Cytochrome c Maturation
2003; Elsevier BV; Volume: 278; Issue: 7 Linguagem: Inglês
10.1074/jbc.m211124200
ISSN1083-351X
AutoresOliver Daltrop, Stuart J. Ferguson,
Tópico(s)Protist diversity and phylogeny
ResumoC-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. 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. C-type cytochromes are found in almost all organisms, being mainly involved in electron transport (1Scott R.A. Mauk A.G. Cytochrome C: A Multidisciplinary Approach. University Science Books, Mill Valley, CA1995Google Scholar, 2Ferguson S.J. Biochem. Soc. Trans. 2001; 29: 629-640Crossref PubMed Google Scholar). They contain a characteristic CXXCH motif, whereby the conserved cysteine residues are involved in forming a covalent thioether bond between the thiol functionalities and the vinyl groups of the prosthetic heme moiety (3Barker P.D. Ferguson S.J. Structure Fold. Des. 1999; 7: R281-R290Abstract Full Text Full Text PDF Scopus (141) Google Scholar). Remarkably, three different systems have evolved to facilitate cytochrome c formation in vivo (4Kranz R. Lill R. Goldman B. Bonnard G. Merchant S. Mol. Microbiol. 1998; 29: 383-396Crossref PubMed Scopus (237) Google Scholar, 5Page M.D. Sambongi Y. Ferguson S.J. Trends Biochem. Sci. 1998; 23: 103-108Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). In some eukaryotes, one enzyme, designated heme lyase, catalyzes the attachment of the heme moiety to the apocytochromec (6Steiner H. Kispal G. Zollner A. Haid A. Neupert W. Lill R. J. Biol. Chem. 1996; 271: 32605-32611Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). These enzymes have a high specificity and do not act on bacterial apocytochromes c (7Sanders C. Lill H. Biochim. Biophys. Acta. 2000; 1459: 131-138Crossref PubMed Scopus (47) Google Scholar). Mammalian organisms make use of this system as exemplified by horse (Equus caballus) heart mitochondria.Many Gram-negative bacteria use a system that involves more than ten gene products to ensure correct cytochrome c maturation (ccm) 1The abbreviations used are: ccm, cytochromec maturation; DTT, dithiothreitol; AMS, 4-acetamido-4′-maleimidyl-stilbene-2,2′-disulfonate; ANS, 8-anilino-1-naphthalenesulfonate; CD, circular dichroism; ES-MS, electrospray mass spectrometry 1The abbreviations used are: ccm, cytochromec maturation; DTT, dithiothreitol; AMS, 4-acetamido-4′-maleimidyl-stilbene-2,2′-disulfonate; ANS, 8-anilino-1-naphthalenesulfonate; CD, circular dichroism; ES-MS, electrospray mass spectrometry in the periplasm (5Page M.D. Sambongi Y. Ferguson S.J. Trends Biochem. Sci. 1998; 23: 103-108Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar,8Thöny-Meyer L. Microbiol. Mol. Biol. Rev. 1997; 61: 337-376Crossref PubMed Scopus (314) Google Scholar, 9Thöny-Meyer L. Biochem. Soc. Trans. 2002; 30: 633-638Crossref PubMed Scopus (102) Google Scholar). An example of a cytochrome c that is matured in vivo using this biosynthesis apparatus is cytochromec 550 from P. denitrificans (10Sambongi Y. Ferguson S.J. FEBS Lett. 1994; 340: 65-70Crossref PubMed Scopus (66) Google Scholar).Following our report of the in vitro formation of holocytochrome c 552 from H. thermophilus (11Daltrop O. Allen J.W. Willis A.C. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7872-7876Crossref PubMed Scopus (87) Google Scholar), an important point to be established was whether the spontaneous attachment of heme to apocytochromec is a reaction applicable to other cytochromesc. Because holocytochrome c 552 fromH. thermophilus forms exceptionally in the cytoplasm ofEscherichia coli (12Sinha N. Ferguson S.J. FEMS Microbiol. Lett. 1998; 161: 1-6Crossref PubMed Google Scholar), its spontaneous in vitroformation might not necessarily be anticipated to apply generally to all c-type cytochromes. The previously reported thioether bond formation in holocytochrome c 552 from ofH. thermophilus involved a b-type cytochrome intermediate (11Daltrop O. Allen J.W. Willis A.C. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7872-7876Crossref PubMed Scopus (87) Google Scholar). Prompted by the previously reported similar ability of the horse heart apocytochrome to bind heme to yield a species characteristic of a b-type cytochrome (13Dumont M.E. Corin A.F. Campbell G.A. Biochemistry. 1994; 33: 7368-7378Crossref PubMed Scopus (67) Google Scholar), the reaction of horse heart apocytochrome c with heme has been reinvestigated.The formation of c-type cytochromes is especially interesting in light of the different biosynthetic pathways, which are reviewed extensively elsewhere (4Kranz R. Lill R. Goldman B. Bonnard G. Merchant S. Mol. Microbiol. 1998; 29: 383-396Crossref PubMed Scopus (237) Google Scholar, 5Page M.D. Sambongi Y. Ferguson S.J. Trends Biochem. Sci. 1998; 23: 103-108Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 9Thöny-Meyer L. Biochem. Soc. Trans. 2002; 30: 633-638Crossref PubMed Scopus (102) Google Scholar), by which heme is attached to the apoproteins in vivo. Therefore, a second candidate for studying the reaction of heme with apocytochrome was P. denitrificans cytochrome c 550. The two cytochromes used in this study are matured by two different cytochromec maturation systems in vivo. A comparison of the reactions with respect to the attachment mechanism is anticipated to have important implications for the molecular basis of heme attachment in different organisms in vivo.DISCUSSIONIn this work we show the data obtained from the production of apocytochromes c from a bacterial and a mammalian organism and characterize the reaction of the cysteine thiols with the vinyl moieties of heme. Both proteins have very similar properties with regard to their apoforms and their reaction with heme. We show that the cysteines of the conserved CXXCH motif can form an internal disulfide. This observation raises the question if this oxidation occurs in vivo for the respective proteins in either organism. For the bacterial system it has been suggested that the disulfide is an important intermediate during cytochrome cmaturation due to the presence of the disulfide bond forming (Dsb) proteins in the periplasm, the location of cytochrome cmaturation (2Ferguson S.J. Biochem. Soc. Trans. 2001; 29: 629-640Crossref PubMed Google Scholar). Furthermore, it has been proposed that proteins (CcmG/H and DsbD) required for cytochrome c biogenesis are involved in the reduction of the internal disulfide bond in the apocytochrome (9Thöny-Meyer L. Biochem. Soc. Trans. 2002; 30: 633-638Crossref PubMed Scopus (102) Google Scholar). Why this in situ reduction of an apocytochrome has evolved is unclear, but an internal protection mechanism against metallation of the coordination site of the reduced apocytochrome might offer an explanation.Heme attachment to mitochondrial apocytochrome c occurs in the intermembrane space (29Diekert K. Kispal G. Guiard B. Lill R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11752-11757Crossref PubMed Scopus (106) Google Scholar). The in vivo oxidation state of the cysteines in mitochondrial apocytochromes c has not been investigated to the best of our knowledge. Whether, as we show here for the first time in vitro, a disulfide bond occurs during thein vivo protein maturation process, will depend on both the reduction potential of its locus and kinetic factors. If formation of a disulfide must be avoided, a thioredoxin-like protein could be required in the intermembrane space (30Laloi C. Rayapuram N. Chartier Y. Grienenberger J.M. Bonnard G. Meyer Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14144-14149Crossref PubMed Scopus (222) Google Scholar). A thioredoxin (Trx3) and its reductase (Trr2) are known to be present in yeast mitochondria, but submitochondrial localization to the matrix is suspected rather than to the intermembrane space (31Pedrajas J.R. Kosmidou E. Miranda-Vizuete A. Gustafsson J.A. Wright A.P. Spyrou G. J. Biol. Chem. 1999; 274: 6366-6373Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). These proteins can in any case be deduced to be dispensable for cytochrome c biogenesis in yeast, as mutants carrying specific disruptions in the corresponding genes were able to grow normally under aerobic, respiratory conditions, which require participation of cytochrome c in the mitochondrial respiratory chain. However, in the case of Arabidopsis thaliana, it has been recognized, on the basis of the genome sequence, that there may be a yet to be characterized thioredoxin that is targeted to the intermembrane space (30Laloi C. Rayapuram N. Chartier Y. Grienenberger J.M. Bonnard G. Meyer Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14144-14149Crossref PubMed Scopus (222) Google Scholar). Alternatively, the formation of holocytochrome c may happen sufficiently fast that the two thiols of apocytochrome c cannot form a disulfide during its lifetime in the intermembrane space following delivery from the reducing environment of the cytoplasm. Such a kinetic constraint would suffice if the rate of disulfide bond formation in the intermembrane space were to occur on a similar timescale to thatin vitro (hours as observed in the present work). The presence of the CcmABCEF components of one cytochrome cbiogenesis pathway, but not of the thioredoxin-like CcmG component, in plant mitochondria might support the latter view.The binding of heme to the apoform of mitochondrial cytochromec clearly involves some ordering of the polypeptide chain. This is evident from several biophysical studies (13Dumont M.E. Corin A.F. Campbell G.A. Biochemistry. 1994; 33: 7368-7378Crossref PubMed Scopus (67) Google Scholar, 28Goldberg M.E. Schaeffer F. Guillou Y. Djavadi-Ohaniance L. J. Biol. Chem. 1999; 274: 16052-16061Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). A non-covalent complex between heme and apoprotein of H. thermophilus cytochrome c 552 generates a protein structure that is very similar to that of the native protein with covalently bound heme (32Tomlinson E.J. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5156-5160Crossref PubMed Scopus (65) Google Scholar). Although it appears that the structure of the non-covalent complex between heme and mitochondrial apocytochromec does not fully resemble the native, holo structure (13Dumont M.E. Corin A.F. Campbell G.A. Biochemistry. 1994; 33: 7368-7378Crossref PubMed Scopus (67) Google Scholar,28Goldberg M.E. Schaeffer F. Guillou Y. Djavadi-Ohaniance L. J. Biol. Chem. 1999; 274: 16052-16061Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar), studies with two monoclonal antibodies that scarcely recognize the apoprotein, but bind with comparable affinities to both the non-covalent heme-apoprotein complex and the holoprotein, indicate that the complex must be similar to the holoprotein in respect of quite specific structural features at the epitope regions (28Goldberg M.E. Schaeffer F. Guillou Y. Djavadi-Ohaniance L. J. Biol. Chem. 1999; 274: 16052-16061Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Such similarities are evidently sufficient to permit the vinyl groups of heme to be oriented appropriately, in at least a fraction of the molecules, for reaction with the thiol groups, which have previously been shown to exhibit nucleophilic reactivity (27Kang X. Carey J. Biochemistry. 1999; 38: 15944-15951Crossref PubMed Scopus (28) Google Scholar). The effect of covalently bound hydrophobic moieties on the folding of cytochromec has also been discussed (27Kang X. Carey J. Biochemistry. 1999; 38: 15944-15951Crossref PubMed Scopus (28) Google Scholar).The affinity of the apoproteins for hydrophobic ligands and the ability of both apoproteins to ligate heme in a b-type cytochrome complex has important implications for the catalytic strategy of the enzymes involved during cytochrome c maturation. It is unclear from previous studies how the heme lyase functions in mitochondria, except for the proposal that it binds heme via a conserved CPX motif (6Steiner H. Kispal G. Zollner A. Haid A. Neupert W. Lill R. J. Biol. Chem. 1996; 271: 32605-32611Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). For the bacterial Ccm system, two proteins have been proposed to function as a heme lyase (33Ren Q. Ahuja U. Thöny-Meyer L. J. Biol. Chem. 2002; 277: 7657-7663Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), but the heme attachment is more complex due to the presence of a heme chaperone, CcmE (34Schulz H. Hennecke H. Thöny-Meyer L. Science. 1998; 281: 1197-1200Crossref PubMed Scopus (153) Google Scholar). However, in view of our data showing that theb-type cytochrome can spontaneously react to give thioether bond formation yielding holocytochrome c, two key conditions have to be achieved by the catalytic systems. Firstly, the heme group has to be kept reduced to give optimal reactivity for cysteine-thiol and heme-vinyl group reaction partners. This is in agreement with previous studies (35Nicholson D.W. Neupert W. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4340-4344Crossref PubMed Scopus (85) Google Scholar, 36Tong J. Margoliash E. J. Biol. Chem. 1998; 273: 25695-25702Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). And secondly, the orientation of the heme relative to its rotation around the α,γ meso-axis has to be correct to yield the required stereochemistry at the prochiral α-carbon of the vinyl substituents of the heme moiety (3Barker P.D. Ferguson S.J. Structure Fold. Des. 1999; 7: R281-R290Abstract Full Text Full Text PDF Scopus (141) Google Scholar). McRee et al.(37McRee D.E. Williams P.A. Sridhar V. Pastuszyn A. Bren K.L. Patel K.M. Chen Y. Todaro T.R. Sanders D. Luna E. Fee J.A. J. Biol. Chem. 2001; 276: 6537-6544Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) showed that a recombinant cytochrome c ofThermus thermophilus could be improperly matured in vivo, but without any catalytic assistance in the cytoplasm ofE. coli, because of both heme inversion and an intermolecular disulfide bond between cysteine residues, which are usually involved in heme binding (37McRee D.E. Williams P.A. Sridhar V. Pastuszyn A. Bren K.L. Patel K.M. Chen Y. Todaro T.R. Sanders D. Luna E. Fee J.A. J. Biol. Chem. 2001; 276: 6537-6544Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The latter behavior was clearly reflected in the visible absorption spectrum, which was different from the reported, properly matured holocytochrome c, and the pyridine hemochrome spectrum, which was not indicative of two thioether bonds to the heme-vinyl groups (37McRee D.E. Williams P.A. Sridhar V. Pastuszyn A. Bren K.L. Patel K.M. Chen Y. Todaro T.R. Sanders D. Luna E. Fee J.A. J. Biol. Chem. 2001; 276: 6537-6544Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). For mitochondrial cytochromec, two thioether bonds have formed in the present work. In the case of in vitro reaction of heme with P. denitrificans apocytochrome c 550 it is not evident if the reaction yielded a mixture of only b- andc-type cytochrome complexes or also contained species with only one thioether bond. Due to the presence of DTT during the course of the reaction, no reaction product containing an intermolecular disulfide bond is obtained in vitro.In the in vitro reaction, the oxidation state of the heme can be controlled, but the heme delivery is nonspecific with respect to the stereochemical aspects of the heme orientation. The stereochemistry for the in vitro produced cytochromesc is not determined and will be focus of future work. However, the fact that a fraction of the mitochondrialb-type cytochrome intermediate remained unreactive might hint that incorrect orientation of heme does not lead to thioether bond formation, suggesting the in vitro cytochromec formation to be stereoselective. In the case of thein vitro formation of H. thermophilus c 552 the reaction is stereoselective (11Daltrop O. Allen J.W. Willis A.C. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7872-7876Crossref PubMed Scopus (87) Google Scholar).An interesting point arises from the observation that neither P. denitrificans c 550 (10Sambongi Y. Ferguson S.J. FEBS Lett. 1994; 340: 65-70Crossref PubMed Scopus (66) Google Scholar) nor mitochondrial cytochromec, in the absence of a heme lyase (38Pollock W.B. Rosell F.I. Twitchett M.B. Dumont M.E. Mauk A.G. Biochemistry. 1998; 37: 6124-6131Crossref PubMed Scopus (251) Google Scholar, 39Patel C.N. Lind M.C. Pielak G.J. Protein Expr. Purif. 2001; 22: 220-224Crossref PubMed Scopus (47) Google Scholar), can form in the cytoplasm of E. coli, in contrast to H. thermophilus c 552 (12Sinha N. Ferguson S.J. FEMS Microbiol. Lett. 1998; 161: 1-6Crossref PubMed Google Scholar). These observations might reflect the stability and the folding state of the apoproteins, but not the intrinsic difference in their reactivity toward heme, which is shown from previous (11Daltrop O. Allen J.W. Willis A.C. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7872-7876Crossref PubMed Scopus (87) Google Scholar) and present work to be similar for the three apocytochromes c. The higher stability and partial folding (24Wain R. Pertinhez T.A. Tomlinson E.J. Hong L. Dobson C.M. Ferguson S.J. Smith L.J. J. Biol. Chem. 2001; 276: 45813-45817Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) of H. thermophilus c 552apocytochrome may lead to a slower rate of degradation in the cytoplasm of E. coli and a higher heme affinity compared with the two proteins studied in this work. Therefore, in addition to the aspects mentioned above, another function of the heme lyase may be to increase the heme affinity of the apocytochrome c.In summary, we have shown that two c-type cytochromes, from a mesophilic bacterium and a mammalian mitochondrion, can form in vitro from the reaction of apocytochrome and heme. Remarkably, this reaction of horse heart cytochrome has not been reported despite first having been attempted nearly 30 years ago (15Fisher W.R. Taniuchi H. Anfinsen C.B. J. Biol. Chem. 1973; 248: 3188-3195Abstract Full Text PDF PubMed Google Scholar). The reaction generally seems to proceed from a b-type cytochrome intermediate, provided the potential disulfide between the conserved cysteine residues is avoided and the heme-iron is reduced. Indeed, we suspect that had Dumont et al. (13Dumont M.E. Corin A.F. Campbell G.A. Biochemistry. 1994; 33: 7368-7378Crossref PubMed Scopus (67) Google Scholar) extended their duration, and/or modified the reaction condition for instance by exclusion of oxygen, of incubation of heme with apocytochrome c, they too might have seen covalent bond formation to heme. C-type cytochromes are found in almost all organisms, being mainly involved in electron transport (1Scott R.A. Mauk A.G. Cytochrome C: A Multidisciplinary Approach. University Science Books, Mill Valley, CA1995Google Scholar, 2Ferguson S.J. Biochem. Soc. Trans. 2001; 29: 629-640Crossref PubMed Google Scholar). They contain a characteristic CXXCH motif, whereby the conserved cysteine residues are involved in forming a covalent thioether bond between the thiol functionalities and the vinyl groups of the prosthetic heme moiety (3Barker P.D. Ferguson S.J. Structure Fold. Des. 1999; 7: R281-R290Abstract Full Text Full Text PDF Scopus (141) Google Scholar). Remarkably, three different systems have evolved to facilitate cytochrome c formation in vivo (4Kranz R. Lill R. Goldman B. Bonnard G. Merchant S. Mol. Microbiol. 1998; 29: 383-396Crossref PubMed Scopus (237) Google Scholar, 5Page M.D. Sambongi Y. Ferguson S.J. Trends Biochem. Sci. 1998; 23: 103-108Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). In some eukaryotes, one enzyme, designated heme lyase, catalyzes the attachment of the heme moiety to the apocytochromec (6Steiner H. Kispal G. Zollner A. Haid A. Neupert W. Lill R. J. Biol. Chem. 1996; 271: 32605-32611Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). These enzymes have a high specificity and do not act on bacterial apocytochromes c (7Sanders C. Lill H. Biochim. Biophys. Acta. 2000; 1459: 131-138Crossref PubMed Scopus (47) Google Scholar). Mammalian organisms make use of this system as exemplified by horse (Equus caballus) heart mitochondria. Many Gram-negative bacteria use a system that involves more than ten gene products to ensure correct cytochrome c maturation (ccm) 1The abbreviations used are: ccm, cytochromec maturation; DTT, dithiothreitol; AMS, 4-acetamido-4′-maleimidyl-stilbene-2,2′-disulfonate; ANS, 8-anilino-1-naphthalenesulfonate; CD, circular dichroism; ES-MS, electrospray mass spectrometry 1The abbreviations used are: ccm, cytochromec maturation; DTT, dithiothreitol; AMS, 4-acetamido-4′-maleimidyl-stilbene-2,2′-disulfonate; ANS, 8-anilino-1-naphthalenesulfonate; CD, circular dichroism; ES-MS, electrospray mass spectrometry in the periplasm (5Page M.D. Sambongi Y. Ferguson S.J. Trends Biochem. Sci. 1998; 23: 103-108Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar,8Thöny-Meyer L. Microbiol. Mol. Biol. Rev. 1997; 61: 337-376Crossref PubMed Scopus (314) Google Scholar, 9Thöny-Meyer L. Biochem. Soc. Trans. 2002; 30: 633-638Crossref PubMed Scopus (102) Google Scholar). An example of a cytochrome c that is matured in vivo using this biosynthesis apparatus is cytochromec 550 from P. denitrificans (10Sambongi Y. Ferguson S.J. FEBS Lett. 1994; 340: 65-70Crossref PubMed Scopus (66) Google Scholar). Following our report of the in vitro formation of holocytochrome c 552 from H. thermophilus (11Daltrop O. Allen J.W. Willis A.C. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7872-7876Crossref PubMed Scopus (87) Google Scholar), an important point to be established was whether the spontaneous attachment of heme to apocytochromec is a reaction applicable to other cytochromesc. Because holocytochrome c 552 fromH. thermophilus forms exceptionally in the cytoplasm ofEscherichia coli (12Sinha N. Ferguson S.J. FEMS Microbiol. Lett. 1998; 161: 1-6Crossref PubMed Google Scholar), its spontaneous in vitroformation might not necessarily be anticipated to apply generally to all c-type cytochromes. The previously reported thioether bond formation in holocytochrome c 552 from ofH. thermophilus involved a b-type cytochrome intermediate (11Daltrop O. Allen J.W. Willis A.C. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7872-7876Crossref PubMed Scopus (87) Google Scholar). Prompted by the previously reported similar ability of the horse heart apocytochrome to bind heme to yield a species characteristic of a b-type cytochrome (13Dumont M.E. Corin A.F. Campbell G.A. Biochemistry. 1994; 33: 7368-7378Crossref PubMed Scopus (67) Google Scholar), the reaction of horse heart apocytochrome c with heme has been reinvestigated. The formation of c-type cytochromes is especially interesting in light of the different biosynthetic pathways, which are reviewed extensively elsewhere (4Kranz R. Lill R. Goldman B. Bonnard G. Merchant S. Mol. Microbiol. 1998; 29: 383-396Crossref PubMed Scopus (237) Google Scholar, 5Page M.D. Sambongi Y. Ferguson S.J. Trends Biochem. Sci. 1998; 23: 103-108Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 9Thöny-Meyer L. Biochem. Soc. Trans. 2002; 30: 633-638Crossref PubMed Scopus (102) Google Scholar), by which heme is attached to the apoproteins in vivo. Therefore, a second candidate for studying the reaction of heme with apocytochrome was P. denitrificans cytochrome c 550. The two cytochromes used in this study are matured by two different cytochromec maturation systems in vivo. A comparison of the reactions with respect to the attachment mechanism is anticipated to have important implications for the molecular basis of heme attachment in different organisms in vivo. DISCUSSIONIn this work we show the data obtained from the production of apocytochromes c from a bacterial and a mammalian organism and characterize the reaction of the cysteine thiols with the vinyl moieties of heme. Both proteins have very similar properties with regard to their apoforms and their reaction with heme. We show that the cysteines of the conserved CXXCH motif can form an internal disulfide. This observation raises the question if this oxidation occurs in vivo for the respective proteins in either organism. For the bacterial system it has been suggested that the disulfide is an important intermediate during cytochrome cmaturation due to the presence of the disulfide bond forming (Dsb) proteins in the periplasm, the location of cytochrome cmaturation (2Ferguson S.J. Biochem. Soc. Trans. 2001; 29: 629-640Crossref PubMed Google Scholar). Furthermore, it has been proposed that proteins (CcmG/H and DsbD) required for cytochrome c biogenesis are involved in the reduction of the internal disulfide bond in the apocytochrome (9Thöny-Meyer L. Biochem. Soc. Trans. 2002; 30: 633-638Crossref PubMed Scopus (102) Google Scholar). Why this in situ reduction of an apocytochrome has evolved is unclear, but an internal protection mechanism against metallation of the coordination site of the reduced apocytochrome might offer an explanation.Heme attachment to mitochondrial apocytochrome c occurs in the intermembrane space (29Diekert K. Kispal G. Guiard B. Lill R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11752-11757Crossref PubMed Scopus (106) Google Scholar). The in vivo oxidation state of the cysteines in mitochondrial apocytochromes c has not been investigated to the best of our knowledge. Whether, as we show here for the first time in vitro, a disulfide bond occurs during thein vivo protein maturation process, will depend on both the reduction potential of its locus and kinetic factors. If formation of a disulfide must be avoided, a thioredoxin-like protein could be required in the intermembrane space (30Laloi C. Rayapuram N. Chartier Y. Grienenberger J.M. Bonnard G. Meyer Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14144-14149Crossref PubMed Scopus (222) Google Scholar). A thioredoxin (Trx3) and its reductase (Trr2) are known to be present in yeast mitochondria, but submitochondrial localization to the matrix is suspected rather than to the intermembrane space (31Pedrajas J.R. Kosmidou E. Miranda-Vizuete A. Gustafsson J.A. Wright A.P. Spyrou G. J. Biol. Chem. 1999; 274: 6366-6373Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). These proteins can in any case be deduced to be dispensable for cytochrome c biogenesis in yeast, as mutants carrying specific disruptions in the corresponding genes were able to grow normally under aerobic, respiratory conditions, which require participation of cytochrome c in the mitochondrial respiratory chain. However, in the case of Arabidopsis thaliana, it has been recognized, on the basis of the genome sequence, that there may be a yet to be characterized thioredoxin that is targeted to the intermembrane space (30Laloi C. Rayapuram N. Chartier Y. Grienenberger J.M. Bonnard G. Meyer Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14144-14149Crossref PubMed Scopus (222) Google Scholar). Alternatively, the formation of holocytochrome c may happen sufficiently fast that the two thiols of apocytochrome c cannot form a disulfide during its lifetime in the intermembrane space following delivery from the reducing environment of the cytoplasm. Such a kinetic constraint would suffice if the rate of disulfide bond formation in the intermembrane space were to occur on a similar timescale to thatin vitro (hours as observed in the present work). The presence of the CcmABCEF components of one cytochrome cbiogenesis pathway, but not of the thioredoxin-like CcmG component, in plant mitochondria might support the latter view.The binding of heme to the apoform of mitochondrial cytochromec clearly involves some ordering of the polypeptide chain. This is evident from several biophysical studies (13Dumont M.E. Corin A.F. Campbell G.A. Biochemistry. 1994; 33: 7368-7378Crossref PubMed Scopus (67) Google Scholar, 28Goldberg M.E. Schaeffer F. Guillou Y. Djavadi-Ohaniance L. J. Biol. Chem. 1999; 274: 16052-16061Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). A non-covalent complex between heme and apoprotein of H. thermophilus cytochrome c 552 generates a protein structure that is very similar to that of the native protein with covalently bound heme (32Tomlinson E.J. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5156-5160Crossref PubMed Scopus (65) Google Scholar). Although it appears that the structure of the non-covalent complex between heme and mitochondrial apocytochromec does not fully resemble the native, holo structure (13Dumont M.E. Corin A.F. Campbell G.A. Biochemistry. 1994; 33: 7368-7378Crossref PubMed Scopus (67) Google Scholar,28Goldberg M.E. Schaeffer F. Guillou Y. Djavadi-Ohaniance L. J. Biol. Chem. 1999; 274: 16052-16061Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar), studies with two monoclonal antibodies that scarcely recognize the apoprotein, but bind with comparable affinities to both the non-covalent heme-apoprotein complex and the holoprotein, indicate that the complex must be similar to the holoprotein in respect of quite specific structural features at the epitope regions (28Goldberg M.E. Schaeffer F. Guillou Y. Djavadi-Ohaniance L. J. Biol. Chem. 1999; 274: 16052-16061Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Such similarities are evidently sufficient to permit the vinyl groups of heme to be oriented appropriately, in at least a fraction of the molecules, for reaction with the thiol groups, which have previously been shown to exhibit nucleophilic reactivity (27Kang X. Carey J. Biochemistry. 1999; 38: 15944-15951Crossref PubMed Scopus (28) Google Scholar). The effect of covalently bound hydrophobic moieties on the folding of cytochromec has also been discussed (27Kang X. Carey J. Biochemistry. 1999; 38: 15944-15951Crossref PubMed Scopus (28) Google Scholar).The affinity of the apoproteins for hydrophobic ligands and the ability of both apoproteins to ligate heme in a b-type cytochrome complex has important implications for the catalytic strategy of the enzymes involved during cytochrome c maturation. It is unclear from previous studies how the heme lyase functions in mitochondria, except for the proposal that it binds heme via a conserved CPX motif (6Steiner H. Kispal G. Zollner A. Haid A. Neupert W. Lill R. J. Biol. Chem. 1996; 271: 32605-32611Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). For the bacterial Ccm system, two proteins have been proposed to function as a heme lyase (33Ren Q. Ahuja U. Thöny-Meyer L. J. Biol. Chem. 2002; 277: 7657-7663Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), but the heme attachment is more complex due to the presence of a heme chaperone, CcmE (34Schulz H. Hennecke H. Thöny-Meyer L. Science. 1998; 281: 1197-1200Crossref PubMed Scopus (153) Google Scholar). However, in view of our data showing that theb-type cytochrome can spontaneously react to give thioether bond formation yielding holocytochrome c, two key conditions have to be achieved by the catalytic systems. Firstly, the heme group has to be kept reduced to give optimal reactivity for cysteine-thiol and heme-vinyl group reaction partners. This is in agreement with previous studies (35Nicholson D.W. Neupert W. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4340-4344Crossref PubMed Scopus (85) Google Scholar, 36Tong J. Margoliash E. J. Biol. Chem. 1998; 273: 25695-25702Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). And secondly, the orientation of the heme relative to its rotation around the α,γ meso-axis has to be correct to yield the required stereochemistry at the prochiral α-carbon of the vinyl substituents of the heme moiety (3Barker P.D. Ferguson S.J. Structure Fold. Des. 1999; 7: R281-R290Abstract Full Text Full Text PDF Scopus (141) Google Scholar). McRee et al.(37McRee D.E. Williams P.A. Sridhar V. Pastuszyn A. Bren K.L. Patel K.M. Chen Y. Todaro T.R. Sanders D. Luna E. Fee J.A. J. Biol. Chem. 2001; 276: 6537-6544Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) showed that a recombinant cytochrome c ofThermus thermophilus could be improperly matured in vivo, but without any catalytic assistance in the cytoplasm ofE. coli, because of both heme inversion and an intermolecular disulfide bond between cysteine residues, which are usually involved in heme binding (37McRee D.E. Williams P.A. Sridhar V. Pastuszyn A. Bren K.L. Patel K.M. Chen Y. Todaro T.R. Sanders D. Luna E. Fee J.A. J. Biol. Chem. 2001; 276: 6537-6544Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The latter behavior was clearly reflected in the visible absorption spectrum, which was different from the reported, properly matured holocytochrome c, and the pyridine hemochrome spectrum, which was not indicative of two thioether bonds to the heme-vinyl groups (37McRee D.E. Williams P.A. Sridhar V. Pastuszyn A. Bren K.L. Patel K.M. Chen Y. Todaro T.R. Sanders D. Luna E. Fee J.A. J. Biol. Chem. 2001; 276: 6537-6544Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). For mitochondrial cytochromec, two thioether bonds have formed in the present work. In the case of in vitro reaction of heme with P. denitrificans apocytochrome c 550 it is not evident if the reaction yielded a mixture of only b- andc-type cytochrome complexes or also contained species with only one thioether bond. Due to the presence of DTT during the course of the reaction, no reaction product containing an intermolecular disulfide bond is obtained in vitro.In the in vitro reaction, the oxidation state of the heme can be controlled, but the heme delivery is nonspecific with respect to the stereochemical aspects of the heme orientation. The stereochemistry for the in vitro produced cytochromesc is not determined and will be focus of future work. However, the fact that a fraction of the mitochondrialb-type cytochrome intermediate remained unreactive might hint that incorrect orientation of heme does not lead to thioether bond formation, suggesting the in vitro cytochromec formation to be stereoselective. In the case of thein vitro formation of H. thermophilus c 552 the reaction is stereoselective (11Daltrop O. Allen J.W. Willis A.C. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7872-7876Crossref PubMed Scopus (87) Google Scholar).An interesting point arises from the observation that neither P. denitrificans c 550 (10Sambongi Y. Ferguson S.J. FEBS Lett. 1994; 340: 65-70Crossref PubMed Scopus (66) Google Scholar) nor mitochondrial cytochromec, in the absence of a heme lyase (38Pollock W.B. Rosell F.I. Twitchett M.B. Dumont M.E. Mauk A.G. Biochemistry. 1998; 37: 6124-6131Crossref PubMed Scopus (251) Google Scholar, 39Patel C.N. Lind M.C. Pielak G.J. Protein Expr. Purif. 2001; 22: 220-224Crossref PubMed Scopus (47) Google Scholar), can form in the cytoplasm of E. coli, in contrast to H. thermophilus c 552 (12Sinha N. Ferguson S.J. FEMS Microbiol. Lett. 1998; 161: 1-6Crossref PubMed Google Scholar). These observations might reflect the stability and the folding state of the apoproteins, but not the intrinsic difference in their reactivity toward heme, which is shown from previous (11Daltrop O. Allen J.W. Willis A.C. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7872-7876Crossref PubMed Scopus (87) Google Scholar) and present work to be similar for the three apocytochromes c. The higher stability and partial folding (24Wain R. Pertinhez T.A. Tomlinson E.J. Hong L. Dobson C.M. Ferguson S.J. Smith L.J. J. Biol. Chem. 2001; 276: 45813-45817Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) of H. thermophilus c 552apocytochrome may lead to a slower rate of degradation in the cytoplasm of E. coli and a higher heme affinity compared with the two proteins studied in this work. Therefore, in addition to the aspects mentioned above, another function of the heme lyase may be to increase the heme affinity of the apocytochrome c.In summary, we have shown that two c-type cytochromes, from a mesophilic bacterium and a mammalian mitochondrion, can form in vitro from the reaction of apocytochrome and heme. Remarkably, this reaction of horse heart cytochrome has not been reported despite first having been attempted nearly 30 years ago (15Fisher W.R. Taniuchi H. Anfinsen C.B. J. Biol. Chem. 1973; 248: 3188-3195Abstract Full Text PDF PubMed Google Scholar). The reaction generally seems to proceed from a b-type cytochrome intermediate, provided the potential disulfide between the conserved cysteine residues is avoided and the heme-iron is reduced. Indeed, we suspect that had Dumont et al. (13Dumont M.E. Corin A.F. Campbell G.A. Biochemistry. 1994; 33: 7368-7378Crossref PubMed Scopus (67) Google Scholar) extended their duration, and/or modified the reaction condition for instance by exclusion of oxygen, of incubation of heme with apocytochrome c, they too might have seen covalent bond formation to heme. In this work we show the data obtained from the production of apocytochromes c from a bacterial and a mammalian organism and characterize the reaction of the cysteine thiols with the vinyl moieties of heme. Both proteins have very similar properties with regard to their apoforms and their reaction with heme. We show that the cysteines of the conserved CXXCH motif can form an internal disulfide. This observation raises the question if this oxidation occurs in vivo for the respective proteins in either organism. For the bacterial system it has been suggested that the disulfide is an important intermediate during cytochrome cmaturation due to the presence of the disulfide bond forming (Dsb) proteins in the periplasm, the location of cytochrome cmaturation (2Ferguson S.J. Biochem. Soc. Trans. 2001; 29: 629-640Crossref PubMed Google Scholar). Furthermore, it has been proposed that proteins (CcmG/H and DsbD) required for cytochrome c biogenesis are involved in the reduction of the internal disulfide bond in the apocytochrome (9Thöny-Meyer L. Biochem. Soc. Trans. 2002; 30: 633-638Crossref PubMed Scopus (102) Google Scholar). Why this in situ reduction of an apocytochrome has evolved is unclear, but an internal protection mechanism against metallation of the coordination site of the reduced apocytochrome might offer an explanation. Heme attachment to mitochondrial apocytochrome c occurs in the intermembrane space (29Diekert K. Kispal G. Guiard B. Lill R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11752-11757Crossref PubMed Scopus (106) Google Scholar). The in vivo oxidation state of the cysteines in mitochondrial apocytochromes c has not been investigated to the best of our knowledge. Whether, as we show here for the first time in vitro, a disulfide bond occurs during thein vivo protein maturation process, will depend on both the reduction potential of its locus and kinetic factors. If formation of a disulfide must be avoided, a thioredoxin-like protein could be required in the intermembrane space (30Laloi C. Rayapuram N. Chartier Y. Grienenberger J.M. Bonnard G. Meyer Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14144-14149Crossref PubMed Scopus (222) Google Scholar). A thioredoxin (Trx3) and its reductase (Trr2) are known to be present in yeast mitochondria, but submitochondrial localization to the matrix is suspected rather than to the intermembrane space (31Pedrajas J.R. Kosmidou E. Miranda-Vizuete A. Gustafsson J.A. Wright A.P. Spyrou G. J. Biol. Chem. 1999; 274: 6366-6373Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). These proteins can in any case be deduced to be dispensable for cytochrome c biogenesis in yeast, as mutants carrying specific disruptions in the corresponding genes were able to grow normally under aerobic, respiratory conditions, which require participation of cytochrome c in the mitochondrial respiratory chain. However, in the case of Arabidopsis thaliana, it has been recognized, on the basis of the genome sequence, that there may be a yet to be characterized thioredoxin that is targeted to the intermembrane space (30Laloi C. Rayapuram N. Chartier Y. Grienenberger J.M. Bonnard G. Meyer Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14144-14149Crossref PubMed Scopus (222) Google Scholar). Alternatively, the formation of holocytochrome c may happen sufficiently fast that the two thiols of apocytochrome c cannot form a disulfide during its lifetime in the intermembrane space following delivery from the reducing environment of the cytoplasm. Such a kinetic constraint would suffice if the rate of disulfide bond formation in the intermembrane space were to occur on a similar timescale to thatin vitro (hours as observed in the present work). The presence of the CcmABCEF components of one cytochrome cbiogenesis pathway, but not of the thioredoxin-like CcmG component, in plant mitochondria might support the latter view. The binding of heme to the apoform of mitochondrial cytochromec clearly involves some ordering of the polypeptide chain. This is evident from several biophysical studies (13Dumont M.E. Corin A.F. Campbell G.A. Biochemistry. 1994; 33: 7368-7378Crossref PubMed Scopus (67) Google Scholar, 28Goldberg M.E. Schaeffer F. Guillou Y. Djavadi-Ohaniance L. J. Biol. Chem. 1999; 274: 16052-16061Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). A non-covalent complex between heme and apoprotein of H. thermophilus cytochrome c 552 generates a protein structure that is very similar to that of the native protein with covalently bound heme (32Tomlinson E.J. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5156-5160Crossref PubMed Scopus (65) Google Scholar). Although it appears that the structure of the non-covalent complex between heme and mitochondrial apocytochromec does not fully resemble the native, holo structure (13Dumont M.E. Corin A.F. Campbell G.A. Biochemistry. 1994; 33: 7368-7378Crossref PubMed Scopus (67) Google Scholar,28Goldberg M.E. Schaeffer F. Guillou Y. Djavadi-Ohaniance L. J. Biol. Chem. 1999; 274: 16052-16061Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar), studies with two monoclonal antibodies that scarcely recognize the apoprotein, but bind with comparable affinities to both the non-covalent heme-apoprotein complex and the holoprotein, indicate that the complex must be similar to the holoprotein in respect of quite specific structural features at the epitope regions (28Goldberg M.E. Schaeffer F. Guillou Y. Djavadi-Ohaniance L. J. Biol. Chem. 1999; 274: 16052-16061Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Such similarities are evidently sufficient to permit the vinyl groups of heme to be oriented appropriately, in at least a fraction of the molecules, for reaction with the thiol groups, which have previously been shown to exhibit nucleophilic reactivity (27Kang X. Carey J. Biochemistry. 1999; 38: 15944-15951Crossref PubMed Scopus (28) Google Scholar). The effect of covalently bound hydrophobic moieties on the folding of cytochromec has also been discussed (27Kang X. Carey J. Biochemistry. 1999; 38: 15944-15951Crossref PubMed Scopus (28) Google Scholar). The affinity of the apoproteins for hydrophobic ligands and the ability of both apoproteins to ligate heme in a b-type cytochrome complex has important implications for the catalytic strategy of the enzymes involved during cytochrome c maturation. It is unclear from previous studies how the heme lyase functions in mitochondria, except for the proposal that it binds heme via a conserved CPX motif (6Steiner H. Kispal G. Zollner A. Haid A. Neupert W. Lill R. J. Biol. Chem. 1996; 271: 32605-32611Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). For the bacterial Ccm system, two proteins have been proposed to function as a heme lyase (33Ren Q. Ahuja U. Thöny-Meyer L. J. Biol. Chem. 2002; 277: 7657-7663Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), but the heme attachment is more complex due to the presence of a heme chaperone, CcmE (34Schulz H. Hennecke H. Thöny-Meyer L. Science. 1998; 281: 1197-1200Crossref PubMed Scopus (153) Google Scholar). However, in view of our data showing that theb-type cytochrome can spontaneously react to give thioether bond formation yielding holocytochrome c, two key conditions have to be achieved by the catalytic systems. Firstly, the heme group has to be kept reduced to give optimal reactivity for cysteine-thiol and heme-vinyl group reaction partners. This is in agreement with previous studies (35Nicholson D.W. Neupert W. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4340-4344Crossref PubMed Scopus (85) Google Scholar, 36Tong J. Margoliash E. J. Biol. Chem. 1998; 273: 25695-25702Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). And secondly, the orientation of the heme relative to its rotation around the α,γ meso-axis has to be correct to yield the required stereochemistry at the prochiral α-carbon of the vinyl substituents of the heme moiety (3Barker P.D. Ferguson S.J. Structure Fold. Des. 1999; 7: R281-R290Abstract Full Text Full Text PDF Scopus (141) Google Scholar). McRee et al.(37McRee D.E. Williams P.A. Sridhar V. Pastuszyn A. Bren K.L. Patel K.M. Chen Y. Todaro T.R. Sanders D. Luna E. Fee J.A. J. Biol. Chem. 2001; 276: 6537-6544Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) showed that a recombinant cytochrome c ofThermus thermophilus could be improperly matured in vivo, but without any catalytic assistance in the cytoplasm ofE. coli, because of both heme inversion and an intermolecular disulfide bond between cysteine residues, which are usually involved in heme binding (37McRee D.E. Williams P.A. Sridhar V. Pastuszyn A. Bren K.L. Patel K.M. Chen Y. Todaro T.R. Sanders D. Luna E. Fee J.A. J. Biol. Chem. 2001; 276: 6537-6544Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The latter behavior was clearly reflected in the visible absorption spectrum, which was different from the reported, properly matured holocytochrome c, and the pyridine hemochrome spectrum, which was not indicative of two thioether bonds to the heme-vinyl groups (37McRee D.E. Williams P.A. Sridhar V. Pastuszyn A. Bren K.L. Patel K.M. Chen Y. Todaro T.R. Sanders D. Luna E. Fee J.A. J. Biol. Chem. 2001; 276: 6537-6544Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). For mitochondrial cytochromec, two thioether bonds have formed in the present work. In the case of in vitro reaction of heme with P. denitrificans apocytochrome c 550 it is not evident if the reaction yielded a mixture of only b- andc-type cytochrome complexes or also contained species with only one thioether bond. Due to the presence of DTT during the course of the reaction, no reaction product containing an intermolecular disulfide bond is obtained in vitro. In the in vitro reaction, the oxidation state of the heme can be controlled, but the heme delivery is nonspecific with respect to the stereochemical aspects of the heme orientation. The stereochemistry for the in vitro produced cytochromesc is not determined and will be focus of future work. However, the fact that a fraction of the mitochondrialb-type cytochrome intermediate remained unreactive might hint that incorrect orientation of heme does not lead to thioether bond formation, suggesting the in vitro cytochromec formation to be stereoselective. In the case of thein vitro formation of H. thermophilus c 552 the reaction is stereoselective (11Daltrop O. Allen J.W. Willis A.C. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7872-7876Crossref PubMed Scopus (87) Google Scholar). An interesting point arises from the observation that neither P. denitrificans c 550 (10Sambongi Y. Ferguson S.J. FEBS Lett. 1994; 340: 65-70Crossref PubMed Scopus (66) Google Scholar) nor mitochondrial cytochromec, in the absence of a heme lyase (38Pollock W.B. Rosell F.I. Twitchett M.B. Dumont M.E. Mauk A.G. Biochemistry. 1998; 37: 6124-6131Crossref PubMed Scopus (251) Google Scholar, 39Patel C.N. Lind M.C. Pielak G.J. Protein Expr. Purif. 2001; 22: 220-224Crossref PubMed Scopus (47) Google Scholar), can form in the cytoplasm of E. coli, in contrast to H. thermophilus c 552 (12Sinha N. Ferguson S.J. FEMS Microbiol. Lett. 1998; 161: 1-6Crossref PubMed Google Scholar). These observations might reflect the stability and the folding state of the apoproteins, but not the intrinsic difference in their reactivity toward heme, which is shown from previous (11Daltrop O. Allen J.W. Willis A.C. Ferguson S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7872-7876Crossref PubMed Scopus (87) Google Scholar) and present work to be similar for the three apocytochromes c. The higher stability and partial folding (24Wain R. Pertinhez T.A. Tomlinson E.J. Hong L. Dobson C.M. Ferguson S.J. Smith L.J. J. Biol. Chem. 2001; 276: 45813-45817Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) of H. thermophilus c 552apocytochrome may lead to a slower rate of degradation in the cytoplasm of E. coli and a higher heme affinity compared with the two proteins studied in this work. Therefore, in addition to the aspects mentioned above, another function of the heme lyase may be to increase the heme affinity of the apocytochrome c. In summary, we have shown that two c-type cytochromes, from a mesophilic bacterium and a mammalian mitochondrion, can form in vitro from the reaction of apocytochrome and heme. Remarkably, this reaction of horse heart cytochrome has not been reported despite first having been attempted nearly 30 years ago (15Fisher W.R. Taniuchi H. Anfinsen C.B. J. Biol. Chem. 1973; 248: 3188-3195Abstract Full Text PDF PubMed Google Scholar). The reaction generally seems to proceed from a b-type cytochrome intermediate, provided the potential disulfide between the conserved cysteine residues is avoided and the heme-iron is reduced. Indeed, we suspect that had Dumont et al. (13Dumont M.E. Corin A.F. Campbell G.A. Biochemistry. 1994; 33: 7368-7378Crossref PubMed Scopus (67) Google Scholar) extended their duration, and/or modified the reaction condition for instance by exclusion of oxygen, of incubation of heme with apocytochrome c, they too might have seen covalent bond formation to heme. We thank Julie Stevens, Mark Bushell, and James Allen for help and discussions. Christopher Higham and Carsten Richter are gratefully acknowledged for help with the production of P. denitrificans c 550.
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