Comparing the substrate specificities of cytochrome c biogenesis Systems I and II

Bioenergetics

Authors


S. J. Ferguson, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
Fax: +44 1865 613201
Tel: +44 1865 613299
E-mail: stuart.ferguson@bioch.ox.ac.uk

Abstract

c-Type cytochromes require specific post-translational protein systems, which vary in different organisms, for the characteristic covalent attachment of heme to the cytochrome polypeptide. Cytochrome c biogenesis System II, found in chloroplasts and many bacteria, comprises four subunits, two of which (ResB and ResC) are the minimal functional unit. The ycf5 gene from Helicobacter pylori encodes a fusion of ResB and ResC. Heterologous expression of ResBC in Escherichia coli lacking its own biogenesis machinery allowed us to investigate the substrate specificity of System II. ResBC is able to attach heme to monoheme c-type cytochromes c550 from Paracoccus denitrificans and c552 from Hydrogenobacter thermophilus, both normally matured by System I. The production of holocytochrome is enhanced by the addition of exogenous reductant. Single-cysteine variants of these cytochromes were not efficiently matured by System II, but System I was able to produce detectable amounts of AXXCH variants; this adds to evidence that there is no obligate requirement for a disulfide-bonded intermediate for the latter c-type cytochrome biogenesis system. In addition, System II was able to mature an AXXAH-containing variant into a b-type cytochrome, with implications for both heme supply to the periplasm and substrate recognition by System II.

Abbreviations
Ccm

cytochrome c maturation

IPTG

isopropyl thio-β-d-galactoside

MESA

2-mercaptoethane sulfonate

Introduction

Nature employs at least five distinct systems for the biogenesis of c-type cytochromes [1–3]; this post-translational modification process covalently links the heme cofactor to, normally, two cysteines in a CXXCH motif. System I is found in many Gram-negative bacteria and various mitochondria, including from plants [4,5]; System II appears in Gram-positive and some Gram-negative bacteria, and chloroplasts [6]; System III occurs in many non-plant mitochondria [5]; System IV is specific for the unusual cytochrome b6 involved in photosynthesis [7], and a fifth system, which remains to be characterized, exists in trypanosomatids [8]. Very unusually, some thermophilic cytochromes c are able to form spontaneously in vitro or in the cytoplasm of Escherichia coli [9], although it is believed that they are naturally matured by one of the biogenesis systems above.

The experimental amenability of E. coli has allowed the heterologous replacement of its own cytochrome c maturation (Ccm) machinery (encoded by the ccmABCDEFGH operon, called System I, Fig. 1) with systems from other organisms to facilitate their analysis. The enzyme heme lyase (System III) has been shown to function in E. coli cytoplasm [10] and to produce mitochondrial holocytochrome c. System II from Helicobacter pylori has been substituted for the E. coli Ccm machinery, enabling a comparison of the heme delivery activities of the two systems towards a diheme cytochrome c [11]. In E. coli, natural cytochrome c biogenesis requires at least 10 proteins, contrasting with the single protein constituting System III. System II is of intermediate complexity and comprises four proteins in, for example, Bacillus subtilis and Bordetella pertussis, namely ResA, ResB, ResC and CcdA [12,13] (Fig. 1). Notably, the genomes of some Bordetella species, e.g. B. parapertussis, encode both Systems I and II [1].

Figure 1.

 Schematic representation of cytochrome c biogenesis Systems I and II. The systems illustrated are System I (the Ccm system) from E. coli and System II from B. subtilis. Note that ResBC is a fusion protein in H. pylori. The two systems can each be subdivided into proteins which contribute to the handling of heme and ligation of heme to the apocytochrome, and those involved in the provision of reductant to the apocytochrome in order to reduce a potential disulfide bond in the CXXCH heme-binding site. CcmH, which in some organisms is two separate proteins CcmH and CcmI [50], appears to be involved in both heme handling/ligation and reductant provision [2].

In common with System I, it is not clear whether heme is transported across the membrane by System II itself or by some other process. Heme must then be attached to the CXXCH apocytochrome motif, the cysteine side-chains of which need to be in the reduced state. CcdA (or, in some organisms, DsbD) is a membrane protein that provides the required reducing power to the thioredoxin-like protein ResA, which is thought to reduce the apocytochrome [14]. ResB (also called Ccs1 and CcsB) is a membrane protein of unknown function with a large lumenal/extracytoplasmic domain [15,16]. ResC (also called CcsA) is also membranous with a soluble domain, and contains a tryptophan-rich signature motif found in various cytochrome c biogenesis proteins (the System I proteins CcmC and CcmF), which has been proposed to function in heme handling [17–19]. The biogenesis machinery from H. pylori appears to be a single protein that is a fusion of the proteins ResB and ResC, making it a useful minimal System II model. The expression of H. pylori ResBC in E. coli allowed the heterologous maturation of B. pertussis cytochrome c4, a cytochrome normally matured by System II [11]. In addition, this approach allowed the identification of essential histidine residues within ResBC proposed to act as axial ligands to heme iron [20]. However, little is known about the substrate recognition or specificity of System II.

A particularly notable point for examination is the ability of Systems I and II to mature single-cysteine-containing c-type cytochromes (XXXCH or CXXXH motifs). XXXCH cytochromes occur in nature in the mitochondria of Euglenozoan organisms, such as Crithidia fasciculata, and it has been demonstrated that the overall structure of cytochrome c from this organism bears remarkable similarity to yeast cytochrome c [21]. It is believed that organisms which possess such single-cysteine c-type cytochromes exhibit an as yet unidentified, novel biogenesis system. All fully sequenced genomes of organisms expressing such single-cysteine cytochromes lack identifiable homologues of known c-type cytochrome biogenesis proteins. The ability (or otherwise) of System I or II to mature such cytochromes may shed light on the mechanism of heme attachment in these systems. To date, System I has not been clearly observed to attach heme to such single-cysteine variants [22,23]. Contrastingly, System II has been proposed to attach heme to an SXXCH motif in NrfH [24].

In this work, we have cloned the ResBC-encoding gene from H. pylori (ycf5) into the backbone plasmid (pACYC184) of pEC86 which contains the E. coli ccm operon [25] and which has been very widely used for heterologous cytochrome c production. Heterologous expression of H. pylori ResBC from this new plasmid (pHP86) in E. coli allowed us to explore the substrate specificity of System II in direct comparison with System I.

Results

Various c-type cytochrome proteins were used to probe different aspects of the specificity of the expressed cytochrome c biogenesis systems. Experiments were conducted in a strain of E. coli lacking all known endogenous cytochrome c biogenesis proteins, EC06. In each case, control experiments were performed for spontaneous heme attachment to exogenous cytochromes [using the biogenesis system plasmid backbone containing no cytochrome c biogenesis genes (AD377)]. In addition, correction was performed for the formation of any endogenous E. coli cytochromes catalyzed by the products of the different biogenesis plasmids in the absence of a gene for an exogenous cytochrome.

System II can mature monoheme c-type cytochromes in E. coli

Cytochrome c550 from Paracoccus denitrificans is well characterized as a heterologous holocytochrome produced by the E. coli Ccm system [26]. The ability of System II to attach heme to this monoheme bacterial cytochrome in the periplasm of EC06 cells was tested. Holocytochrome c550 was detected in periplasmic extracts of cells containing pHP86 (H. pylori ResBC) and pKPD1 (cytochrome c550) and was quantified spectroscopically (Fig. 2). The yield was approximately 0.6% of that with System I, which produces very large amounts of the holocytochrome (Table 1). SDS-PAGE analysis followed by heme staining (Fig. 2, right-hand inset) shows a band of the expected mass (∼ 15 kDa for P. denitrificans holocytochrome c550 cleaved of its signal peptide) for the cytochrome produced by System II, the intensity of which is consistent with the amount of cytochrome determined spectroscopically compared with System I. The α-band of the pyridine hemochrome spectrum, which is indicative of the saturation of the heme vinyl groups to which the cysteine residues attach, was found to be at 550 nm for the System II-matured cytochrome c550, as expected for the formation of two thioether bonds (Fig. 2, left-hand inset). Cytochrome c550 made by System II (Fig. 2) was therefore indistinguishable from that made by System I, its natural biogenesis machinery. This is the first demonstration that System II can mature a cytochrome normally matured by System I.

Figure 2.

 Maturation of P. denitrificans cytochrome c550 by System II. Visible absorption spectra reflecting the formation of P. denitrificans cytochrome c550 and variants in periplasmic extracts of E. coli EC06 catalyzed by H. pylori ResBC: wild-type cytochrome c550 (full line), AXXCH-containing variant (broken/dotted line) and CXXAH-containing variant (broken line). The vertical scale bar represents 0.01 absorbance units. The spectra are vertically offset for clarity. Samples were reduced by the addition of a few grains of disodium dithionite. The absorbance maxima for wild-type cytochrome c550 are at 415, 521.5 and 550 nm. The inset spectrum shows the reduced pyridine hemochrome spectrum of cytochrome c550 produced by H. pylori ResBC. The vertical line indicates 550 nm. The inset gel illustrates the detection of c-type cytochromes via SDS-PAGE analysis, and subsequent heme staining of the gel, of periplasmic fractions from cells expressing P. denitrificans cytochrome c550 and the indicated biogenesis system (I or II). The periplasmic fraction from cells expressing System I and cytochrome c550 was diluted 20-fold before analysis (equating to 0.25–0.5 μg protein loaded, compared with 5–10 μg for the undiluted System II-produced sample). The left-most lane (M) contains See-Blue Plus 2 protein markers of the indicated molecular weights (kDa).

Table 1.   Levels of holocytochrome production for biogenesis Systems I and II expressed in E. coli strain EC06. These values have been corrected to account for any spontaneous formation of the respective cytochromes and for background levels of endogenous cytochrome production. The units are milligrams of holocytochrome per gram of wet cell pellet. The values in parentheses are standard deviations. ND, not detectable.
 System ISystem II
Cytochrome c550
CXXCH4.07 (0.55)0.024 (0.009)
AXXCH0.045 (0.002)ND
CXXAH0.023 (0.005)ND
Cytochrome c552
CXXCH0.99 (0.42)0.16 (0.054)
AXXCH0.030 (0.004)0.009 (0.002)
CXXAHNDND

We also examined the biogenesis of Hydrogenobacter thermophilus cytochrome c552. This System I-matured thermophilic cytochrome has also been used as a substrate to test the properties of the E. coli cytochrome c biogenesis system [22]. System I is able to produce large quantities of the c552 holocytochrome (Table 1 and Fig. 3). Co-expression of cytochrome c552 with the System II plasmid resulted in approximately 16% of the level produced by System I, a much higher proportion than that observed with the mesophilic cytochrome c550. The spectroscopic features and mobility on SDS-PAGE of the System II-produced cytochrome c552 are identical to those of the same cytochrome produced by System I (Fig. 3 and inset). Spontaneous periplasmic assembly of cytochrome c552 appears to occur, as some holocytochrome is detected in the absence of any biogenesis system (Fig. 3). The data presented in Table 1 have been corrected for the mean level of spontaneous heme attachment. Uncatalyzed heme attachment to cytochrome c552 is known to occur in the E. coli cytoplasm [9], and a small amount of cytoplasmic contamination of periplasmic extracts can occur during preparation [22]. However, SDS-PAGE analysis of the periplasmic fractions in this study demonstrated that the spontaneous holocytochrome formation detected was essentially all periplasmic, as the observed mass was consistent with that of the cytochrome polypeptide cleaved of its periplasmic targeting sequence. The mass of H. thermophilus holocytochrome c552 cleaved of its signal peptide is approximately 9 kDa, whereas the uncleaved product has a mass of approximately 11 kDa.

Figure 3.

 Maturation of H. thermophilus cytochrome c552 by Systems I and II. Visible absorption spectra reflecting the formation of H. thermophilus cytochrome c552 in periplasmic extracts of E. coli EC06 catalyzed by E. coli System I (full line), H. pylori System II (broken line) and in the absence of any biogenesis system (AD377) (broken/dotted line) (showing the level of spontaneous, i.e. uncatalyzed, holocytochrome formation). The vertical scale bar represents 0.2 absorbance units. The spectra are vertically offset for clarity and normalized for wet cell weight. Samples were reduced by the addition of a few grains of disodium dithionite. The absorbance maxima are at 417, 521 and 552 nm. The inset gel illustrates the detection of c-type cytochromes via SDS-PAGE analysis of periplasmic fractions from cells expressing H. thermophilus cytochrome c552 and the indicated biogenesis system (I or II), and subsequent heme-staining of the gel. Loading was normalized for total protein content. The left-most lane (M) contains See-Blue Plus 2 protein markers of the indicated molecular weights (kDa).

Maturation of single-cysteine holocytochromes

There are natural examples of cytochromes in which heme is attached via a single thioether bond to a cysteine in the protein [8,27], which raises questions about the purpose of covalent heme attachment via two bonds [21,28]. The determination of whether the presence of two cysteine thiols is essential could also address the requirement for an intramolecular disulfide bond, known to occur within apocytochromes [29], in the heme attachment reaction. It has been argued that System II can attach heme to one SXXCH motif generated by site-directed mutagenesis in the tetraheme cytochrome NrfH from Wolinella succinogenes [24]. However, this is an important point requiring further investigation.

Cytochrome c550 containing an AXXCH motif (C35A) acquired approximately 1.1% of the level of heme attachment observed for the wild-type CXXCH protein when expressed with System I (Table 1). The values in Table 1 are based on the absorption values at single wavelengths. However, they are only taken to indicate the presence of the specific holocytochrome under investigation if the features of the spectrum, in terms of wavelength maxima, and the position and intensity of heme-staining bands on SDS-PAGE gels, also appropriately demonstrate holocytochrome formation. The AXXCH variant produced by System I has spectroscopic features indicative of single-cysteine holocytochrome formation, and a band of the expected mass is observed on heme-stained gels (data not shown). The values in Table 1 imply that a small amount of the C38A (CXXAH) variant may have undergone heme attachment by System I. However, using the criteria described above (spectra and heme staining), we conclude that the single-wavelength absorption intensity is not in fact indicative of C38A holocytochrome. Effectively, therefore, the value in Table 1 for the C38A variant of cytochrome c550 matured by System I represents the lower limit of detectability and the experimental error. Notably, the production of the AXXCH variant of cytochrome c550 was detected by western blotting using anti-cytochrome c550 serum, whereas the CXXAH variant was not (Fig. S1, see Supporting Information).

Although it is possible that the CXXAH variant of P. denitrificans cytochrome c550 is unstable and cannot be made, the two single-cysteine-containing (and AXXAH) variants of H. thermophilus cytochrome c552 can form stably in the E. coli cytoplasm [30,31]. System I was unable to produce the CXXAH variant cytochrome c552, but some System I-dependent formation of the AXXCH variant (approximately 3% relative to CXXCH) was detected (this is the value after subtraction to account for the level of spontaneous holocytochrome formation). Figure 4 shows that the heme-staining band corresponding to holo-c552 AXXCH matured by System I is significantly more intense than the band observed when no biogenesis genes were co-expressed (i.e. with spontaneous holocytochrome formation). This is a significant observation regarding the substrate specificity of the Ccm system.

Figure 4.

 Maturation of H. thermophilus cytochrome c552 AXXCH variant. SDS-PAGE analysis and subsequent heme staining of periplasmic extracts from E. coli EC06 cells containing the H. thermophilus cytochrome c552 AXXCH variant and the indicated biogenesis system [I or II (with the lane marked - being periplasm from cells containing empty vector, AD377)]. Loading was normalized for total protein content. The left-most lane (M) contains See-Blue Plus 2 protein markers of the indicated molecular weights (kDa).

Neither single-cysteine holocytochrome c550 was detected with the coexpression of the System II plasmid, as shown in the spectra of the periplasmic extracts in Fig. 2, which have no features indicative of holocytochrome formation. It should be noted that pEC86 (System I) complements the Ccm deletion of E. coli strain EC06, whereas pHP86 (System II) does not; thus our experimental errors as a result of background (endogenous) cytochrome production are much larger with System I than with System II. Although cultures grown in this work are considered to be aerobic, some microanaerobicity can occur, which causes low-level expression of the endogenous E. coli c-type cytochromes. System II appears to produce a very low level of the AXXCH holocytochrome c552 (Table 1), compared with the CXXCH form. The analysis of 12 independent experiments revealed the production of spectroscopically detectable AXXCH above the level of spontaneous cytochrome formation in two of the cultures. These data are responsible for the apparent formation of AXXCH by System II when compared with AD377 (reported as mean values in Table 1). It is possible that in the majority of our observations single-cysteine cytochromes were formed by System II at such low levels that they were undetectable either by spectroscopic analysis of periplasmic fractions or heme staining of appropriate SDS-PAGE gels.

System II mediates the formation of a b-type cytochrome

Unexpectedly, the spectra of periplasmic extracts of cells containing the System II plasmid and the double-alanine cytochrome c550 (AXXAH motif, C35A/C38A) indicated the presence of small amounts of a typical low-spin, b-type cytochrome (Fig. 5). The Soret band is red shifted by 4 nm and the α-band by 5 nm compared with the wild-type cytochrome c550, as would be expected for noncovalently bound heme (saturation of each heme vinyl group on formation of a c-type cytochrome causes a blue shift of 2–3 nm in the α-band of the absorption spectrum). To confirm the presence of variant cytochrome c550, we performed a western blot of periplasmic extracts from this strain and a strain containing only pKK223-3 (i.e. no cytochrome). A band consistent with the molecular weight of cytochrome c550 was evident in the strain expressing AXXAH-containing cytochrome c550, but not in the control strain containing pKK223-3 (Fig. S1, see Supporting Information). It was not possible to detect low levels of b-type complexes (if they exist) made with System I because of the relatively high levels of endogenous cytochromes that are produced (see above) would mask the spectroscopic features of the b-type cytochrome. However, we were unable to detect the formation of a b-type AXXAH variant by System I using western blot analysis and anti-cytochrome c550 serum (Fig. S1, see Supporting Information). In addition, no b-type AXXAH cytochrome was detected when no System II biogenesis proteins were present. These observations have implications for the provision of heme to the periplasm by ResBC, and suggest that it may facilitate heme delivery from the cytoplasm, in agreement with a recent study by Frawley & Kranz [20].

Figure 5.

 Maturation of a b-type cytochrome AXXAH variant of P. denitrificans cytochrome c550. Visible absorption spectra of periplasmic extracts from E. coli EC06 cells expressing H. pylori ResBC and P. denitrificans cytochrome c550 (broken-dotted line), cytochrome c550 AXXAH variant (full line) and no cytochrome (pKK223-3) (dotted line). The vertical scale bar represents 0.005 absorbance units. Samples were reduced by the addition of a few grains of disodium dithionite. The Soret and α-band absorbance maxima are at 415 and 550 nm, respectively, for wild-type cytochrome c550, and at around 419 and 555 nm for the AXXAH-containing variant. The spectrum of the wild-type cytochrome c550 has been reduced by a factor of seven for clarity, and the spectra are vertically offset. The vertical line indicates 550 nm.

Provision of reductant increases significantly System II-mediated c-type cytochrome formation

As the H. pylori biogenesis system expressed in this study lacks the thiol-disulfide oxidoreductase components that are thought to reduce the cysteine thiols in the cytochrome heme-binding motif (neither ResA of System II nor CcmG of System I are present), the effect of the addition of a chemical reductant to the growth medium was tested: 5 mm 2-mercaptoethane sulfonate (MESA) was added to cells containing wild-type (CXXCH) P. denitrificans cytochrome c550, and the System II plasmid and holocytochrome contents were determined. The added reductant caused a two- to three-fold increase in holocytochrome formation (data not shown). Exogenous chemical reductant has been used to recover the phenotypes of strains lacking other oxidoreductases [32,33]. The addition of 5 mm MESA to cells expressing the single-cysteine c550 C35A variant did not result in the formation of detectable holocytochrome, implying that the augmentation in wild-type holocytochrome maturation with the addition of reductant is a result of the reduction of a disulfide in the apocytochrome.

Production of endogenous E. coli cytochromes

Escherichia coli contains a number of endogenous c-type cytochromes that it expresses under different anaerobic growth conditions. Some of these are observed at low levels in periplasmic extracts when the Ccm deletion strain EC06 is complemented with System I (pEC86), but not with System II (pHP86), as shown in Fig. 6. The two bands observed correspond to the masses of the soluble cytochromes NapB (around 15 kDa) and NrfA (around 50 kDa). However, given the relative maturation levels of exogenous cytochromes c (see above), it may be that any endogenous cytochrome matured by System II would be present below the lower limit of detection in our experiments. We have determined that the limit of detection for heme on a heme-stained SDS-PAGE gel is 1 nmol per lane (A. D. Goddard & S. J. Ferguson, unpublished observations). E. coli NapB has two hemes and NrfA five. Therefore, we would expect to detect 0.5 and 0.2 nmol of these cytochromes, respectively.

Figure 6.

 Analysis of endogenous cytochrome production. SDS-PAGE analysis and subsequent heme staining of periplasmic extracts from E. coli EC06 cells containing pKK223-3 (no exogenous cytochrome) and the indicated biogenesis system (I or II). The left-most lane (M) contains See-Blue Plus 2 protein markers of the indicated molecular weights (kDa). Equal amounts of total protein were loaded in each lane.

Discussion

The complex and somewhat unpredictable natural distribution of cytochrome c biogenesis systems does not correlate specifically with the types of cytochrome produced by the organisms concerned [5,34]. Cytochromes c vary widely in terms of overall fold, heme iron ligands, number of hemes per polypeptide, the presence of other cofactors, number of subunits, being membrane-bound or soluble, as well as the way in which the heme is linked to the protein (a few cytochromes have single thioether bonds to heme). The latter group includes the unusual cytochrome b6 and the trypanosomatid cytochromes c. The specificity of E. coli System I has been studied extensively. It can produce cytochromes c from a wide variety of organisms, with many hemes per polypeptide, and even attach heme to peptides as short as 12 residues in length [35,36]. The specificity of some heme lyases (System III) has also been determined; some organisms contain a heme lyase for each mitochondrial cytochrome c (e.g. yeast cytochromes c and c1), whereas others (e.g. animals) contain a single such enzyme that apparently catalyzes heme attachment to both cytochromes [37]. No study has examined the specificity of System II, which in nature produces an array of mono- and multiheme cytochromes.

System II produces monoheme bacterial cytochromes

In this work, we have shown that coexpression of the plasmid pHP86 (expressing System II) in E. coli with the mesophilic cytochrome c550 from P. denitrificans and the thermophilic cytochrome c552 from H. thermophilus, both naturally matured by System I, results in heme attachment to these cytochromes, yielding products that are indistinguishable from those produced by System I. This suggests that System II, in common with System I and in contrast with System III [38], has a broad substrate specificity and is able to mature c-type cytochromes from a variety of sources, including those that it does not naturally encounter. A relatively high level of holocytochrome c552 was produced (16% relative to System I) considering that the System II fusion protein is expressed heterologously and without the remaining (disulfide oxidoreductase) components of the biogenesis system. A previous study has interpreted a reduced level of cytochrome production by System II compared with System I as the former having a lower affinity for heme [11]; as no measurement of the relative abundance of the biogenesis proteins was shown, and there is no reason to believe that they would be equally stable in E. coli, we have reservations about this interpretation.

That higher levels of thermophilic cytochrome c552 are produced by System II compared with a mesophilic cytochrome (c550) is possibly a result of the higher stability of the apocytochrome c552 when it is delivered by the Sec system to the periplasm. Proteolytic degradation of apocytochromes might compete with the heme attachment machinery. In addition, apocytochromes are susceptible to periplasmic oxidation of the heme-binding cysteine residues. In our heterologous System II, the oxidoreductase that would normally reduce such a disulfide bond, ResA, is absent; the oxidation would also slow heme attachment. Our observation that added reductant results in a substantial increase in cytochrome c550 production indicates that oxidation of the heme-binding motif can reduce the efficiency of heme attachment by System II. Nevertheless, it is becoming increasingly clear from this work and others [20] that dithiol/disulfide oxidoreductases are not strictly necessary for cytochrome c maturation in the periplasm of E. coli.

Maturation of single-cysteine-attached cytochromes c

We found no detectable heme attachment to the single-cysteine-containing variants of P. denitrificans c550 with coexpression of the System II plasmid. A very low, variable level of heme attachment was observed with the AXXCH variant of cytochrome c552, but none with the CXXAH form. If there is a capability to attach heme to a single-cysteine cytochrome then, in common with System I, it is to a very low extent compared with heme attachment to CXXCH, below the level of detection of the analysis conducted in this study. It is notable that, in the work of Simon et al. [24], evidence was found for heme attachment to only one SXXCH heme-binding motif of the four possible (and investigated) in NrfH, and that no heme attachment to CXXSH was reported [24]. It may be that the observed heme attachment to one SXXCH motif was not in fact catalyzed by System II, e.g. it was instead spontaneous, perhaps facilitated by substantial folding of the protein around the three other hemes attached to CXXCH motifs by System II.

It has been reported previously that System I cannot produce detectable levels of single-cysteine-containing holocytochrome c552 [22]. In that work, the lower level of detectability was estimated as 2% of the wild-type (CXXCH) holocytochrome yield. Control experiments performed in the present work, to allow a direct comparison of System I and II plasmids (which are identical but for their encoded operons), permit a refinement of the conclusion of the former work. We found here low but detectable levels of the holo-forms of AXXCH variants of both cytochromes c550 and c552 (1 and 3% relative to the wild-type CXXCH cytochromes, respectively) when expressed with pEC86. The difference is presumably partly a result of the different E. coli strains used. Here, we used EC06, a ccm deletion strain, which had a significant effect on the amount of background cytochrome c matured (producing no detectable c-type cytochromes in the absence of a plasmid-borne biogenesis system). The sensitivity of the analytical methods used (e.g. the former work did not use heme-stained gels) may also contribute to the differences. That System I can attach some heme to single-cysteine-containing cytochromes is significant, particularly in the context of a possible relationship between heme attachment and a disulfide bond in the CXXCH motif. The fact that two cysteines are not absolutely essential for the heme attachment reaction demonstrates that the chemistry of such a reaction is not necessarily via an obligate disulfide-containing intermediate. Breaking a disulfide bond could be envisaged as providing the driving force for formation of the thioether bonds to heme. However, successful in vitro thioether bond formation using a phosphine (in the absence of thiol reagents) to reduce the apocytochrome disulfide also indicated that disulfide-linked chemistry is not involved in the heme attachment reaction [39]. The present work implies that the formation of the two thioether bonds is not thermodynamically necessary to release heme from the covalent heme-binding chaperone CcmE. We observe, as might be anticipated [21,31,40], that the single-cysteine variant in which the heme-binding cysteine is directly adjacent to the heme iron-ligating histidine (i.e. XXXCH) is the more likely to be recognized by the system and to undergo heme attachment than is CXXXH. Nevertheless, it remains clear that System I is far more effective and efficient at attaching heme to apocytochrome c with two cysteines, rather than one, in the heme-binding motif.

System II facilitates the formation of a b-type cytochrome in the periplasm

In the absence of any biogenesis proteins, it was not possible to detect the b-type forms (i.e. containing noncovalently bound heme) of c-type cytochromes lacking the heme-binding cysteine residues (i.e. the AXXAH variants). Apocytochromes c appear to be proteolytically degraded when heme is not attached [41]. Because of the clean background observed with the System II plasmid (i.e. the lack of any endogenous c-type cytochromes), it was possible to detect some b-type cytochrome when cytochrome c550 AXXAH was coexpressed with pHP86. It is possible that an equivalent cytochrome is produced by System I, but that it is rendered undetectable as a result of the production of endogenous cytochromes c which mask the b-type spectra [b-type cytochromes generally lose heme in SDS-PAGE and therefore cannot be detected by the heme staining of gels; western blotting with anti-cytochrome c550 serum failed to detect the presence of any cytochrome (Fig. S1, see Supporting Information)]. Alternatively, it is possible that, as a result of a covalent intermediate (CcmE–heme) [42], System I is unable to pass heme to an AXXAH variant apocytochrome; System II (ResBC) from Helicobacter hepaticus does not appear to covalently bind heme [20]. However, a recent study with Bacillus subtilis ResB and ResC (unfused proteins in that organism) revealed covalent binding of heme to the cytoplasmic side of ResB [43]. It is therefore possible that an initial covalent attachment of heme to ResB occurs, followed by trafficking through ResC, before insertion of heme into the periplasmic cytochrome. However, the residue covalently bound to the heme of ResB was found to be nonessential for cytochrome c biogenesis. The function, if any, of System II proteins covalently binding heme therefore remains to be resolved.

That expression of the System II protein in E. coli allows the formation of a b-type cytochrome suggests that heme provision from the cytoplasm to the periplasm might be performed by ResBC, concurrent with recent observations [20]. The study by Frawley & Kranz [20] also demonstrated the essentiality of H858 of H. hepaticus ResBC in holocytochrome formation, and proposed that this residue, together with H77, is involved in supplying heme to the periplasm. We note that a H857E mutant in H. pylori ResBC (equivalent to H858 in the H. hepaticus protein) is unable to mature the b-type cytochrome described above (A. D. Goddard & S. J. Ferguson, unpublished observations). This is consistent with H857 playing a role in heme transport. It is not known how heme is transported across the inner membrane by System I, but it has been shown conclusively that, contrary to earlier suggestions, CcmA and CcmB are not involved in heme transport in E. coli [44,45]. Notably, maturation of an AXXAH-containing variant b-type cytochrome c550 in the present study indicates a nascent heme-binding site, even in this mesophilic apocytochrome c (see also [46]), as well as possible recognition features in the apocytochrome, at least for cytochrome c biogenesis System II, other than the CXXCH heme-binding motif. These data also suggest that heme delivery to apocytochrome and thioether bond formation by System II are independent processes.

Materials and methods

Strains, plasmids and culture conditions

Escherichia coli strain EC06 [47] contains a chromosomal deletion of the ccm operon and was used to examine holocytochrome formation in the presence of the plasmid-encoded biogenesis systems. E. coli strain DH5α (Invitrogen, Paisley, UK) was used for routine molecular biology. KOD polymerase (Merck Chemicals Ltd, Nottingham, UK) was used for PCRs. All oligonucleotides used in this study are listed in Table S1 (see Supporting Information).

Biogenesis plasmids

The plasmids used in this work are listed in Table S2. The E. coli ccmABCDEFGH operon (System I) was expressed from pEC86 [25]. To create a comparable plasmid lacking any biogenesis system, inverse PCR was performed on pEC86 using the primers AG234 and AG235, and the product was self-ligated. This removed the entire ccm operon, and the plasmid created is AD377 (no biogenesis system). To create a suitable plasmid for the expression of other biogenesis systems, a XhoI site was introduced immediately after the initiating ATG of ccmA in pEC86 via Quikchange mutagenesis using the primers WC1 and WC2. The resultant construct is pEC86x, in which the entire ccm operon can be excised by digestion with XhoI and StuI. The ycf5 gene was amplified from H. pylori (strain 26695) genomic DNA using oligonucleotides HelF and HelR. The PCR product was cloned into XhoI/StuI-digested pEC86x. The resultant plasmid for the expression of H. pylori ResBC is pHP86 (System II).

Cytochrome c plasmids

P. denitrificans cytochrome c550 was expressed from the isopropyl thio-β-d-galactoside (IPTG)-inducible promoter of pKPD1 [26]. Mutations within the CXXCH heme-binding motif of c550 were created by Quikchange using the following: c550 C35A, C35AF and C35AR; c550 C38A, C38AF and C38AR; c550 C35AC38A, C35AC38AF and C35AC38AR. H. thermophilus cytochrome c552 and its AXXCH, AXXAH and CXXAH variants were expressed from the plasmids pEST210, pEST211, pEST212 and pEST213, respectively [22].

In each case, the plasmid bearing the biogenesis system confers resistance to chloramphenicol and the expression of the proteins is constitutive. The plasmids bearing the cytochromes are inducible with IPTG and confer resistance to carbenicillin. All constructs were sequenced before use.

Routine cell growth was conducted using Luria–Bertani medium supplemented with appropriate antibiotics. Growth on solid medium used Luria–Bertani medium supplemented with 1.5% bacteriological agar. For the preparation of periplasmic fractions, single colonies containing appropriate plasmids were picked into 500 mL 2× TY medium (16 g·L−1 peptone, 10 g·L−1 yeast extract, 5 g·L−1 NaCl), supplemented with 1 mm IPTG and appropriate antibiotics, in 2 L flasks. Cultures were grown at 37 °C with shaking at 200 r.p.m. for 16–20 h before harvesting. Carbenicillin was used at 100 μg·mL−1 and chloramphenicol at 34 μg·mL−1.

Analysis of cytochrome production

Periplasmic extractions were performed as described previously [22]. Extracts were analyzed by SDS-PAGE (Invitrogen pre-cast 10% Bis-Tris gels), followed by heme staining [48], which stains proteins with covalently bound heme. Samples were normalized for wet cell weight, and equal amounts of protein were loaded per lane (5–10 μg). Western blots to detect P. denitrificans cytochrome c550 were performed according to the manufacturer's instructions using anti-cytochrome c550 rabbit serum and a commercial alkaline-phosphatase-conjugated anti-rabbit secondary IgG raised in goat. The marker used was SeeBlue Plus 2 (Invitrogen).

UV–visible spectroscopy was performed using a Perkin-Elmer (Waltham, MA, USA) Lambda 2 spectrophotometer; samples were reduced by the addition of a few grains of disodium dithionite (Sigma-Aldrich Company Ltd, Poole, UK). Pyridine hemochrome spectra were determined according to the method described by Bartsch [49]. The normalized cytochrome content of each extract is presented as the number of milligrams of holocytochrome c per gram of cell pellet. The data are averages of at least five growths. The extinction coefficients used to calculate the yields of holocytochromes were as follows: wild-type H. thermophilus cytochrome c552, ε = 182 mm−1·cm−1 at 417 nm; C11A c552, ε = 179.5 mm−1·cm−1 at 422 nm; C14A c552, ε = 174.5 mm−1·cm−1 at 420 nm; C11A/C14A c552, ε = 145 mm−1·cm−1 at 425 nm; P. denitrificans cytochrome c550 wild-type and variants, 140 mm−1·cm−1 at 415 nm [22]. The extinction coefficients for the cytochrome c550 variants are unknown; the wild-type value was therefore used. Corrections of the average normalized values for each dataset were performed by subtracting the value observed when no biogenesis genes were expressed (i.e. with plasmid AD377 and the relevant cytochrome plasmid, to correct for spontaneous holocytochrome production) and subtracting the value observed when no heterologous cytochrome gene was expressed (i.e. with plasmid pKK223-3 and the relevant biogenesis plasmid, to correct for the production of endogenous E. coli cytochromes). Finally, the values for cells expressing the two empty vectors AD377 and pKK223-3 were added back, so that any corrections were for endogenous or spontaneous cytochrome c production only. Cultures for the corrections were grown and analyzed at least three times, and the mean values were used for the corrections.

Acknowledgements

This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC; grant numbers BB/D523019/1, BB/E004865/1 and BB/D019753/1). J.W.A.A. is a BBSRC David Phillips Fellow. A.D.G. gratefully acknowledges the E. P. Abraham Cephalosporin Fund. We thank Professor David Kelly (University of Sheffield) for kindly providing H. pylori genomic DNA.

Since the submission of this manuscript Kern et al. [50a] have also shown that System II cannot attach heme to a single-cysteine motif in a cytochrome at detectable levels [sentence added at proof stage].

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