c-Type cytochromes are characterized by covalent attachment of haem to protein through thioether bonds between the vinyl groups of the haem and the thiols of a Cys-Xaa-Xaa-Cys-His motif. Proteins of this type play crucial roles in the biochemistry of the nitrogen cycle. Many Gram-negative bacteria use the Ccm (cytochrome c maturation) proteins for the post-translational haem attachment to their c-type cytochromes. The Ccm system can correctly mature c-type cytochromes with CCXXCH, CCXCH, CXCCH and CXXCHC motifs, even though these are not found naturally and the extra cysteine might, in principle, disrupt the biogenesis proteins. The non-occurrence of these motifs probably relates to the destructive chemistry that can occur if a free thiol reacts with haem iron to generate a radical.
c-Type cytochromes are characterized by covalent attachment of haem to protein through thioether bonds between the vinyl groups of the haem and the thiols of a CXXCH (Cys-Xaa-Xaa-Cys-His) motif . Proteins of this type play crucial roles in the biochemistry of the nitrogen cycle. c-Type cytochrome centres are, for example, found in nitric oxide reductase, hydroxylamine oxidoreductase, a recently discovered nitrous oxide reductase and two types of nitrite reductase; anammox organisms are rich in cytochromes c. In cells, haem attachment to the cytochrome is a catalysed post-translational process, requiring dedicated biogenesis proteins [1,2–3]. Many important nitrogen cycle bacteria, e.g. Paracoccus pantotrophus, Nitrosomonas europaea and Pseudomonas aeruginosa, use the Ccm (cytochrome c maturation) proteins  for this biogenesis of their c-type cytochromes.
Notably, there is great variation in the ‘XX’ residues of the CXXCH motifs of Ccm matured c-type cytochromes. Indeed, the only residue not naturally observed to date in either of the X positions is cysteine. We hypothesized that this is because additional cysteine residues would inhibit or disrupt the Ccm biogenesis apparatus which is known to interact intricately with the disulphide bond oxidation/reduction proteins of Gram-negative bacteria . We have investigated this hypothesis using variants of Escherichia coli cytochrome b562. Following introduction of cysteines by mutagenesis to create a c-type cytochrome CXXCH haem-binding motif, this protein can be matured correctly as a homogeneous c-type cytochrome by the E. coli Ccm apparatus (shown by various techniques, including NMR) . In the absence of the Ccm proteins, a heterogeneous mixture of improperly matured c-type cytochromes was observed [4,5]. In each case, a large amount of stable apo (haem-free) protein was also obtained. Our cytochrome b562 derivative thus has several experimental advantages, as follows. (i) The apoprotein and various forms of the holoprotein are, unlike almost all other apocytochromes c, stable. This allows for the positive detection of expressed proteins and confirmation of the correct mutations by MS. (ii) Correctly and incorrectly matured c-type holocytochrome products can be readily assessed and distinguished using absorption spectroscopy (see [4,6] for further details). Expanding on this approach, we have constructed variants of cytochrome b562 containing CCXXCH, CCXCH, CXCCH and CXXCHC motifs and have investigated their maturation by the E. coli Ccm proteins. Our results are summarized below.
Absorption and 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 were indistinguishable from those of extracts of cells producing correctly matured b562 CXXCH . Each had absorbance α- and β-band maxima at 556 and 526 nm respectively, and the pyridine haemochrome α-band maximum at 550 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). Such spectral data are characteristic of properly matured c-type cytochromes with haem bound to protein through two thioether bonds (see, e.g.,  for details). The holocytochrome yields were high (approx. 5 mg of holocytochrome c per g of wet cells) for each variant.
When the b562 variants containing three cysteines were analysed using SDS/PAGE gels stained for covalently bound haem, two major bands were apparent, one corresponding to a monomer (e.g. purified holocytochrome b562 CXXCH) and one corresponding to a dimer. When DTT (dithiothreitol) was added for approx. 30 min after the samples were boiled but before running the gel, the amount of dimer decreased dramatically. These data suggest that the variants can all form disulphide-linked dimers.
ES-MS (electrospray MS) of the purified holocytochrome showed, for each b562 variant (b562 CCXXCH, CCXCH, CXCCH and CXXCHC), peaks with masses corresponding to holocytochrome monomer, apocytochrome–holocytochrome dimer (i.e. dimer with one haem attached) and holocytochrome–holocytochrome dimer. These data both confirm the presence of the desired mutations and imply that the dimerizations probably occurred more slowly than (and hence subsequent to) haem attachment to the cytochromes.
In the absence of expression of the Ccm proteins, incorrectly matured holocytochrome was observed for each of the b562 variants judging by absorption spectra, pyridine haemochrome spectra and SDS/PAGE analysis. Very similar observations were made previously for b562 CXXCH [4,5] and represent haem attachment without the aid of enzyme catalysis. Haem-stained SDS/PAGE gels for each of the triple cysteine variants expressed in the absence of the Ccm proteins showed the formation of dimers, trimers and higher polymers which were predominantly DTT-reducible, as well as monomers. This polymerization is consistent with non-Ccm-dependent haem attachment to b562 CXXCH through only a single cysteine–haem bond [4,5] (hence the variants with three cysteines being able to form disulphides with up to two other protein molecules).
Our data suggest that cytochromes with CCXXCH, CCXCH, CXCCH and CXXCHC motifs can be matured homogeneously and with correct haem attachment by the E. coli Ccm apparatus. Thus our hypothesis that extra cysteine residues in or around the consensus CXXCH haem-binding motif would disrupt the biogenesis proteins has apparently been proved incorrect. Note, however, that, for each variant, the haem could (in principle) be attached to any two of the three cysteines. The absorption spectroscopic methods used in our studies 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 cysteines. This is of particular relevance for the CCXXCH variant, since the Ccm proteins are known to be able to attach haem to both CXXCH and CXXXCH motifs . It is commonly believed that a disulphide bond in the apocytochrome CXXCH motif is an intermediate in bacterial cytochrome maturation by the Ccm system. Such a disulphide forms most easily within a CXXC grouping (this is the classic thioredoxin motif), thus haem attachment to the two ‘normal’ (i.e. CXXCH) cysteines seems the most likely outcome for each variant.
So, why are extra cysteine residues not observed in the XX positions of natural c-type cytochrome haem-binding motifs? More generally, why are cysteines other than those in the CXXCH motif very rare overall in the soluble domains of c-type cytochromes? Given that our results suggest that such proteins could be matured (at least by one biogenesis apparatus), the most likely explanation is that free cysteine thiols could react with haem iron and promote destructive chemistry. In particular, a thiol could react with 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 (see, e.g., [8,9–10]). 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 disulphide bonds in the oxidizing environment of the bacterial periplasm, which was clearly apparent in each of our mutants. Any functionally undesirable consequences of dimer formation can be avoided if there are no free cysteine residues available to form disulphide bonds.
The 11th Nitrogen Cycle Meeting 2005: Independent Meeting held at Estación Experimental del Zaidín, Granada, Spain, 15–17 September 2005. Organized and Edited by E.J. Bedmar (Granada, Spain), M.J. Delgado (Granada, Spain) and C. Moreno-Vivián (Córdoba, Spain).
This work was supported by the BBSRC (Biotechnology and Biological Sciences Research Council). We thank Dr Paul Barker (University of Cambridge) for very helpful discussions.