Oliver Daltrop

In vitro formation of a c-type cytochrome

C-type cytochromes are essential for almost all organisms; they are characterized by the covalent attachment of heme to protein through two thioether bonds to a Cys-Xaa-Xaa-Cys-His peptide motif. Here we show, contrary to opinion of 30 years standing, that a c-type cytochrome can form from heme and apoprotein in vitro under mild conditions and in the absence of any biosynthesis apparatus. This reaction occurs provided formation of a disulfide bond within the Cys-Xaa-Xaa-Cys-His motif is avoided. There are important implications for understanding in vivo cytochrome c assembly.

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 Maturation THE IN VITRO REACTIONS OF HORSE HEART APOCYTOCHROMEc AND PARACOCCUS DENITRIFICANS APOCYTOCHROMEc550 WITH HEME

C-type cytochromes are characterized by having the heme moiety covalently attached via thioether bonds between the heme vinyl groups and the thiols of conserved cysteine residues of the polypeptide chain. Previously, we have shown the in vitro formation of Hydrogenobacter thermophilus cytochrome c 552 (Daltrop, O., Allen, J. W. A., Willis, A. C., and Ferguson, S. J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7872–7876). In this work we report that thioether bonds can form spontaneously in vitro between heme and the apocytochromes c from horse heart and Paracoccus denitrificans via b-type cytochrome intermediates. Both apocytochromes, but not the holo forms, bind 8-anilino-1-naphthalenesulfonate, indicating that the apoproteins each have an affinity for a hydrophobic ligand. Furthermore, for both apocytochromes c an intramolecular disulfide can form between the cysteines of the CXXCH motif that is characteristic of c-type cytochromes. In vitroreaction of these apocytochromes c with heme to yield holocytochromes c, and the tendency to form a disulfide, have implications for the different systems responsible for cytochrome c maturation in vivo in various organisms.

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.

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.

Dynamic Ligation Properties of the Escherichia coli Heme Chaperone CcmE to Non-covalently Bound Heme

The cytochrome c maturation protein CcmE is an essential membrane-anchored heme chaperone involved in the post-translational covalent attachment of heme to c-type cytochromes in Gram-negative bacteria such as Escherichia coli. Previous in vitro studies have shown that CcmE can bind heme both covalently (via a histidine residue) and non-covalently. In this work we present results on the latter form of heme binding to a soluble form of CcmE. Examination of a number of site-directed mutants of E. coli CcmE by resonance Raman spectroscopy has identified ligands of the heme iron and provided insight into the initial steps of heme binding by CcmE before it binds the heme covalently. The heme binding histidine (His-130) appears to ligate the heme iron in the ferric oxidation state, but two other residues ligate the iron in the ferrous form, thereby freeing His-130 to undergo covalent attachment to a heme vinyl group. It appears that the heme ligation in the non-covalent form is different from that in the holo-form, suggesting that a change in ligation could act as a trigger for the formation of the covalent bond and showing the dynamic and oxidation state-sensitive ligation properties of CcmE.

Tuning the formation of a covalent haem-protein link by selection of reductive or oxidative conditions as exemplified by ascorbate peroxidase.

Previous work [Metcalfe, Ott, Patel, Singh, Mistry, Goff and Raven (2004) J. Am. Chem. Soc. 126, 16242-16248] has shown that the introduction of a methionine residue (S160M variant) close to the 2-vinyl group of the haem in ascorbate peroxidase leads to the formation of a covalent haem-methionine linkage under oxidative conditions (i.e. on reaction with H2O2). In the present study, spectroscopic, HPLC and mass spectrometric evidence is presented to show that covalent attachment of the haem to an engineered cysteine residue can also occur in the S160C variant, but, in this case, under reducing conditions analogous to those used in the formation of covalent links in cytochrome c. The data add an extra dimension to our understanding of haem to protein covalent bond formation because they show that different types of covalent attachment (one requiring an oxidative mechanism, the other a reductive pathway) are both accessible within same protein architecture.

Semisynthetic Nanoreactor for Reversible Single-Molecule Covalent Chemistry

Protein engineering has been used to remodel pores for applications in biotechnology. For example, the heptameric α-hemolysin pore (αHL) has been engineered to form a nanoreactor to study covalent chemistry at the single-molecule level. Previous work has been confined largely to the chemistry of cysteine side chains or, in one instance, to an irreversible reaction of an unnatural amino acid side chain bearing a terminal alkyne. Here, we present four different αHL pores obtained by coupling either two or three fragments by native chemical ligation (NCL). The synthetic αHL monomers were folded and incorporated into heptameric pores. The functionality of the pores was validated by hemolysis assays and by single-channel current recording. By using NCL to introduce a ketone amino acid, the nanoreactor approach was extended to an investigation of reversible covalent chemistry on an unnatural side chain at the single-molecule level.