Báo cáo khoa học: Comparing the substrate specificities of cytochrome c biogenesis Systems I and II docx

Comparing the substrate specificities of cytochrome c biogenesis Systems I and II Bioenergetics Alan D. Goddard*, Julie M. Stevens*, Arnaud Rondelet, Elena Nomerotskaia, James W. A. Allen and Stuart J. Ferguson Department of Biochemistry, University of Oxford, UK Introduction Nature employs at least five distinct systems for the biogenesis of c-type cytochromes [1–3]; this post-trans- lational modification process covalently links the heme cofactor to, normally, two cysteines in a CXXCH motif. System I is found in many Gram-negative bacte- ria 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 b 6 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 analy- sis. The enzyme heme lyase (System III) has been shown to function in E. coli cytoplasm [10] and to Keywords cytochrome c; cytochrome c maturation; heme; heme provision; System II Correspondence 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 *These authors contributed equally to this work (Received 28 July 2009, revised 12 October 2009, accepted 25 November 2009) doi:10.1111/j.1742-4658.2009.07517.x c-Type cytochromes require specific post-translational protein systems, which vary in different organisms, for the characteristic covalent attach- ment of heme to the cytochrome polypeptide. Cytochrome c biogenesis System II, found in chloroplasts and many bacteria, comprises four subun- its, 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 bio- genesis machinery allowed us to investigate the substrate specificity of Sys- tem II. ResBC is able to attach heme to monoheme c-type cytochromes c 550 from Paracoccus denitrificans and c 552 from Hydrogenobacter thermo- philus, both normally matured by System I. The production of holocyto- chrome 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 peri- plasm and substrate recognition by System II. Abbreviations Ccm, cytochrome c maturation; IPTG, isopropyl thio-b- D-galactoside; MESA, 2-mercaptoethane sulfonate. 726 FEBS Journal 277 (2010) 726–737 ª 2009 The Authors Journal compilation ª 2009 FEBS 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 cyto- chrome c biogenesis requires at least 10 proteins, con- trasting 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]. 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 cys- teine side-chains of which need to be in the reduced state. CcdA (or, in some organisms, DsbD) is a mem- brane 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 biogene- sis 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 pro- teins 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 c 4 , 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 Cri- thidia fasciculata, and it has been demonstrated that the overall structure of cytochrome c from this organ- ism 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 sin- gle-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 Heme handling/ligation System I System II CcmE CcmF ResA ResB ResC CcdA CcmH CcmG DsbD D CcmB CcmA CcmC p-side p-side Disulfide isomerization Fig. 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]. A. D. Goddard et al. Specificity of cytochrome c biogenesis System II FEBS Journal 277 (2010) 726–737 ª 2009 The Authors Journal compilation ª 2009 FEBS 727 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 cyto- chromes [using the biogenesis system plasmid back- bone 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 c 550 from Paracoccus denitrificans is well characterized as a heterologous holocytochrome pro- duced 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 c 550 was detected in periplasmic extracts of cells containing pHP86 (H. pylori ResBC) and pKPD1 (cytochrome c 550 ) 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 c 550 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 a-band of the pyridine hemo- chrome 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 c 550 , as expected for the forma- tion of two thioether bonds (Fig. 2, left-hand inset). Cytochrome c 550 made by System II (Fig. 2) was there- fore indistinguishable from that made by System I, its natural biogenesis machinery. This is the first demon- stration that System II can mature a cytochrome nor- mally matured by System I. We also examined the biogenesis of Hydro- genobacter thermophilus cytochrome c 552 . This System 0.035 0.030 0.025 Absorbance 14 550 560 570540530 0.020 MI II 6 Δ Absorbance 400 450 500 550 600 650 Wavelen g th (nm) Fig. 2. Maturation of P. denitrificans cytochrome c 550 by System II. Visible absorption spectra reflecting the formation of P. denitrificans cytochrome c 550 and variants in periplasmic extracts of E. coli EC06 catalyzed by H. pylori ResBC: wild-type cytochrome c 550 (full line), AXXCH-containing variant (broken ⁄ dotted line) and CXXAH-contain- ing variant (broken line). The vertical scale bar represents 0.01 absorbance units. The spectra are vertically offset for clarity. Sam- ples were reduced by the addition of a few grains of disodium dithi- onite. The absorbance maxima for wild-type cytochrome c 550 are at 415, 521.5 and 550 nm. The inset spectrum shows the reduced pyridine hemochrome spectrum of cytochrome c 550 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 c 550 and the indicated biogenesis system (I or II). The periplasmic fraction from cells expressing System I and cytochrome c 550 was diluted 20-fold before analysis (equating to 0.25–0.5 lg protein loaded, compared with 5–10 lg for the undiluted System II-produced sam- ple). The left-most lane (M) contains See-Blue Plus 2 protein mark- ers 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 endoge- nous cytochrome production. The units are milligrams of holocyto- chrome per gram of wet cell pellet. The values in parentheses are standard deviations. ND, not detectable. System I System II Cytochrome c 550 CXXCH 4.07 (0.55) 0.024 (0.009) AXXCH 0.045 (0.002) ND CXXAH 0.023 (0.005) ND Cytochrome c 552 CXXCH 0.99 (0.42) 0.16 (0.054) AXXCH 0.030 (0.004) 0.009 (0.002) CXXAH ND ND Specificity of cytochrome c biogenesis System II A. D. Goddard et al. 728 FEBS Journal 277 (2010) 726–737 ª 2009 The Authors Journal compilation ª 2009 FEBS I-matured thermophilic cytochrome has also been used as a substrate to test the properties of the E. coli cyto- chrome c biogenesis system [22]. System I is able to produce large quantities of the c 552 holocytochrome (Table 1 and Fig. 3). Co-expression of cytochrome c 552 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 c 550 . The spectroscopic features and mobil- ity on SDS-PAGE of the System II-produced cyto- chrome c 552 are identical to those of the same cytochrome produced by System I (Fig. 3 and inset). Spontaneous periplasmic assembly of cytochrome c 552 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. Uncata- lyzed heme attachment to cytochrome c 552 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 holocyto- chrome formation detected was essentially all periplas- mic, as the observed mass was consistent with that of the cytochrome polypeptide cleaved of its periplasmic targeting sequence. The mass of H. thermophilus holo- cytochrome c 552 cleaved of its signal peptide is approx- imately 9 kDa, whereas the uncleaved product has a mass of approximately 11 kDa. Maturation of single-cysteine holocytochromes There are natural examples of cytochromes in which heme is attached via a single thioether bond to a cys- teine 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 pres- ence 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 gen- erated by site-directed mutagenesis in the tetraheme cytochrome NrfH from Wolinella succinogenes [24]. However, this is an important point requiring further investigation. Cytochrome c 550 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 forma- tion. The AXXCH variant produced by System I has spectroscopic features indicative of single-cysteine holo- cytochrome 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 c 550 matured by System I represents the lower limit of detectability and the experimental error. Notably, the production of the AXXCH variant of cytochrome c 550 was detected by western blotting using anti-cytochrome 6 14 Δ Absorbance MIII Wavelength (nm) 400 450 500 550 600 Fig. 3. Maturation of H. thermophilus cytochrome c 552 by Systems I and II. Visible absorption spectra reflecting the formation of H. thermophilus cytochrome c 552 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. uncata- lyzed, 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 periplas- mic fractions from cells expressing H. thermophilus cytochrome c 552 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). A. D. Goddard et al. Specificity of cytochrome c biogenesis System II FEBS Journal 277 (2010) 726–737 ª 2009 The Authors Journal compilation ª 2009 FEBS 729 c 550 serum, whereas the CXXAH variant was not (Fig. S1, see Supporting Information). Although it is possible that the CXXAH variant of P. denitrificans cytochrome c 550 is unstable and cannot be made, the two single-cysteine-containing (and AXXAH) variants of H. thermophilus cytochrome c 552 can form stably in the E. coli cytoplasm [30,31]. Sys- tem I was unable to produce the CXXAH variant cytochrome c 552 , but some System I-dependent forma- tion of the AXXCH variant (approximately 3% rela- tive 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-c 552 AXXCH matured by System I is significantly more intense than the band observed when no biogenesis genes were co-expressed (i.e. with spontaneous holocy- tochrome formation). This is a significant observation regarding the substrate specificity of the Ccm system. Neither single-cysteine holocytochrome c 550 was detected with the coexpression of the System II plas- mid, 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 back- ground (endogenous) cytochrome production are much larger with System I than with System II. Although cultures grown in this work are considered to be aero- bic, 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 c 552 (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 c 550 (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 a-band by 5 nm com- pared with the wild-type cytochrome c 550 , as would be expected for noncovalently bound heme (saturation of each heme vinyl group on formation of a c-type cyto- chrome causes a blue shift of 2–3 nm in the a-band of the absorption spectrum). To confirm the presence of variant cytochrome c 550 , 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 14 6 M I II - Fig. 4. Maturation of H. thermophilus cytochrome c 552 AXXCH vari- ant. SDS-PAGE analysis and subsequent heme staining of periplas- mic extracts from E. coli EC06 cells containing the H. thermophilus cytochrome c 552 AXXCH variant and the indicated biogenesis sys- tem [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). Δ Absorbance Wavelength (nm) 400 450 500 550 600 650 Fig. 5. Maturation of a b-type cytochrome AXXAH variant of P. denitrificans cytochrome c 550 . Visible absorption spectra of peri- plasmic extracts from E. coli EC06 cells expressing H. pylori ResBC and P. denitrificans cytochrome c 550 (broken-dotted line), cyto- chrome c 550 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 a-band absorbance maxima are at 415 and 550 nm, respectively, for wild-type cytochrome c 550 , and at around 419 and 555 nm for the AXXAH-containing variant. The spectrum of the wild-type cytochrome c 550 has been reduced by a factor of seven for clarity, and the spectra are vertically offset. The vertical line indicates 550 nm. Specificity of cytochrome c biogenesis System II A. D. Goddard et al. 730 FEBS Journal 277 (2010) 726–737 ª 2009 The Authors Journal compilation ª 2009 FEBS cytochrome c 550 was evident in the strain expressing AXXAH-containing cytochrome c 550 , but not in the control strain containing pKK223-3 (Fig. S1, see Sup- porting 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 endog- enous 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 c 550 serum (Fig. S1, see Supporting Information). In addi- tion, 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]. 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 compo- nents 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 c 550 , 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 c 550 C35A variant did not result in the formation of detectable holocytochrome, implying that the augmentation in wild-type holocytochrome maturation with the addi- tion of reductant is a result of the reduction of a disul- fide 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. Discussion The complex and somewhat unpredictable natural distribution of cytochrome c biogenesis systems does not correlate specifically with the types of cyto- chrome 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 cyto- chromes have single thioether bonds to heme). The latter group includes the unusual cytochrome b 6 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 organ- isms, 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 62 49 38 14 28 17 MIII 6 Fig. 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 cyto- chrome) 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. A. D. Goddard et al. Specificity of cytochrome c biogenesis System II FEBS Journal 277 (2010) 726–737 ª 2009 The Authors Journal compilation ª 2009 FEBS 731 contain a heme lyase for each mitochondrial cyto- chrome c (e.g. yeast cytochromes c and c 1 ), whereas others (e.g. animals) contain a single such enzyme that apparently catalyzes heme attachment to both cyto- chromes [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 c 550 from P. denitrificans and the thermophilic cytochrome c 552 from H. thermo- philus, both naturally matured by System I, results in heme attachment to these cytochromes, yielding prod- ucts 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 c 552 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 inter- preted a reduced level of cytochrome production by System II compared with System I as the former hav- ing 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 c 552 are produced by System II compared with a mesophilic cytochrome (c 550 ) is possibly a result of the higher sta- bility of the apocytochrome c 552 when it is delivered by the Sec system to the periplasm. Proteolytic degra- dation of apocytochromes might compete with the heme attachment machinery. In addition, apocyto- chromes 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 oxi- dation would also slow heme attachment. Our observa- tion that added reductant results in a substantial increase in cytochrome c 550 production indicates that oxidation of the heme-binding motif can reduce the efficiency of heme attachment by System II. Neverthe- less, 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 sin- gle-cysteine-containing variants of P. denitrificans c 550 with coexpression of the System II plasmid. A very low, variable level of heme attachment was observed with the AXXCH variant of cytochrome c 552 , 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 com- pared 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 attach- ment 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 c 552 [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 com- parison of System I and II plasmids (which are identi- cal 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 c 550 and c 552 (1 and 3% relative to the wild-type CXXCH cytochromes, respec- tively) 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 plas- mid-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 dif- ferences. That System I can attach some heme to sin- gle-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 Specificity of cytochrome c biogenesis System II A. D. Goddard et al. 732 FEBS Journal 277 (2010) 726–737 ª 2009 The Authors Journal compilation ª 2009 FEBS demonstrates that the chemistry of such a reaction is not necessarily via an obligate disulfide-containing intermediate. Breaking a disulfide bond could be envis- aged 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 apocy- tochrome disulfide also indicated that disulfide-linked chemistry is not involved in the heme attachment reac- tion [39]. The present work implies that the formation of the two thioether bonds is not thermodynamically neces- sary 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 attach- ment 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 non- covalently bound heme) of c-type cytochromes lacking the heme-binding cysteine residues (i.e. the AXXAH variants). Apocytochromes c appear to be proteolyti- cally 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 c 550 AXXAH was coex- pressed with pHP86. It is possible that an equivalent cytochrome is produced by System I, but that it is ren- dered 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-cyto- chrome c 550 serum failed to detect the presence of any cytochrome (Fig. S1, see Supporting Information)]. Alternatively, it is possible that, as a result of a cova- lent intermediate (CcmE–heme) [42], System I is unable to pass heme to an AXXAH variant apocyto- chrome; System II (ResBC) from Helicobacter hepati- cus 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 cova- lent 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 func- tion, 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 peri- plasm 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 sug- gestions, CcmA and CcmB are not involved in heme transport in E. coli [44,45]. Notably, maturation of an AXXAH-containing variant b -type cytochrome c 550 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 holocy- tochrome formation in the presence of the plasmid-encoded biogenesis systems. E. coli strain DH5 a (Invitrogen, Paisley, UK) was used for routine molecular biology. KOD poly- merase (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 A. D. Goddard et al. Specificity of cytochrome c biogenesis System II FEBS Journal 277 (2010) 726–737 ª 2009 The Authors Journal compilation ª 2009 FEBS 733 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 prod- uct 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 bio- genesis 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 resul- tant 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 c 550 was expressed from the iso- propyl thio-b-d-galactoside (IPTG)-inducible promoter of pKPD1 [26]. Mutations within the CXXCH heme-binding motif of c 550 were created by Quikchange using the follow- ing: c 550 C35A, C35AF and C35AR; c 550 C38A, C38AF and C38AR; c 550 C35AC38A, C35AC38AF and C35AC38AR. H. thermophilus cytochrome c 552 and its AX- XCH, 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 cyto- chromes 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 sup- plemented with 1.5% bacteriological agar. For the prepara- tion of periplasmic fractions, single colonies containing appropriate plasmids were picked into 500 mL 2· TY medium (16 gÆL )1 peptone, 10 gÆL )1 yeast extract, 5 gÆL )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 l g Æ mL )1 and chlorampheni- col at 34 lg Æ mL )1 . Analysis of cytochrome production Periplasmic extractions were performed as described previ- ously [22]. Extracts were analyzed by SDS-PAGE (Invitro- gen 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 lg). Western blots to detect P. denitrificans cytochrome c 550 were performed according to the manufacturer’s instruc- tions using anti-cytochrome c 550 rabbit serum and a commercial alkaline-phosphatase-conjugated anti-rabbit secondary IgG raised in goat. The marker used was See- Blue 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 pre- sented 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 c 552 , e = 182 mm )1 Æcm )1 at 417 nm; C11A c 552 , e = 179.5 mm )1 Æcm )1 at 422 nm; C14A c 552 , e = 174.5 mm )1 Æcm )1 at 420 nm; C11A ⁄ C14A c 552 , e = 145 mm )1 Æcm )1 at 425 nm; P. denitrificans cytochrome c 550 wild-type and variants, 140 mm )1 Æcm )1 at 415 nm [22]. The extinction coefficients for the cytochrome c 550 variants are unknown; the wild-type value was therefore used. Cor- rections 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 sponta- neous holocytochrome production) and subtracting the value observed when no heterologous cytochrome gene was expressed (i.e. with plasmid pKK223-3 and the relevant bio- genesis plasmid, to correct for the production of endoge- nous 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 endoge- nous 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. Abra- ham 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]. Specificity of cytochrome c biogenesis System II A. D. Goddard et al. 734 FEBS Journal 277 (2010) 726–737 ª 2009 The Authors Journal compilation ª 2009 FEBS References 1 Stevens JM, Daltrop O, Allen JW & Ferguson SJ (2004) C-type cytochrome formation: chemical and biological enigmas. Acc Chem Res 37, 999–1007. 2 Ferguson SJ, Stevens JM, Allen JW & Robertson IB (2008) Cytochrome c assembly: a tale of ever increasing variation and mystery? Biochim Biophys Acta 1777, 980–984. 3 Bowman SE & Bren KL (2008) The chemistry and biochemistry of heme c: functional bases for covalent attachment. Nat Prod Rep 25, 1118–1130. 4 Hamel P, Corvest V, Giege P & Bonnard G (2009) Biochemical requirements for the maturation of mitochondrial c-type cytochromes. Biochim Biophys Acta 1793, 125–138. 5 Allen JW, Jackson AP, Rigden DJ, Willis AC, Fergu- son SJ & Ginger ML (2008) Order within a mosaic dis- tribution of mitochondrial c-type cytochrome biogenesis systems? Febs J 275, 2385–2402. 6 Kranz RG, Beckett CS & Goldman BS (2002) Geno- mic analyses of bacterial respiratory and cytochrome c assembly systems: Bordetella as a model for the system II cytochrome c biogenesis pathway. Res Microbiol 153, 1–6. 7 Kuras R, Saint-Marcoux D, Wollman FA & de Vitry C (2007) A specific c-type cytochrome maturation sys- tem is required for oxygenic photosynthesis. Proc Natl Acad Sci USA 104, 9906–9910. 8 Allen JW, Ginger ML & Ferguson SJ (2004) Matura- tion of the unusual single-cysteine (XXXCH) mito- chondrial c-type cytochromes found in trypanosomatids must occur through a novel biogene- sis pathway. Biochem J 383, 537–542. 9 Sanbongi Y, Yang JH, Igarashi Y & Kodama T (1991) Cloning, nucleotide sequence and expression of the cytochrome c-552 gene from Hydrogenobacter thermo- philus. Eur J Biochem 198, 7–12. 10 Pollock WB, Rosell FI, Twitchett MB, Dumont ME & Mauk AG (1998) Bacterial expression of a mitochon- drial cytochrome c. Trimethylation of lys72 in yeast iso-1-cytochrome c and the alkaline conformational transition. Biochemistry 37, 6124–6131. 11 Feissner RE, Richard-Fogal CL, Frawley ER, Lough- man JA, Earley KW & Kranz RG (2006) Recombinant cytochromes c biogenesis systems I and II and analysis of haem delivery pathways in Escherichia coli. Mol Microbiol 60, 563–577. 12 Le Brun NE, Bengtsson J & Hederstedt L (2000) Genes required for cytochrome c synthesis in Bacillus subtilis. Mol Microbiol 36, 638–650. 13 Hodson CT, Lewin A, Hederstedt L & Le Brun NE (2008) The active-site cysteinyls and hydrophobic cav- ity residues of ResA are important for cytochrome c maturation in Bacillus subtilis. J Bacteriol 190, 4697– 4705. 14 Crow A, Le Brun NE & Oubrie A (2005) The role of ResA in type II cytochrome c maturation. Biochem Soc Trans 33, 149–151. 15 Dreyfuss BW, Hamel PP, Nakamoto SS & Merchant S (2003) Functional analysis of a divergent system II pro- tein, Ccs1, involved in c-type cytochrome biogenesis. J Biol Chem 278, 2604–2613. 16 Beckett CS, Loughman JA, Karberg KA, Donato GM, Goldman WE & Kranz RG (2000) Four genes are required for the system II cytochrome c biogenesis pathway in Bordetella pertussis, a unique bacterial model. Mol Microbiol 38, 465–481. 17 Hamel PP, Dreyfuss BW, Xie Z, Gabilly ST & Merchant S (2003) Essential histidine and tryptophan residues in CcsA, a system II polytopic cytochrome c biogenesis protein. J Biol Chem 278, 2593–2603. 18 Lee JH, Harvat EM, Stevens JM, Ferguson SJ & Saier MH Jr (2007) Evolutionary origins of members of a superfamily of integral membrane cytochrome c bio- genesis proteins. Biochim Biophys Acta 1768, 2164– 2181. 19 Richard-Fogal CL, Frawley ER, Bonner ER, Zhu H, San Francisco B & Kranz RG (2009) A conserved haem redox and trafficking pathway for cofactor attachment. EMBO J 28, 2349–2359. 20 Frawley ER & Kranz RG (2009) CcsBA is a cyto- chrome c synthetase that also functions in heme trans- port. Proc Natl Acad Sci USA 106, 10201–10206. 21 Fulop V, Sam KA, Ferguson SJ, Ginger ML & Allen JW (2009) Structure of a trypanosomatid mitochon- drial cytochrome c with heme attached via only one thioether bond and implications for the substrate rec- ognition requirements of heme lyase. Febs J 276, 2822– 2832. 22 Allen JWA, Tomlinson EJ, Hong L & Ferguson SJ (2002) The Escherichia coli cytochrome c maturation (Ccm) system does not detectably attach heme to single cysteine variants of an apocytochrome c. J Biol Chem 277, 33559–33563. 23 Sambongi Y, Stoll R & Ferguson SJ (1996) Alteration of haem-attachment and signal-cleavage sites for Paracoccus denitrificans cytochrome c550 probes path- way of c-type cytochrome biogenesis in Escherichia coli. Mol Microbiol 19, 1193–1204. 24 Simon J, Eichler R, Pisa R, Biel S & Gross R (2002) Modification of heme c binding motifs in the small subunit (NrfH) of the Wolinella succinogenes cyto- chrome c nitrite reductase complex. FEBS Lett 522, 83–87. 25 Arslan E, Schulz H, Zufferey R, Kunzler P & Tho ¨ ny- Meyer L (1998) Overproduction of the Bradyrhizobium japonicum c-type cytochrome subunits of the cbb3 A. D. Goddard et al. Specificity of cytochrome c biogenesis System II FEBS Journal 277 (2010) 726–737 ª 2009 The Authors Journal compilation ª 2009 FEBS 735 [...]... deficiency in c- type cytochrome biogenesis in Escherichia coli carrying a mutation in a membrane-bound disulphide isomerase-like protein FEBS Lett 353, 235–238 Bertini I, Cavallaro G & Rosato A (2006) Cytochrome c: occurrence and functions Chem Rev 106, 90–115 Braun M & Thony-Meyer L (2004) Biosynthesis of ¨ artificial microperoxidases by exploiting the secretion and cytochrome c maturation apparatuses of Escherichia...Specificity of cytochrome c biogenesis System II 26 27 28 29 30 31 32 33 34 35 36 37 38 736 A D Goddard et al oxidase in Escherichia coli Biochem Biophys Res Commun 251, 744–747 Sambongi Y & Ferguson SJ (1994) Synthesis of holo Paracoccus denitrificans cytochrome c5 50 requires targeting to the periplasm whereas that of holo Hydrogenobacter thermophilus cytochrome c5 52 does not Implications for c- type cytochrome. .. hydrolysis activity by CcmAB results in loss of c- type cytochrome synthesis and incomplete processing of CcmE Febs J 274, 2322–2332 46 Daltrop O & Ferguson SJ (2003) Cytochrome c maturation The in vitro reactions of horse heart apocytochrome c and Paracoccus denitrificans apocytochrome c5 50 with heme J Biol Chem 278, 4404–4409 47 Thony-Meyer L, Fischer F, Kunzler P, Ritz D & ¨ Hennecke H (1995) Escherichia... a class I c- type cytochrome has a surprisingly small effect on physicochemical properties J Biol Chem 275, 32530–32534 Fabianek RA, Hofer T & Thony-Meyer L (1999) ¨ Characterization of the Escherichia coli CcmH protein reveals new insights into the redox pathway required for cytochrome c maturation Arch Microbiol 171, 92–100 Sambongi Y & Ferguson SJ (1994) Speci c thiol compounds complement deficiency... Expression of prokaryotic and eukaryotic cytochromes c in Escherichia coli Biochim Biophys Acta 1459, 131–138 39 Daltrop O & Ferguson SJ (2004) In vitro studies on thioether bond formation between Hydrogenobacter thermophilus apocytochrome c5 52 with metalloprotoporphyrin derivatives J Biol Chem 279, 45347– 45353 40 Rosell FI & Mauk AG (2002) Spectroscopic properties of a mitochondrial cytochrome c with a single... Escherichia coli Proc Natl Acad Sci USA 101, 12830–12835 Allen JW & Ferguson SJ (2006) What is the substrate specificity of the System I cytochrome c biogenesis apparatus? Biochem Soc Trans 34, 150–151 Bernard DG, Gabilly ST, Dujardin G, Merchant S & Hamel PP (2003) Overlapping specificities of the mitochondrial cytochrome c and c1 heme lyases J Biol Chem 278, 49732–49742 Sanders C & Lill H (2000) Expression... maturation components CcmF, CcmH, and CcmI form a membrane-integral multisubunit heme ligation complex J Biol Chem 283, 29715–29722 50a Kern M, Eisel F, Scheithauer J, Kranz RG & Simon J (2009) Substrate specificity of three cytochrome c haem lyase isoenzymes from Wolinella succinogenes: unconventional haem c binding motifs are not sufficient for haem c attachment by NrfI and CcsA1 Mol Microbiol, doi:10.1111/j.1365-2958.2009.06965.x... 2009 The Authors Journal compilation ª 2009 FEBS A D Goddard et al Supporting Information The following supplementary material is available: Fig S1 Detection of P denitrificans cytochrome c5 50 and variants via western blot Table S1 Oligonucleotides used in this work Table S2 Plasmids used in this work This supplementary material can be found in the online version of this article Specificity of cytochrome. .. (2002) In vitro formation of a c- type cytochrome Proc Natl Acad Sci USA 99, 7872–7876 Tomlinson EJ & Ferguson SJ (2000) Conversion of a c type cytochrome to a b type that spontaneously forms in vitro from apo protein and heme: implications for c type cytochrome biogenesis and folding Proc Natl Acad Sci USA 97, 5156–5160 Tomlinson EJ & Ferguson SJ (2000) Loss of either of the two heme-binding cysteines... with a single thioether bond to the heme prosthetic group Biochemistry 41, 7811–7818 41 Gao T & O’Brian MR (2007) Control of DegP-dependent degradation of c- type cytochromes by heme and the cytochrome c maturation system in Escherichia coli J Bacteriol 189, 6253–6259 42 Schulz H, Hennecke H & Thony-Meyer L (1998) ¨ Prototype of a heme chaperone essential for cytochrome c maturation Science 281, 1197–1200 . CcdA CcmH CcmG DsbD D CcmB CcmA CcmC p-side p-side Disulfide isomerization Fig. 1. Schematic representation of cytochrome c biogenesis Systems I and II. 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