c-type cytochromes are normally characterized by covalent attachment of the iron cofactor haem to protein through two thioether bonds between the vinyl groups of the haem and the thiol groups of a CXXCH (Cys–Xaa–Xaa–Cys–His) motif. In cells, the haem attachment is an enzyme-catalysed post-translational modification. We have previously shown that co-expression of a variant of Escherichia coli cytochrome b562 containing a CXXCH haem-binding motif with the E. coli Ccm (cytochrome c maturation) proteins resulted in homogeneous maturation of a correctly formed c-type cytochrome. In contrast, in the absence of the Ccm apparatus, the product holocytochrome was heterogeneous, the main species having haem inverted and attached through only one thioether bond. In the present study we use further variants of cytochrome b562 to investigate the substrate specificity of the E. coli Ccm apparatus. The system can mature c-type cytochromes with CCXXCH, CCXCH, CXCCH and CXXCHC motifs, even though these are not found naturally and the extra cysteine residue might, in principle, disrupt the biogenesis proteins which must interact intricately with disulfide-bond oxidizing and reducing proteins in the E. coli periplasm. The Ccm proteins can also attach haem to motifs of the type CXnCH where n ranges from 2 to 6. For n=3 and 4, the haem attachment was correct and homogeneous, but for higher values of n the holocytochromes displayed oxidative addition of sulfur and/or oxygen atoms associated with the covalent haem-attachment process. The implications of our observations for the haem-attachment reaction, for genome analyses and for the substrate specificity of the Ccm system, are discussed.
- c-type cytochrome
- cytochrome b562
- cytochrome c maturation
- haem-binding motif
- post-translational modification
Cytochrome b562 is a haemoprotein naturally expressed in the periplasm of Escherichia coli . It has a four α-helix bundle structure [2,3], and its apoprotein (the haem-free form) is also stable and structurally relatively ordered . Other periplasmic cytochromes in E. coli are of the c-type, i.e. they contain haem(s) covalently attached to polypeptide through thioether bonds between the vinyl groups of haem and the cysteine residue sulfurs of a CXXCH (Cys–Xaa–Xaa–Cys–His) peptide motif [5–8]. Biogenesis of c-type cytochromes in E. coli and many other Gram-negative bacteria is a complex post-translational process requiring multiple proteins. These are the Ccm (cytochrome c maturation) system (often called System I), CcmABCDEFGH, and various disulfide-bond oxidizing and reducing proteins, including DsbA, DsbB, DsbD and TrxA [5,7,9,10]. The Dsb and Ccm systems are believed to work in series during cytochrome c biogenesis such that as the unfolded apocytochrome is exported to the periplasm it is first oxidized by DsbA (whose oxidant is DsbB). The apocytochrome disulfide bond must be reduced before haem can be attached, and this reduction is achieved by CcmG (with the possible involvement of CcmH). CcmG is, in turn, reduced by the transmembrane protein DsbD, whose electron donor is the cytoplasmic TrxA [7,9,11].
E. coli cytochrome b562 can be altered by site-directed mutagenesis such that it will form c-type cytochromes in the periplasm [12,13]. In cytochrome b562, a histidine residue, residue 102, is the proximal ligand to the haem iron; an analogous histidine is a virtually universal feature of both b- and c-type cytochromes. We constructed a R98C/Y101C variant of b562 resulting in a CXXCH haem-binding motif . When this protein was expressed in the presence of the E. coli Ccm proteins (also expressed from a plasmid), the product was a homogeneous bona fide c-type cytochrome with haem attached through two thioether bonds (which was characterized extensively by NMR) . In contrast, b562 CXXCH expressed in the absence of the Ccm system resulted in heterogeneous products; in the main species, haem was attached by only one thioether bond and was inverted relative to its orientation in the Ccm-matured cytochrome [12,13]. A large amount of apocytochrome was also present in each case. Cytochrome b562 derivatives thus have major experimental advantages in the analysis of c-type cytochrome biogenesis: (i) the apoproteins and various forms of the holoprotein are, unlike almost all other apocytochromes c, stable in vivo and can be isolated. This allows for the positive detection of expressed proteins and confirmation of the correct mutations by MS; and (ii) correctly and incorrectly matured c-type holocytochrome products can be readily assessed and distinguished using absorption spectroscopy (see [13,14] for further details).
The E. coli Ccm apparatus has been shown to mature c-type cytochromes from diverse prokaryotic and eukaryotic origins with many different folds and functions (e.g. [15–18]). However, it is essentially unable to mature potential c-type cytochromes with only one cysteine residue in the haem-binding motif, or those lacking the histidine residue of the CXXCH sequence [14,19,20]. In the present study, we use other variants of E. coli cytochrome b562 to further investigate the substrate specificity of the E. coli Ccm apparatus. Notably, there is great variation in the ‘XX’ residues of the CXXCH motifs of Ccm-matured c-type cytochromes. The only residue not naturally observed to date in either of the XX positions is cysteine. We hypothesized that this is because additional cysteine residues would inhibit or disrupt the Ccm biogenesis system, which (see above) interacts intricately with the disulfide-bond oxidation/reduction proteins of Gram-negative bacteria. Thus we have constructed variants of cytochrome b562 containing CCXXCH, CCXCH, CXCCH and CXXCHC motifs and investigated their maturation by the E. coli Ccm proteins. The Ccm system can, at least in some organisms, mature c-type cytochromes with three or four residues between the cysteine residues of the haem-binding motif (e.g. Pseudomonas stutzeri cytochrome c552 , cytochrome c3 from sulfate-reducing bacteria , and a variant of Rhodobacter sphaeroides cytochrome c2 ). However, the specificity of the biogenesis system for such proteins is not clear, nor is there any evidence on possible limits for the number of ‘X’ residues. Thus we have investigated maturation of a series of b562 variants with amino acid insertions to create haem-binding motifs ranging from CX2CH to CX6CH.
Bacterial strains, plasmids and growth conditions
E. coli strain JCB387 (a ΔnirB Δlac derivative of strain RV) , a gift of Professor Jeff Cole (School of Biosciences, University of Birmingham, Birmingham, U.K.), was used in all experiments. This strain was chosen because we have found in previous studies that it reliably produces large amounts of holocytochromes c including b562 CXXCH (and also apocytochrome b562 CXXCH), and to ensure consistency with our previous work (e.g. [13,14,19]). The plasmid for the R98C/Y101C (CXXCH haem-binding motif) variant of E. coli cytochrome b562 was as described previously . As required, cells were also transformed with plasmid pEC86  encoding the E. coli cytochrome c maturation genes, ccmABCDEFGH. Transformants were initially grown on LB (Luria broth)-agar plates with the appropriate antibiotics (100 μg of ampicillin·ml−1 in each case, plus 34 μg of chloramphenicol·ml−1 where pEC86 was co-transformed). Single colonies were picked into 500 ml of 2×TY medium (16 g of peptone·l−1, 10 g of yeast extract·l−1 and 5 g of NaCl·l−1), supplemented with 1 mM IPTG (isopropyl β-D-thiogalactoside), in 2.5 litre flasks. Cultures were grown at 37 °C with shaking at 200 rev./min for 20 h, before harvesting. Such aerobic growth conditions repress expression of the FNR (fumarate and nitrate reductase regulatory protein)-regulated endogenous Ccm proteins of E. coli.
The sequence of wild-type cytochrome b562 around the site of incorporation of the CXXCH haem-binding motif  is TRNAYHQ (where the histidine residue, the proximal ligand to the haem iron, is residue 102). Thus, after introduction of the CXXCH motif, the sequence is TCNACHQ. The following plasmids encoding new b562 variants with additional cysteine residues around the haem-binding motif were prepared using the QuikChange® method (Stratagene) and Pfx polymerase (Invitrogen): T97C/R98C/Y101C (b562 CCXXCH), R98C/N99C/Y101C (b562 CCXCH), R98C/A100C/Y101C (b562 CXCCH) and R98C/Y101C/Q103C (b562 CXXCHC). Other variants were prepared from b562 CXXCH by inserting amino acids (see the Results section) between residues 100 and 101 of the starting sequence to give: CNAACH (b562 CX3CH), CNAAGCH (b562 CX4CH), CNA-AGSCH (b562 CX5CH) and CNAAGSQCH (b562 CX6CH). The presence of the desired mutations and the lack of secondary mutations were confirmed both by DNA sequencing and, as a consequence of the characterization of the purified proteins, by MS (see the Results section).
Cell fractionation and biochemical procedures
Periplasmic fractions were obtained from cells using procedures described previously . Cytochrome content was determined by recording absorption spectra of the crude or purified periplasmic fractions to which a small amount of dithionite had been added to reduce the cytochromes. E. coli produces small amounts of endogenous c-type cytochromes in the periplasm in the growth conditions used in the present study; for details of how these were handled during data analysis, see . Cytochrome b562 derivatives were initially purified on a DEAE–Sepharose anion-exchange column. Chromatography was conducted at room temperature (21 °C) in 50 mM Tris/HCl buffer (pH 8.0). The column (Amersham Pharmacia XK26/20 with a resin bed volume of 70 ml) was eluted with a 0–0.5 M NaCl gradient at a flow rate of 9 ml·min−1 and 7 ml fractions were collected. Apocytochrome b562 eluted before, and largely separated from, the holocytochrome during this chromatographic step. Apocytochrome-containing fractions were identified from their absorption spectra following the addition of DTT (dithiothreitol) and haem (producing a species with a characteristic b-type cytochrome spectrum), and were nearly pure as judged by SDS/PAGE. Where necessary, the spectrophotometrically purest holocytochrome fractions were pooled, diluted 4-fold and loaded on to a Q-Sepharose column (XK26/20), which was eluted with a 0–0.25 M NaCl gradient in the same buffer and conditions. After this step, the holocytochrome b562 derivatives were electrophoretically pure.
SDS/PAGE was conducted using the NuPage system (Invitrogen) with 10% Bis-Tris gels and Mes running buffer, used according to the manufacturer's protocols (Invitrogen). Samples for analysis by SDS/PAGE did not include reductant unless stated. Gels were stained for protein using Simply Blue Safestain (Invitrogen) according to the manufacturer's protocol. Gels were stained for proteins with haem covalently bound as follows: after electrophoresis, the gel was soaked in 50 mM sodium acetate solution (pH 5) for 30 min with gentle rocking, after which 33 mg of TMBZ (3,3′,5,5′-tetramethylbenzidine) dissolved in 30 ml of methanol was added. The gel was allowed to incubate for a further 10 min, after which 600 μl of 30% hydrogen peroxide was added. The stain developed in <10 min. Ellman's reagent was used as described by Riddles et al. . Reduced pyridine haemochrome spectra were obtained as described by Bartsch . ESI–MS (electrospray ionization MS) was performed on a Micromass Bio-Q II-2S triple quadrupole atmospheric pressure instrument equipped with an electrospray interface. Samples were introduced via a loop injector as a solution [20 pmol·μl−1 in 1:1 water/acetonitrile and 0.2% (v/v) formic acid] at a flow rate of 10 μl·min−1. Data were analysed using the maximum entropy-based component of the MassLynx suite of software (Micromass) .
NMR spectra of Ccm-matured holocytochrome b562 CCXXCH protein were collected as described previously  using a Bruker DRX 500 spectrometer. All spectra were acquired at 300 K. Data processing was as described previously , except that it was performed using XWINNMR (Bruker) or SPARKY. Samples contained 0.5 mM protein in 20 mM potassium phosphate buffer in 2H2O (p2H 6.6) and 0.5 M KCl. The protein was reduced by the addition of sodium dithionite to a concentration of 5 mM from anaerobic stock solutions in the same buffer.
The effect of extra cysteine residues around the haem-binding motif
Absorption and reduced pyridine haemochrome spectra of crude periplasmic extracts from E. coli cells co-transformed with a plasmid for b562 CCXXCH, CCXCH, CXCCH or CXXCHC and a plasmid expressing the ccm genes (Figure 1 and Table 1) were indistinguishable from those of extracts of cells producing correctly (i.e. Ccm) matured b562 CXXCH . Each construct included the natural signal sequence of cytochrome b562 which directs the apoprotein to the periplasm, where both wild-type b562 and all Gram-negative bacterial cytochromes c are naturally assembled. Each product holocytochrome had absorbance α- and β-band maxima at 556 and 526 nm respectively, and the pyridine haemochrome α-band maximum at 550 nm (Figure 1 and Table 1). [Reduced pyridine haemochrome spectra provide information about the nature of any modifications to the haem (such as covalent attachment to protein through thioether bonds), but the spectrum is otherwise independent of the protein environment because the protein is denatured. Their absorption maxima thus give strong evidence about proper or improper maturation of a c-type cytochrome: for a cytochrome with haem attached through two thioether bonds the α-band maximum is at 549–550 nm, for attachment through one thioether bond it is at 552–553 nm, and for a b-type cytochrome (no covalent attachment) it is at 556 nm.] These results were the same for the purified cytochromes, which were shown chromatographically to be ≥95% homogeneous in each case (based on absorption and haemochrome data). The spectral data are characteristic of c-type cytochrome variants of cytochrome b562 with haem bound to protein through two thioether bonds (see e.g. ). The holocytochrome yields were high (approx. 5–6 mg of holocytochrome c per g of wet cells) for each variant (calculated by assuming a molar absorption coefficient of 22900 M−1·cm−1 at 556 nm, as for Component II described by Barker and co-workers [12,13]). As was the case with b562 CXXCH, a large amount of apo- (haem-free) cytochrome could be observed (by adding haem, DTT and dithionite to periplasmic extracts to generate reduced b-type cytochromes ) following expression of each of the CCXXCH, CCXCH, CXCCH or CXXCHC cytochromes.
When the b562 variants containing three cysteine residues were analysed using SDS/PAGE gels stained for covalently bound haem, two major bands were apparent in each case, one corresponding to monomer (running at the same molecular mass as the positive control, purified holocytochrome b562 CXXCH) and one corresponding to dimer. When DTT was added for approx. 30 min after the samples were boiled, but before running the gel, the amount of dimer dramatically decreased. These results suggest that these variants can all form disulfide-bond-linked dimers. ESI–MS of holocytochrome purified by DEAE–Sepharose chromatography showed, for each ‘extra-cysteine’ b562 variant (b562 CCXXCH, CCXCH, CXCCH and CXXCHC), peaks with masses corresponding to holocytochrome monomer, apocytochrome–holocytochrome dimer (i.e. protein dimer with one haem attached) and holocytochrome–holocytochrome dimer (Supplementary Table S1 at http://www.BiochemJ.org/bj/419/bj4190177add.htm). [Apocytochrome–apocytochrome dimers were also observed (Supplementary Table S1)]. The dimerizations probably occurred more slowly than haem attachment to the cytochromes, possibly during cell fractionation and protein isolation; it seems unlikely that the Ccm system could attach haem to a dimer already formed via cysteine residues adjacent to, or within, the CXXCH haem-binding motif.
In the absence of expression of the Ccm proteins, incorrectly matured holocytochrome was observed for each of the b562 extra cysteine variants, judging by absorption spectra, pyridine haemochrome spectra and SDS/PAGE gels (Figure 1 and Table 1). Absorption maxima were at 558–560 nm and 528–529 nm with pyridine haemochrome maxima at 552–553 nm. Very similar observations were previously made for b562 CXXCH matured in the absence of the Ccm proteins [12,13] and represent haem attachment apparently without the aid of enzyme catalysis. The yield for the cytochrome b562 ‘extra cysteine’ variants expressed in the absence of the Ccm proteins ranged from 1.5 to 2.8 mg of holocytochrome per g of wet cells (calculated assuming a molar absorption coefficient of 25800 M−1·cm−1 for the reduced α-band peak in each case, as for Component I in ). Thus, for each holocytochrome, the yield was lower than when the corresponding cytochrome was matured by the Ccm apparatus (see above and Table 1). SDS/PAGE gels stained for protein or for covalently bound haem for each of the triple cysteine variants expressed in the absence of the Ccm proteins showed the formation of dimers, trimers and small amounts of tetramer which were predominantly DTT reducible, as well as monomers. This polymerization (as well as the pyridine haemochrome maxima) is consistent with non-Ccm-dependent haem attachment to b562 CXXCH being through only a single cysteine–haem bond [12,13] (hence the variants with three cysteine residues being able to oligomerize).
For the Ccm-matured cytochrome b562 extra cysteine variants, the haem could, in principle, be attached to any two of the three cysteine residues. The absorption spectroscopic methods described so far demonstrate that the haem is attached to two of them (i.e. that the two original haem vinyl groups are both saturated), but cannot determine which cysteine residues. This is of particular relevance for the CCXXCH variant, since the Ccm proteins can attach haem readily to both CXXCH and CXXXCH motifs [including a cytochrome b562 CX3CH variant (see below and Figure 2A)]. NMR data indicate that the vast majority of the haem attachment to the CCXXCH protein by the Ccm apparatus was to the CCXXCH cysteine residues (shown in bold), rather than to CCXXCH. 1H-NMR spectra (Figure 3) of the purified holoprotein are consistent with one predominant species. The haem proton resonances from this species, together with resonances from some amino acid side-chain protons of residues in the vicinity of the haem, were assigned using homonuclear NOESY and TOCSY experiments (Figure 3B) as previously described [12,13]. These chemical shifts are compared with those of Ccm-matured holocytochrome b562 CXXCH [12,13] in Supplementary Table S2 (at http://www.BiochemJ.org/bj/419/bj4190177add.htm). The close similarity of these resonances from the two proteins suggests that the structure at the haem-attachment sites, and the electronic structure of the haem, is very similar in the CXXCH and CCXXCH cytochromes. In the spectra of the reduced [Fe(II)] CCXXCH protein, one major resonance from the ε-methyl group of the haem iron ligand Met7 is observed (Figure 3A). Also observed are resonances (marked by asterisks in Figure 3A) from the ε-methyl group of the same residue but in other, very closely related, species. These species are not abundant enough to be able to identify the origin of this alteration to the chemical shift of the ligand methyl protons, but such alternative species have been observed before in both cytochrome b562  and cytochrome c552 from Hydrogenobacter thermophilus , and we do not believe that they arise from species with alternative modes of haem attachment. Indeed, in the NOESY and TOCSY NMR spectra of Ccm-matured holocytochrome b562 CCXXCH (Figure 3B), we can find only the expected two resonances from protons at the 2- and 4-methine positions of the haem. Highlighted in the expanded region of Figure 3(B) are the TOCSY cross-peaks from the 2- and 4-methine and methyl haem substituents that result from the reaction of the cysteine residues of the apocytochrome haem-binding motif with the haem 2- and 4-vinyl groups. There is no heterogeneity revealed in this region of the spectrum, and the chemical shifts of the relevant protons are very close to those reported in our previous studies for Ccm-matured cytochrome b562 CXXCH (Supplementary Table S2; Component II in [12,13]), which has the same stereo- and regio-specific covalent attachment of haem as is observed in all natural c-type cytochromes for which relevant data are available. The sensitivity of our NMR experiments is such that we can therefore confidently say that at least 90% of the protein has haem attached to the CCXXCH motif, but we cannot rule out that <10% has attachment to the alternative CCXXCH cysteine residues (shown in bold). Protein digestion followed by HPLC isolation of peptides, cysteine derivatization and N-terminal sequencing suggested some haem attachment to both the first and second cysteine residues of the CCXXCH motif. This analysis was not quantitative, but it was consistent with the significant majority of haem attachment being to the CCXXCH cysteine residues. It is possible that oxidation of the CCXXCH apocytochrome by the E. coli periplasmic disulfide oxidase DsbA, and/or interaction with the thiol-containing proteins of the Ccm system (CcmG, CcmH), selects preferentially the CXXCH cysteine residues for haem attachment.
The effect of extra residues between the cysteine residues of the haem-binding motif
We also constructed variants of E. coli cytochrome b562 with potential CX3CH, CX4CH, CX5CH and CX6CH haem-binding motifs (‘CXnCH variants’). This was achieved by inserting additional amino acids between the cysteine residues of the b562 R98C/Y101C (i.e. CXXCH) variant. The inserted amino acids were chosen so as to avoid bulky side chains and proline, and to include a mixture of polar and somewhat hydrophobic residues; we did not try to optimize the selection of inserted residues. The resulting sequences were CNAACH (b562 CX3CH), CNAAGCH (b562 CX4CH), CNAAGSCH (b562 CX5CH) and CNAAGSQCH (b562 CX6CH).
Each CXnCH variant was expressed with the Ccm proteins and the holocytochromes were purified. In each case, the absorption and pyridine haemochrome spectra of the resulting holocyto-chromes were indicative of haem attachment through two thioether bonds (Figure 2A and Table 2). The yield is calculated assuming all the holocytochromes have the same molar absorption coefficients as Ccm-matured cytochrome b562 CXXCH (Component II in ; see also ).
Notably, the CX3CH and CX4CH variants were in each measured respect, including by MS, properly matured c-type cytochromes (Figure 2A and Table 2). It is also clear that the Ccm system acted upon the CX5CH and CX6CH apocytochromes. The presence of the Ccm proteins increased the holocytochrome yield and produced absorption and pyridine haemochrome spectra characteristic of double-thioether-bond attachment of haem to protein (Figure 2A and Table 2). In the absence of the Ccm proteins, the b562 CX5CH and CX6CH holocytochromes both showed the characteristics of uncatalysed haem attachment through a single thioether bond (Figure 2B and Table 2) [12,13]. However, even though the Ccm system matured the CX5CH and CX6CH cytochromes, it is clear from the mass spectra that the products did not show the usual ultra-high level of quality control observed for the Ccm apparatus, and species of different mass were observed in various proportions.
Analysis by ESI–MS showed that for the CX5CH variant, approx. 60% of the holocytochrome was of the expected molecular mass, with approx. 10% at 32 Da above the expected value and approx. 30% 64 Da heavier than expected (Table 2). In the CX6CH case, very little of the holocytochrome was at the expected mass; the major species (approx. 55%) was 64 Da above the expected mass, with the remainder at +48 Da (approx. 30%) or +32 Da. Note that the relative populations of each species given are estimates based on the intensities of peaks in the transformed mass spectrum, assuming that each form of the protein flies equally well in the electrospray experiment; the precise proportions also varied between different holocytochrome preparations. (In the extreme case, approx. 75% of the CX5CH holocytochrome had the correct mass.) The peaks at heavier masses than expected presumably arise from modified holocytochromes. These modifications may result from the addition of sulfur (32 Da) and/or oxygen (16 Da) atoms in some proportion(s), but the method of MS used is not precise enough to distinguish between these possibilities. The likely sites of any oxidation in the cytochromes are the cysteine and methionine residues.
Notably, mass spectra of apoproteins containing CX5CH and CX6CH sequences purified from cells also expressing the Ccm proteins from plasmid pEC86 did not show any evidence of significant extra mass (≤10% of the protein), suggesting that the oxidation observed for the holocytochromes (Table 2) is associated with, or a result of, haem attachment. Incubation of apocytochrome b562 CX5CH and CX6CH with DTT before recording the mass spectra resulted in mass increases of 2.1 and 1.8 Da respectively, indicating that reduction of a disulfide bond within the apoprotein had taken place. Analysis with Ellman's reagent confirmed this, showing that >95% of the cysteine residues in the purified apocytochromes CX5CH and CX6CH were present in disulfide bonds (as seen previously for the CXXCH variant ). For the analysis with Ellman's reagent, the apocytochrome concentration was estimated from the theoretical molar absorption coefficient at 280 nm calculated from the protein sequence, and by titrating the reduced apoprotein with haem (to form a b-type cytochrome) until it was saturated.
Our results suggest that cytochromes with CCXXCH, CCXCH, CXCCH and CXXCHC motifs can be matured by the E. coli Ccm apparatus (Figure 1 and Table 1). Thus our hypothesis that extra cysteine residues in or around the consensus CXXCH haem-binding motif would disrupt the biogenesis proteins has proven incorrect. These results therefore beg the question why are extra cysteine residues not observed in the ‘XX’ positions of, or adjacent to, the CXXCH motifs of natural c-type cytochrome haem-binding motifs? More generally, why are cysteine residues other than those in the CXXCH motif rare overall in the soluble domains of c-type cytochromes? Given that our results suggest that such proteins could be matured (at least by the type of cytochrome c biogenesis apparatus investigated in the present study), one likely explanation is that free cysteine thiols could react with haem iron and promote destructive chemistry. In particular, a thiol can be oxidized by ferric haem to generate a thiyl radical, which could then attack the porphyrin ring or the protein. Such reactions have been observed in various conditions (e.g. [30–33]). Clearly this is undesirable in the cell, where haem proteins have the potential to do significant oxidative damage and their formation and properties must be tightly controlled. Another factor is the potential for dimerization via formation of disulfide bonds in the oxidizing environment of the bacterial periplasm, which was clearly apparent in each of our mutants containing extra cysteine residues (e.g. Supplementary Table S1), and the potential to form mixed disulfides with other periplasmic thiol proteins. Any functionally undesirable consequences of such interactions can be avoided if there are no free cysteine residues available to form disulfide bonds. Thirdly, as our results for the CCXXCH variant of cytochrome b562 suggest, extra cysteine residues around the haem-binding motif introduce the possibility of heterogeneity in haem attachment, which could have negative consequences for the function of c-type cytochromes and hence for the cell.
It is also clear from our results that the Ccm system can act on apocytochromes with at least six residues between the haem-binding cysteine residues (Figure 2A and Table 2). All methods used showed that the CX3CH and CX4CH variants were matured properly. Examples of such haem-attachment motifs are, although rare, known in Nature (e.g. [21,22], and reviewed in ). Maturation of the cytochrome b562 CX5CH and CX6CH variants was also enhanced by the Ccm apparatus (Figure 2), although in these cases the products were neither perfect nor homogeneous. The Ccm-matured CXnCH holocytochromes had absorption α-band maxima at 555 nm and pyridine haemochrome α-bands at 549.5 nm (Figure 2A), both characteristic of haem attachment to protein with saturation of both haem vinyl groups. In contrast, the equivalent cytochromes produced in the absence of the Ccm proteins had red-shifted spectra, indicative of (incorrect) haem attachment through only one thioether bond (Figure 2B) (reminiscent of our earlier observations for the CXXCH variant [12,13]). Moreover, the Ccm-matured holocytochromes were present at 6–7-fold higher yields than those produced in the absence of the Ccm proteins (Table 2). Nevertheless, ESI–MS revealed additional mass in ≤40% of the Ccm-matured CX5CH holocytochrome and approx. 100% of the CX6CH equivalent. The holocytochromes formed by these variants in the absence of the Ccm proteins are far more heterogeneous and, despite extensive attempts to do so, we have been unable to obtain consistent mass data by ESI–MS of these samples. More detailed analysis will be required to identify the origin of the extra mass in the modified holocytochromes, but this represents a challenging problem since relevant isotopic labels (18O, 35S) cannot be incorporated specifically into the cytochromes.
The results of the present study are a rare, clear example of the Ccm system failing to exercise ultra-high-fidelity quality control in the maturation of a c-type cytochrome. One interpretation of the data is that a CX4CH motif is the upper limit for the Ccm system to be able to mature the resulting c-type cytochrome with perfect control. However, the Ccm system may be able to properly mature natural (evolved) cytochromes with five (or more) residues between the haem-binding cysteine residues, since the imperfections in our cytochrome b562 variants were small (Table 2 and Figure 2A) and, in the CX5CH holocytochrome, affected ≤40% of the protein. Variations in the Ccm proteins themselves in organisms with putative long CXnCH haem-binding motifs could also alleviate any constraint imposed by the E. coli Ccm apparatus which naturally processes CXXCH as its only (known) endogenous c-type cytochrome haem-binding motif. Simon and co-workers  recently showed that an exceptional cytochrome, MccA from Wolinella succinogenes, has a CX15CH haem-binding motif, but in that case a special, apparently dedicated, System II biogenesis protein was also required for the haem attachment. However, they also pointed out that, in Shewanella species, organisms which use the Ccm system to mature their c-type cytochromes, the gene for a MccA homologue is near to genes encoding CcmF and CcmH homologues, which they postulated are required for haem attachment to the CX15CH motif of MccA. Their results, in combination with our own, have important implications for genome analyses. It has generally been assumed that the defining requirements for a protein to be a c-type cytochrome are a periplasmic (or extracytoplasmic) signal sequence and one or more CXXCH motifs. It is now clear that the latter is not a sufficient basis on which to discriminate, and motifs of the type CXnCH should more generally be considered as plausible cytochromes c. On the other hand, the c-type cytochromes found in the protein structure databases, selected essentially randomly, indicate that haem-attachment motifs other than CXXCH are rare.
The chemical origin of the extra mass that we observed for our Ccm-matured CX5CH and CX6CH holocytochromes (Table 2) is unclear. However, the modification appears related to the covalent attachment of haem, since the corresponding purified apocytochromes showed little evidence of oxidation. If the modification occurred, as seems likely, during the haem-attachment process rather than afterwards, it may reflect kinetic competition in the E. coli periplasm between the chemical processes that lead to incorporation of the extra mass and the haem-attachment reaction. No increased mass holocytochrome species was observed where n=2, 3 or 4; only 40% of the protein showed extra mass for n=5, but ∼100% did for n=6. Thus haem attachment may be relatively fast when n is small and slower as n increases. Notably, the overall Ccm-matured holocytochrome yield decreased as n increased (Table 2).
The results of the present study reaffirm that the specificity determinant of the Ccm system is still, apparently, simply the two cysteine residues and the histidine residue of the haem-binding motif (e.g. [13,14,16,18,19,23,35]). The spacing between the cysteine residues does not affect the ability of the Ccm system to act on the apocytochrome, nor do the residues in the XX positions of the haem-binding motif. In Nature, there is great variation in the ‘XX’ residues of the CXXCH motifs of Ccm-matured c-type cytochromes. These residues can be charged (e.g. lysine), bulky (e.g. tryptophan), or even proline. Indeed, the only residue not naturally observed to date in either of the XX positions is cysteine. However, as shown in the present study, the Ccm system can properly mature variant c-type cytochromes with CCXXCH, CCXCH, CXCCH and CXXCHC motifs. Given the choice of two cysteine residues, e.g. in a CCXXCH motif, the Ccm system clearly selects for haem attachment the N-terminal cysteine residue that is separated by two ‘Xaa’ residues from the C-terminal cysteine residue of the haem-binding motif. We have previously discussed possible mechanistic reasons for the need for two cysteine residues and for the histidine residue of the motif elsewhere .
This work was supported by the Biotechnology and Biological Sciences Research Council [grant numbers BB/C508118/1, BB/D019753/1] and the Engineering and Physical Sciences Research Council (studentship to E. B. S.). J. W. A. A. is a Biotechnology and Biological Sciences Research Council David Phillips Fellow and M. L. G. is a Royal Society University Research Fellow.
We thank Professor David J. Richardson, Dr Antony C. Willis and Dr Christopher J. Andrews for very helpful discussions prior to submission.
Abbreviations: Ccm, cytochrome c maturation; DTT, dithiothreitol; ESI–MS, electrospray ionization MS
- © The Authors Journal compilation © 2009 Biochemical Society