Julie M. Stevens

The CcmE protein of the c-type cytochrome biogenesis system: unusual in vitro heme incorporation into apo-CcmE and transfer from holo-CcmE to apocytochrome.

Three key steps of cytochrome c biogenesis in many Gram-negative bacteria, the uptake of heme by the heme chaperone CcmE, the covalent attachment of heme to CcmE, and its subsequent release from CcmE to an apocytochrome c, have been achieved in vitro. apo-CcmE from Escherichia coli preferentially bound to ferric, with high affinity (Kd, 200 nM), rather than ferrous heme. The preference for ferric heme was confirmed by competition with 8-anilino-1-naphthalenesulfonate, which bound to a hydrophobic pocket in apo-CcmE. Reduction under certain conditions of the ferric heme–CcmE complex, which has characteristics of a b-type cytochrome, resulted in covalent attachment of heme to the protein. The resulting in vitro-produced holo-CcmE was identical to the in vivo-produced holo-CcmE, proving that unmodified Fe-protoporphyrin IX is incorporated into CcmE. Only noncovalent binding of mesoheme to CcmE was observed, thus implicating at least one vinyl group in covalent binding of heme to CcmE. Heme transferred in vitro from holo-CcmE to apocytochrome c, provided the heme was reduced. The necessity for reduced holo-CcmE might explain the role of the heme chaperone, i.e., prevention of reaction of ferric heme with apocytochrome and thus avoidance of incorrect side products. In addition, an AXXAH mutant of the CXXCH binding motif in the apocytochrome c was unable to accept heme from holo-CcmE. These in vitro results mimic, and thus have implications for, the molecular pathway of heme transfer during c-type cytochrome maturation in many species of bacteria in vivo.

Cytochrome c biogenesis System I

Cytochromes c are widespread respiratory proteins characterized by the covalent attachment of heme. The formation of c‐type cytochromes requires, in all but a few exceptional cases, the formation of two thioether bonds between the two cysteine sulfurs in a –CXXCH– motif in the protein and the vinyl groups of heme. The vinyl groups of the heme are not particularly activated and therefore the addition reaction does not physiologically occur spontaneously in cells. There are several diverse post‐translational modification systems for forming these bonds. Here, we describe the complex multiprotein cytochrome c maturation (Ccm) system (in Escherichia coli comprising the proteins CcmABCDEFGH), also called System I, that performs the heme attachment. System I is found in plant mitochondria, archaea and many Gram‐negative bacteria; the systems found in other organisms and organelles are described elsewhere in this minireview series.

Cytochrome c assembly: A tale of ever increasing variation and mystery?

Formation of cytochromes c requires a deceptively simple post-translational modification, the formation of two thioether bonds (or rarely one) between the thiol groups of two cysteine residues found in a CXXCH motif (with some occasional variations) and the vinyl groups of heme. There are three partially characterised systems for facilitating this post-translational modification; within these systems there is also variation. In addition, there are clear indications for two other distinct systems. Here some of the current issues in understanding the systems are analysed.

Loss of ATP hydrolysis activity by CcmAB results in loss of c-type cytochrome synthesis and incomplete processing of CcmE

The proteins CcmA and CcmB have long been known to be essential for cytochrome c maturation in Escherichia coli. We have purified a complex of these proteins, and found it to have ATP hydrolysis activity. CcmA, which has the features of a soluble ATP hydrolysis subunit, is found in a membrane‐bound complex only when CcmB is present in the membrane. Mutation of the Walker A motif in CcmA(K40D) results in loss of the in vitro ATPase activity and in loss of cytochrome c biogenesis in vivo. The same mutation does not prevent covalent attachment of heme to the heme chaperone CcmE, but holo‐CcmE is, for some unidentified reason, incompetent for heme transfer to an apocytochrome c or for release into the periplasm as a soluble variant. Addition of exogenous heme to heme‐permeable E. coli with a ccmA deletion did not restore cytochrome c production. Our results suggest a role for CcmAB in the handling of heme by CcmE, which is chemically complex and involves an unusual histidine–heme covalent bond.

Cytochrome c assembly

Cytochromes c are central proteins in energy transduction processes by virtue of their functions in electron transfer in respiration and photosynthesis. They have heme covalently attached to a characteristic CXXCH motif via protein‐catalyzed post‐translational modification reactions. Several systems with diverse constituent proteins have been identified in different organisms and are required to perform the heme attachment and associated functions. The necessary steps are translocation of the apocytochrome polypeptide to the site of heme attachment, transport and provision of heme to the appropriate compartment, reduction and chaperoning of the apocytochrome, and finally, formation of the thioether bonds between heme and two cysteines in the cytochrome. Here we summarize the established classical models for these processes and present recent progress in our understanding of the individual steps within the different cytochrome c biogenesis systems.

A variant System I for cytochrome c biogenesis in archaea and some bacteria has a novel CcmE and no CcmH.

C‐type cytochromes are characterized by post‐translational covalent attachment of heme to thiols that occur in a Cys‐Xxx‐Xxx‐Cys‐His motif. Three distinct biogenesis systems are known for this heme attachment. Archaea are now shown to contain a significantly modified form of cytochrome c maturation System I (the Ccm system). The most notable adaptation relative to the well‐studied apparatus from proteobacteria and plants is a novel form of the heme chaperone CcmE, lacking the highly conserved histidine that covalently binds heme and is essential for function in Escherichia coli. In most archaeal CcmEs this histidine, normally found in a His‐Xxx‐Xxx‐Xxx‐Tyr motif, is replaced by a cysteine residue that occurs in a Cys‐Xxx‐Xxx‐Xxx‐Tyr motif. The CcmEs from two halobacteria contain yet another form of CcmE, having HxxxHxxxH approximately corresponding in alignment to the H/CxxxY motif. The CxxxY‐type of CcmE is, surprisingly, also found in some bacterial genomes (including Desulfovibrio species). All of the modified CcmEs cluster together in a phylogenetic tree, as do other Ccm proteins from the same organisms. Significantly, CcmH is absent from all of the complete archaeal genomes we have studied, and also from most of the bacterial genomes that have CxxxY‐type CcmE.

Covalent cofactor attachment to proteins: cytochrome c biogenesis

Haem (Fe-protoporphyrin IX) is a cofactor found in a wide variety of proteins. It confers diverse functions, including electron transfer, the binding and sensing of gases, and many types of catalysis. The majority of cofactors are non-covalently attached to proteins. There are, however, some proteins in which the cofactor binds covalently and one of the major protein classes characterized by covalent cofactor attachment is the c-type cytochromes. The characteristic haem-binding mode of c-type cytochromes requires the formation of two covalent bonds between two cysteine residues in the protein and the two vinyl groups of haem. Haem attachment is a complex post-translational process that, in bacteria such as Escherichia coli, occurs in the periplasmic space and involves the participation of many proteins. Unexpectedly, it has been found that the haem chaperone CcmE (cytochrome c maturation), which is an essential intermediate in the process, also binds haem covalently before transferring the haem to apocytochromes. A single covalent bond is involved and occurs between a haem vinyl group and a histidine residue of CcmE. Several in vitro and in vivo studies have provided insight into the function of this protein and into the overall process of cytochrome c biogenesis.

Probing the heme-binding site of the cytochrome c maturation protein CcmE.

Maturation of c-type cytochromes in many bacterial species and plant mitochondria requires the participation of the heme chaperone CcmE that binds heme covalently via a His residue (H130 in Escherichia coli) before transferring it stereospecifically to the apo form of cytochromes c. Only the structure of the apo form of CcmE is known; the heme-binding site has been modeled on the surface of the protein in the vicinity of H130. We have determined the reduction potential of CcmE, which suggests that heme bound to CcmE is not as exposed to solvent as was initially thought. Alanine insertions in the vicinity of the heme-binding histidine (which we showed by NMR do not perturb the protein fold) strikingly abolish formation of both holo-CcmE and cytochrome c, whereas previously reported point mutations of residues adjacent to H130 gave only a partial attenuation. The heme iron coordinating residue Y134 proved to be strictly required for axial ligation of both ferrous and ferric heme. These results indicate the existence of a conformationally well-defined heme pocket that involves amino acids located in the proximity of H130. However, mutation of Y134 affected neither heme attachment to CcmE nor cytochrome c maturation, suggesting that heme binding and release from CcmE are hydrophobically driven and relatively indifferent to axial ligation.

Why isn't ‘standard’ heme good enough for c-type and d1-type cytochromes?

This perspective seeks to discuss why biology often modifies the fundamental iron-protoporphyrin IX moiety that is the very versatile cofactor of many heme proteins. A very common modification is the attachment of this cofactor via covalent bonds to two (or rarely one) sulfur atoms of cysteine residue side chains. This modification results in c-type cytochromes, which have diverse structures and functions. The covalent bonds are made in different ways depending on the cell type. There is little understanding of the reasons for this complexity in assembly routes but proposals for the rationale behind the covalent modification are presented. In contrast to the widespread c-type cytochromes, the d1 heme is restricted to a single enzyme, the cytochrome cd1 nitrite reductase that catalyses the one-electron reduction of nitrite to nitric oxide. This is an extensively derivatised heme; a comparison is drawn with another type of respiratory nitrite reductase in which the active site is a c-type heme, but the product ammonia.

c-Type Cytochrome Biogenesis Can Occur via a Natural Ccm System Lacking CcmH, CcmG, and the Heme-binding Histidine of CcmE

The Ccm cytochrome c maturation System I catalyzes covalent attachment of heme to apocytochromes c in many bacterial species and some mitochondria. A covalent, but transient, bond between heme and a conserved histidine in CcmE along with an interaction between CcmH and the apocytochrome have been previously indicated as core aspects of the Ccm system. Here, we show that in the Ccm system from Desulfovibrio desulfuricans, no CcmH is required, and the holo-CcmE covalent bond occurs via a cysteine residue. These observations call for reconsideration of the accepted models of System I-mediated c-type cytochrome biogenesis.