXXCH), in addition to several single cysteine residues and a conserved CH signature. Mass spectrometric analysis of purified MccA from Wolinella succinogenes suggests that two of the single cysteine residues are actually part of an unprecedented CX15CH sequence involved in haem c binding. Spectroscopic characterization of MccA identified an unusual high-potential haem c with a red-shifted absorption maximum, not unlike that of certain eukaryotic cytochromes c that exceptionally bind haem via only one thioether bridge. A haem lyase gene was found to be specifically required for the maturation of MccA in W. succinogenes. Equivalent haem lyase-encoding genes belonging to either the bacterial cytochrome c biogenesis system I or II are present in the vicinity of every known mccA gene suggesting a dedicated cytochrome c maturation pathway. The results necessitate reconsideration of computer-based prediction of putative haem c-binding motifs in bacterial proteomes.     

Biosynthesis and functional role of haem O and haem A

Haem O and/or haem A are specifically synthesized for the haem-copper respiratory oxidases. A 17-carbon hydroxyethylfarnesyl chain at the pyrrole ring A of the haems seems essential for catalytic functions at the oxygen-reduction site. The discovery of haem O in the cytochrome bo complex from Escherichia coli was a breakthrough in the studies on haem A biosynthesis. Molecular biological and biochemical studies in the past three years demonstrated that the cyoE/ctaB/COX10 genes are indispensable for functional expression of the terminal oxidases and encode a novel enzyme haem O synthase (protohaem IX farnesyltransferase). It has recently been suggested that the ctaA gene adjacent to the ctaB-ctaCDEF gene cluster in Bacillus subtilis encodes haem A synthase (haem O monooxygenase). In this article, we review current knowledge of the genes for haem O and haem A biosyntheses, the location and regulation of haem O synthase, the possible enzymatic mechanism of farnesyl transfer to haem B and the possible roles of the farnesylated haems.     

ABC transporter-mediated release of a haem chaperone allows cytochrome c biogenesis

Although organisms from all kingdoms have either the system I or II cytochrome c biogenesis pathway, it has remained a mystery as to why these two distinct pathways have developed. We have previously shown evidence that the system I pathway has a higher affinity for haem than system II for cytochrome c biogenesis. Here, we show the mechanism by which the system I pathway can utilize haem at low levels. The mechanism involves an ATP-binding cassette (ABC) transporter that is required for release of the periplasmic haem chaperone CcmE to the last step of cytochrome c assembly. This ABC transporter is composed of the ABC subunit CcmA, and two membrane proteins, CcmB and CcmC. In the absence of CcmA or CcmB, holo(haem)CcmE binds to CcmC in a stable dead-end complex, indicating high affinity binding of haem to CcmC. Expression of CcmA and CcmB facilitates formation of the CcmA2B1C1 complex and ATP-dependent release of holoCcmE. We propose that the CcmA2B1C1 complex represents a new subgroup within the ABC transporter superfamily that functions to release a chaperone.     

Sporulation properties of cytochrome a-deficient mutants of Bacillus subtilis

Three classes of cytochrome a-deficient mutants of Bacillus subtilis have been found to be asporogenic or oligosporogenic. All three classes showed declines in adenosine 5'-triphosphate (ATP) concentrations during early sporulation, at a time when ATP levels in wild-type strains are constant. Class III mutants were found to be deficient in aconitase and isocitric dehydrogenase, and showed reduced maximum growth in nutrient sporulation medium. These mutants also suffered the most rapid decline in ATP concentration in early sporulation, and exhibited neither the biphasic oxygen consumption curve nor the increase in pH normally observed at the end of logarithmic growth in nutrient sporulation medium. Nicotinamide adenine dinucleotide oxidase activities of purified membrane preparations were approximately normal for mutants in all classes, except for two of the class II mutants and one class III mutant. Neither cytochrome a nor cytochrome c appears to be an obligatory intermediate in cyanide-sensitive nicotinamide adenine dinucleotide oxidation in B. subtilis.     

Escherichia coli genes required for cytochrome c maturation.

The so-called aeg-46.5 region of Escherichia coli contains genes whose expression is induced under anaerobic growth conditions in the presence of nitrate or nitrite as the terminal electron acceptor. In this work, we have examined more closely several genes of this cluster, here designated ccmABCDEFGH, that are homologous to two separate Bradyrhizobium japonicum gene clusters required for the biogenesis of c-type cytochromes. A deletion mutant of E. coli which lacked all of these genes was constructed. Maturation of indigenous c-type cytochromes synthesized under anaerobic respiratory conditions, with nitrite, nitrate, or trimethylamine N-oxide as the electron acceptor, was found to be defective in the mutant. The biogenesis of foreign cytochromes, such as the soluble B. japonicum cytochrome c550 and the membrane-bound Bacillus subtilis cytochrome c550, was also investigated. None of these cytochromes was synthesized in its mature form when expressed in the mutant, as opposed to the situation in the wild type. The results suggest that the E. coli ccm gene cluster present in the aeg-46.5 region is required for a general pathway involved in cytochrome c maturation.     

Expression, purification, and characterization of the CuA-cytochrome c domain from subunit II of the Bacillus subtilis cytochrome caa3 complex in Escherichia coli

Cytochrome caa3 from Bacillus subtilis is a member of the heme-copper oxidase family of integral membrane enzymes that includes mitochondrial cytochrome c oxidase. Subunit II of cytochrome caa3 has an extra 100 amino acids at its C-terminus, relative to its mitochondrial counterpart, and this extension encodes a heme C binding domain. Cytochrome caa3 has many of the properties of the complex formed between mitochondrial cytochrome c and mitochondrial cytochrome c oxidase. To examine more closely the interaction between cytochrome c and the oxidase we have cloned and expressed the Cu(A)-cytochrome c portion of subunit II from the cytochrome caa3 complex of B. subtilis. We are able to express about 2000 nmol, equivalent to 65 mg, of the Cu(A)-cytochrome c protein per litre of Escherichia coli culture. About 500 nmol is correctly targeted to the periplasmic space and we purify 50% of that by a combination of affinity chromatography and ammonium sulfate fractionation. The cytochrome c containing sub-domain is well-folded with a stable environment around the heme C center, as its mid-point potential and rates of reduction are indistinguishable from values for the cytochrome c domain of the holo-enzyme. However, the Cu(A) site lacks copper leading to an inherent instability in this sub-domain. Expression of B. subtilis cytochrome c, as exemplified by the Cu(A)-cytochrome c protein, can be achieved in E. coli, and we conclude that the cytochrome c and Cu(A) sub-domains behave independently despite their close physical and functional association.     

Haem O2 can replace haem A in the active site of cytochrome c oxidase from thermophilic bacterium PS3.

Thermophilic bacterium PS3 cultured under slightly air-limited conditions showed a mitochondrion-like cytochrome pattern similar to that in vigorously aerated cells, but an o-type cytochrome replaced cytochrome a3 as the CO-binding centre. Cytochrome cao-type oxidase was purified from the cell membranes by almost the same procedure as used for cytochrome caa3. The turnover number of cytochrome cao was higher than that of cytochrome caa3, but the Km's of the two enzymes for cytochrome c and O2 were almost the same. Gel electrophoresis in the presence of sodium dodecyl sulfate gave bands of four subunits at the identical positions both for cytochrome cao and cytochrome caa3. Cytochrome cao contained a novel kind of haem in addition to haems C and A. This novel haem is likely to be haem O, very recently found as the chromophore of the cytochrome bo complex in Escherichia coli. These data suggest that cytochrome cao is an alternative form of cytochrome c oxidase (cytochrome caa3), in which the cytochrome a3 centre of the enzyme is replaced with cytochrome o.