Interaction of Heme with Variants of the Heme Chaperone CcmE Carrying Active Site Mutations and a Cleavable N-terminal His Tag

Abstract

Cytochrome c maturation in the periplasms of many bacteria requires the heme chaperone CcmE, which binds heme covalently both in vivo and in vitro via a histidine residue before transferring the heme to apocytochromes c. To investigate the mechanism and specificity of heme attachment to CcmE, we have mutated the conserved histidine 130 of a soluble C-terminally His-tagged version of CcmE (CcmEsol-C-His6) from Escherichia coli to alanine or cysteine. Remarkably, covalent bond formation with heme occurs with the protein carrying the cysteine mutation, and the process occurs both in vivo and in vitro. The yield of holo-H130C CcmEsol-C-His6 produced in vivo is low compared with the wild type. In vitro heme attachment occurs only under reducing conditions. We demonstrate the involvement of one of the heme vinyl groups and a side chain at residue 130 in the bond formation by showing that in vitro attachment does not occur either with the heme analogue mesoheme or when alanine is present at residue 130. These results have implications for the mechanism of heme attachment to the histidine of CcmE. In vitro, CcmEsol lacking a His tag binds 8-anilino-1-naphthalenesulphonate and heme, the latter both noncovalently and via a covalent bond from the histidine side chain, similarly to the tagged proteins, thus countering a recent proposal that the His tag causes the heme binding. However, the His tag does appear to enhance the rate of in vitro covalent heme binding and to affect the heme ligation in the ferric b-type cytochrome form. Cytochrome c maturation in the periplasms of many bacteria requires the heme chaperone CcmE, which binds heme covalently both in vivo and in vitro via a histidine residue before transferring the heme to apocytochromes c. To investigate the mechanism and specificity of heme attachment to CcmE, we have mutated the conserved histidine 130 of a soluble C-terminally His-tagged version of CcmE (CcmEsol-C-His6) from Escherichia coli to alanine or cysteine. Remarkably, covalent bond formation with heme occurs with the protein carrying the cysteine mutation, and the process occurs both in vivo and in vitro. The yield of holo-H130C CcmEsol-C-His6 produced in vivo is low compared with the wild type. In vitro heme attachment occurs only under reducing conditions. We demonstrate the involvement of one of the heme vinyl groups and a side chain at residue 130 in the bond formation by showing that in vitro attachment does not occur either with the heme analogue mesoheme or when alanine is present at residue 130. These results have implications for the mechanism of heme attachment to the histidine of CcmE. In vitro, CcmEsol lacking a His tag binds 8-anilino-1-naphthalenesulphonate and heme, the latter both noncovalently and via a covalent bond from the histidine side chain, similarly to the tagged proteins, thus countering a recent proposal that the His tag causes the heme binding. However, the His tag does appear to enhance the rate of in vitro covalent heme binding and to affect the heme ligation in the ferric b-type cytochrome form. c-type cytochromes are important ubiquitous proteins that bind heme covalently via two thioether bonds between the cysteines in a conserved CXXCH motif in the protein and the vinyl groups of heme. In many Gram-negative bacteria, formation of these covalent bonds occurs in the periplasm in a multistep process involving the so-called Ccm (cytochrome cmaturation) proteins A–H, all of which are essential (1Thöny-Meyer L. Fischer F. Kunzler P. Ritz D. Hennecke H. J. Bacteriol. 1995; 177: 4321-4326Crossref PubMed Google Scholar, 2Thöny-Meyer L. Biochim. Biophys. Acta. 2000; 1459: 316-324Crossref PubMed Scopus (76) Google Scholar). The apocytochromes are synthesized in the cytoplasm and transported to the periplasm via the Sec pathway (3Thöny-Meyer L. Kunzler P. Eur. J. Biochem. 1997; 246: 794-799Crossref PubMed Scopus (66) Google Scholar). The mechanism by which heme, which is also synthesized cytoplasmically, is transported to the periplasm is as yet uncertain (4Goldman B.S. Beck D.L. Monika E.M. Kranz R.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5003-5008Crossref PubMed Scopus (93) Google Scholar, 5Schulz H. Fabianek R.A. Pellicioli E.C. Hennecke H. Thöny-Meyer L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6462-6467Crossref PubMed Scopus (103) Google Scholar, 6Cook G.M. Poole R.K. Microbiology. 2000; 146: 527-536Crossref PubMed Scopus (35) Google Scholar). However, it has been shown that the membrane-bound protein CcmC is required for presentation of heme to the membrane-anchored periplasmic protein CcmE (7Ren Q. Thöny-Meyer L. J. Biol. Chem. 2001; 276: 32591-32596Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). CcmE has been identified as the heme chaperone and binds heme covalently via a conserved histidine residue (His130 in Escherichia coli) before transferring the heme to apocytochromes (8Schulz H. Hennecke H. Thöny-Meyer L. Science. 1998; 281: 1197-1200Crossref PubMed Scopus (156) Google Scholar). Recently, structures of the CcmE apoproteins from two different bacterial species have been reported (9Arnesano F. Banci L. Barker P.D. Bertini I. Rosato A. Su X.C. Viezzoli M.S. Biochemistry. 2002; 41: 13587-13594Crossref PubMed Scopus (40) Google Scholar, 10Enggist E. Thöny-Meyer L. Guntert P. Pervushin K. Structure. 2002; 10: 1551-1557Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), which shows that the heme-binding histidine is exposed on the protein surface but provides no clue as to the unusual properties of this residue. CcmE has been shown to be part of a complex with CcmF in vivo (11Ren Q. Ahuja U. Thöny-Meyer L. J. Biol. Chem. 2002; 277: 7657-7663Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), which together with CcmH forms what is proposed to be a bacterial heme lyase. CcmH interacts with CcmG, which is part of the system that provides reductant to the periplasm, specifically for reducing the cysteines in the CXXCH motif of the apocytochromes (12Reid E. Cole J. Eaves D.J. Biochem. J. 2001; 355: 51-58Crossref PubMed Scopus (60) Google Scholar). The nature of the novel bond between the histidine of CcmE and the heme remains unknown, although it has been shown that one of the vinyl groups of heme is involved (13Daltrop O. Stevens J.M. Higham C.W. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9703-9708Crossref PubMed Scopus (59) Google Scholar). CcmE has been identified in a number of bacteria as well as in Arabidopsis and presumably other plant mitochondria (14Spielewoy N. Schulz H. Grienenberger J.M. Thöny-Meyer L. Bonnard G. J. Biol. Chem. 2001; 276: 5491-5497Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In vitro studies of a soluble version of CcmE (CcmE′) have shown that the covalent attachment occurs under reducing conditions and that release of the heme to apocytochromes will only occur if heme is in its ferrous state (13Daltrop O. Stevens J.M. Higham C.W. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9703-9708Crossref PubMed Scopus (59) Google Scholar). To probe further the covalent attachment of heme in vitro or in vivo, we have altered by site-directed mutagenesis the histidine 130 that has been shown previously to be the site of heme attachment in vivo (8Schulz H. Hennecke H. Thöny-Meyer L. Science. 1998; 281: 1197-1200Crossref PubMed Scopus (156) Google Scholar). Replacement of this histidine by cysteine or alanine was expected to generate proteins with either a potentially reactive or an unreactive side chain at position 130. The CcmE′ protein used in previous in vitro studies (13Daltrop O. Stevens J.M. Higham C.W. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9703-9708Crossref PubMed Scopus (59) Google Scholar) had a His tag at the C terminus. Very recently it has been suggested that such a His tag may significantly influence the binding of heme to CcmE′ in vitro to the extent that protein from Shewanella putrefaciens lacking a tag is reported to be unable to bind heme in vitro (9Arnesano F. Banci L. Barker P.D. Bertini I. Rosato A. Su X.C. Viezzoli M.S. Biochemistry. 2002; 41: 13587-13594Crossref PubMed Scopus (40) Google Scholar). Any possibility that a His tag might heme to a covalent bond to other residue 130 also be by the present mutagenesis of a His tag the binding of heme has been by the tagged and proteins with histidine at 130. the implications of studies are and coli was used for and was used for protein The for CcmEsol-C-His6 (CcmE′) was as (13Daltrop O. Stevens J.M. Higham C.W. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9703-9708Crossref PubMed Scopus (59) Google Scholar) and the soluble periplasmic of the protein from with a for periplasmic of the in this the site-directed mutagenesis the and for the and and for the in The for the of the His-tagged version of the protein was by of the from the was by L. the and which and The the these the in this the The the and the and as well as of the His and to that on the protein the and of the to that only the had been used for mutagenesis and to in a and of the of the periplasmic proteins, the and with the E. Schulz H. Kunzler P. Thöny-Meyer L. Biochem. Biophys. 1998; PubMed Scopus Google Scholar), which the Ccm proteins of these proteins is essential for of (13Daltrop O. Stevens J.M. Higham C.W. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9703-9708Crossref PubMed Scopus (59) Google Scholar). of the apoproteins in the periplasm in the of E. coli as and the proteins with as (13Daltrop O. Stevens J.M. Higham C.W. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9703-9708Crossref PubMed Scopus (59) Google Scholar). The proteins the and in the that the on for to the and the of and was a to the protein was by the to the a of a to that the His had been or PubMed Scopus Google Scholar) was used to the proteins, and for covalently heme was to the of Biochim. Biophys. Acta. Scopus Google Scholar), to noncovalently heme. on a between and in either or to the of R.G. Scopus Google Scholar) protein in and used was a protein in at a of the at a rate of or mesoheme in was to in of the proteins with heme was by of protein with of heme at used to heme from the protein was used to heme. The a and the for heme and as (13Daltrop O. Stevens J.M. Higham C.W. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9703-9708Crossref PubMed Scopus (59) Google Scholar). and to protein and heme from in which at The by with The in the of CcmEsol a His it was suggested that the His tag on a soluble CcmE in previous (13Daltrop O. Stevens J.M. Higham C.W. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9703-9708Crossref PubMed Scopus (59) Google Scholar), causes binding of heme and that the heme binding is (9Arnesano F. Banci L. Barker P.D. Bertini I. Rosato A. Su X.C. Viezzoli M.S. Biochemistry. 2002; 41: 13587-13594Crossref PubMed Scopus (40) Google Scholar). To this we have a of CcmE that be with and the His a site between the His tag and the soluble CcmE The protein with the His tag was by and had a of the protein as a and of the protein had a of The in the of the protein also be by The of the His tag was also by the of ferric heme to lacking a His by by no in the of a low compared with the His protein was a of these as we have for the protein with the His tag (13Daltrop O. Stevens J.M. Higham C.W. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9703-9708Crossref PubMed Scopus (59) Google Scholar). in the of ferric heme and a species was that is to that the of ferric heme to c Biochemistry. PubMed Scopus Google Scholar), showing of a the of the complex a His tag was different from for the proteins with the His tag at either the C or both of which have a at These that the His tag affect the of the ferric complex to a However, in of the of on the of heme, this is not K. Biochem. Biophys. PubMed Scopus Google Scholar). with the of the complex not altered by the of the His tag for forms of of vivo vivo vitro O. Stevens J.M. Higham C.W. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9703-9708Crossref PubMed Scopus (59) Google in a In further of the protein the His tag that it has a to the protein with the tag and the at not it was to from CcmEsol by the of heme, as we have shown for the tagged protein (13Daltrop O. Stevens J.M. Higham C.W. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9703-9708Crossref PubMed Scopus (59) Google Scholar). The of ferrous heme with to a protein with covalently heme that had from CcmEsol-C-His6 (13Daltrop O. Stevens J.M. Higham C.W. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9703-9708Crossref PubMed Scopus (59) Google Scholar) and not of the His heme was shown to be covalently to CcmEsol as shown in of CcmEsol with heme in the of the His tag to the but the of was by at to of the protein with His at either of the protein from two on the of the histidine and To heme ligation by the protein by of the His-tagged these mutated to The proteins with and the His tag as by not and had the expected as by the of ferric heme to the His the compared with the of ferric heme The complex to be in the ferric state and to low in the state the of the of an and from the had no on with heme. of the and the and of CcmEsol-C-His6 well as in the E. coli The of the proteins by which also that the periplasmic had been The for the and for the proteins also in E. coli with of the other Ccm proteins, and the covalent attachment of heme to the proteins was by by heme shows that the proteins to and for and the not appear to bind heme covalently in vivo as by heme of the which is in with previous (8Schulz H. Hennecke H. Thöny-Meyer L. Science. 1998; 281: 1197-1200Crossref PubMed Scopus (156) Google Scholar). The was to heme when with the other Ccm proteins that the protein was to extent by the Ccm system for heme and To the from covalently heme, the had to be such that a was was that the protein bonds in vitro when as shown in was by with which and of for and of the protein at a of to a covalently which was also identified by In is produced at a low of the protein is in the covalently heme, as from the of the to the at In to the and the of ferric heme to the both and b-type cytochrome as by The the and to be in this for The for the of both proteins with ferrous heme and mesoheme as well as the are shown in is a heme analogue that has in the of the vinyl The of the complex of with ferric heme shows the of a b-type with an at with and of the it was not to an of the b-type complex of heme from the protein was was also with the complex (13Daltrop O. Stevens J.M. Higham C.W. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9703-9708Crossref PubMed Scopus (59) Google Scholar). The of the b-type of both with heme have at which is of vinyl groups and shows that covalent attachment does not occur under these conditions. The of the proteins for ferric heme by as for the protein (13Daltrop O. Stevens J.M. Higham C.W. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9703-9708Crossref PubMed Scopus (59) Google Scholar). The of the binding these are to be the The of the was to be which shows that it has a for heme the protein (13Daltrop O. Stevens J.M. Higham C.W. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9703-9708Crossref PubMed Scopus (59) Google is not the of the histidine side chain is to have the of the heme-binding site in the The of the was to be also the wild type. proteins, have a for heme. be that the of heme binding for this protein that a occurs in the heme binding E. Thöny-Meyer L. Guntert P. Pervushin K. Structure. 2002; 10: 1551-1557Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). of to by of the His-tagged b-type with a of protein heme, the a cytochrome to a covalent bond between heme and The from to and the and also with the the of produced in this was to the of the in protein The of the in vitro produced of the protein a which is with the of a vinyl as has been for cysteine of c-type cytochromes Ferguson S.J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). the in vivo produced of this had in the and that as a of the of histidine 130 with a cysteine heme attachment is not and that the formation of side The state of heme and the cysteine in vivo might not be as the periplasmic attachment of heme to the protein compared with the reducing conditions of the in vitro To that the of in vivo and in vitro covalent bond formation between heme and the by heme was shows the of the b-type complex of heme and with The that the protein for covalently heme as does the in vivo produced holo-H130C in that in vitro and in vivo covalent attachment of heme to the protein had The for this are shown in and These are with ferric heme by with with ferric heme, and the of ferric mesoheme to protein by with and for These that covalent attachment of heme to the CcmEsol protein not occur under these no heme be for The results that one of the vinyl groups of heme is involved in formation of the covalent attachment was not with The results also that the bond forms with the cysteine residue of the it is not with the alanine it is shown that covalent heme binding only occur with a reactive side chain of 130. was for the protein (13Daltrop O. Stevens J.M. Higham C.W. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9703-9708Crossref PubMed Scopus (59) Google Scholar), these results also the for of the heme in the covalent bond no heme was with the To the of the His tag on the covalent heme attachment to the and the of the to bind heme covalently in vitro, proteins with these site also with a His The proteins shown to be by as shown in and for and the of heme under conditions by of the His tag by the was to covalently heme as by by heme and The well with the in vivo produced The to for covalently heme and not of a protein in the not of holo-H130C CcmEsol produced in vitro, showing the and the the of protein in of the His of heme to bind covalently to the of CcmEsol that the covalent binding of heme with the and proteins the of the or groups of histidine or cysteine at this residue is to that the binding of ferric heme to of the proteins, and the heme for the covalent bond formation with the two is an important in the that the His tag might to the binding site for heme. Recently, (9Arnesano F. Banci L. Barker P.D. Bertini I. Rosato A. Su X.C. Viezzoli M.S. Biochemistry. 2002; 41: 13587-13594Crossref PubMed Scopus (40) Google Scholar) have that CcmE from S. putrefaciens does not bind heme and that a His tag be for a heme-binding site this and other In the present we that the CcmEsol protein from E. coli a heme-binding site at which a covalent bond forms between His 130 and a vinyl in vitro under the conditions as before (13Daltrop O. Stevens J.M. Higham C.W. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9703-9708Crossref PubMed Scopus (59) Google Scholar). In this results are at with of (9Arnesano F. Banci L. Barker P.D. Bertini I. Rosato A. Su X.C. Viezzoli M.S. Biochemistry. 2002; 41: 13587-13594Crossref PubMed Scopus (40) Google Scholar). However, we have that the of the His tag the of the ferric with the heme a histidine by one of the of the In this the His tag to the in vitro covalent of heme His tag at either the or at the C (13Daltrop O. Stevens J.M. Higham C.W. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9703-9708Crossref PubMed Scopus (59) Google Scholar) to similarly in this However, in the present we that the heme is covalently to CcmEsol the of ferric heme to CcmEsol-C-His6 by under conditions and which has had the His tag by also bind heme both covalently and we that the of a His tag at either of the protein does not the nature of the covalent bond Any of the of heme with CcmEsol by the His tag may be a of the on the CcmC protein that is to in the binding of heme to CcmE in vivo H. Fabianek R.A. Pellicioli E.C. Hennecke H. Thöny-Meyer L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6462-6467Crossref PubMed Scopus (103) Google Scholar). is to the with the results with the protein from Shewanella that that CcmE bind heme, a that is to be by of the of the which does not have a for binding heme (9Arnesano F. Banci L. Barker P.D. Bertini I. Rosato A. Su X.C. Viezzoli M.S. Biochemistry. 2002; 41: 13587-13594Crossref PubMed Scopus (40) Google Scholar). the other E. Thöny-Meyer L. Guntert P. Pervushin K. Structure. 2002; 10: 1551-1557Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), the for the from E. coli as (9Arnesano F. Banci L. Barker P.D. Bertini I. Rosato A. Su X.C. Viezzoli M.S. Biochemistry. 2002; 41: 13587-13594Crossref PubMed Scopus (40) Google Scholar), have a heme-binding site a on the surface of the We that this provides the previously site (13Daltrop O. Stevens J.M. Higham C.W. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9703-9708Crossref PubMed Scopus (59) Google Scholar), which the present is also present in CcmEsol that a His from the at this site is not as as be in proteins, with binding in the exposed heme-binding site by E. Thöny-Meyer L. Guntert P. Pervushin K. Structure. 2002; 10: 1551-1557Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). previous that heme from CcmEsol this it be if the of a His tag the binding of that this probe has a for and that histidine is a However, it be that a His tag the C of the The of the His tag on the of the covalent bond formation between heme and vitro that either the and of the complex by the His tag is or that the His tag may an on the bond to previous in vitro studies have shown that covalent bond formation occur between the histidine of the heme chaperone CcmEsol-C-His6 and ferrous heme (13Daltrop O. Stevens J.M. Higham C.W. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9703-9708Crossref PubMed Scopus (59) Google Scholar). the of ferric heme, the binds heme noncovalently with a and the covalent bond forms only when the heme is To investigate this process we have studies on this protein with the heme-binding histidine mutated to a cysteine. we that the covalent bond both in vitro and in vivo, although in the latter to a extent compared with the The unusual between the heme binding process with the wild and the to of the present an in vivo was in with showing that a His-tagged of CcmE similarly by bind heme covalently on this to a low E. Schulz H. Thöny-Meyer L. J. Bacteriol. PubMed Scopus Google Scholar). for the of Ccm results also the of the protein which has been shown to bind heme and to present CcmE with heme in the periplasm H. Fabianek R.A. Pellicioli E.C. Hennecke H. Thöny-Meyer L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6462-6467Crossref PubMed Scopus (103) Google Scholar, Q. Thöny-Meyer L. J. Biol. Chem. 2001; 276: 32591-32596Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The that the covalently of the is produced in vivo in the of the other Ccm proteins to a extent the in to the in vitro, that CcmC is not in the attachment of the heme to the cysteine compared with the attachment to may that specifically requires the heme-binding histidine residue on the to the heme the in the of the heme chaperone might the vinyl reactive to histidine cysteine The that the covalent via a between the cysteines at position 130 that residue 130 is in CcmE, which is in with the for this protein E. Thöny-Meyer L. Guntert P. Pervushin K. Structure. 2002; 10: 1551-1557Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). the heme is to the histidine residue in this position of the it is expected that the heme is also which the c to the heme from the heme by a from either the Ccm proteins or the to a also enhance the heme ligation by the c a attachment of the heme to cysteine at residue both in vivo and in vitro, provides heme attachment to CcmE. The in vitro to the formation from and heme of a c-type or its carrying one cysteine in the heme-binding motif that was recently reported Ferguson S.J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The reported that CcmEsol is to the c-type cytochromes with a thioether this we have been unable to the for either the or In vitro formation of thioether bonds of a c-type cytochrome presumably requires of the of the vinyl groups of heme and formation of the thioether bond between the cysteine and the of the vinyl groups of heme O. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar). mechanism for this process is to of the of the cysteine on the thioether bond formation to it was shown that ferrous heme is required O. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar). In we have shown that the of either the histidine (13Daltrop O. Stevens J.M. Higham C.W. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9703-9708Crossref PubMed Scopus (59) Google Scholar) or the cysteine residue to the heme requires ferrous heme. the of the in vitro heme binding of either histidine or cysteine of it is that occur by a the mechanism of attachment is suggested to be to the thioether bond formation in c-type The histidine residue of CcmE the of one of the vinyl a covalent bond of this nature has been reported for an unusual of which is in vitro under conditions J. Chem. 2002; PubMed Scopus (55) Google Scholar). the heme attachment to CcmE occurs with a vinyl and heme bind to CcmE in one to the of the heme of the heme to c be The proposed process of heme binding and heme release from CcmE is in However, a mechanism as proposed for in vitro thioether bond formation between heme and cysteine S. S. J. Chem. I. Google Scholar) be on the of the The release of the heme from the heme chaperone has with a a as a of a Chem. Scopus Google Scholar). studies will be required to further the involved heme with to which vinyl with CcmE the nature of the will also be to the proposed with the cysteine of the c cytochrome c maturation L. Ferguson S.J. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar) will affect the heme However, this to the of the of a in cytochrome c We for on the mechanism and Barker for and and for and