Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Architecture of the membrane-bound cytochrome c heme lyase CcmF


The covalent attachment of one or multiple heme cofactors to cytochrome c protein chains enables cytochrome c proteins to be used in electron transfer and redox catalysis in extracytoplasmic environments. A dedicated heme maturation machinery, whose core component is a heme lyase, scans nascent peptides after Sec-dependent translocation for CXnCH-binding motifs. Here we report the three-dimensional (3D) structure of the heme lyase CcmF, a 643-amino acid integral membrane protein, from Thermus thermophilus. CcmF contains a heme b cofactor at the bottom of a large cavity that opens toward the extracellular side to receive heme groups from the heme chaperone CcmE for cytochrome maturation. A surface groove on CcmF may guide the extended apoprotein to heme attachment at or near a loop containing the functionally essential WXWD motif, which is situated above the putative cofactor binding pocket. The structure suggests heme delivery from within the membrane, redefining the role of the chaperone CcmE.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Schematic architecture of heme maturation system I.
Fig. 2: Structure of T. thermophilus CcmF.
Fig. 3: Heme binding to CcmF and structural clues to protein function.
Fig. 4: A buoy model for heme delivery to CcmF.

Data availability

The structural model and structure factors for TtCcmF have been deposited in the Protein Data Bank at under the accession number 6ZMQ.


  1. 1.

    Anson, M. L. & Mirsky, A. E. The heme compounds in nature and biological oxidations. Science 68, 647–648 (1928).

    CAS  PubMed  Google Scholar 

  2. 2.

    Pauling, L. & Coryell, C. D. The magnetic properties and structure of hemoglobin, oxyhemoglobin and carbonmonoxyhemoglobin. Proc. Natl Acad. Sci. USA 22, 210–216 (1936).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Fita, I. & Rossmann, M. G. The active center of catalase. J. Mol. Biol. 185, 21–37 (1985).

    CAS  PubMed  Google Scholar 

  4. 4.

    Einsle, O. et al. Structure of cytochrome c nitrite reductase. Nature 400, 476–480 (1999).

    CAS  PubMed  Google Scholar 

  5. 5.

    Hermann, B., Kern, M., La Pietra, L., Simon, J. & Einsle, O. The octaheme MccA is a heme c-copper sulfite reductase. Nature 520, 706–709 (2015).

    CAS  PubMed  Google Scholar 

  6. 6.

    Schlichting, I. et al. The catalytic pathway of cytochrome P450cam at atomic resolution. Science 287, 1615–1622 (2000).

    CAS  PubMed  Google Scholar 

  7. 7.

    Margoliash, E., Barlow, G. H. & Byers, V. Differential binding properties of cytochrome c: possible relevance for mitochondrial ion transport. Nature 228, 723–726 (1970).

    CAS  PubMed  Google Scholar 

  8. 8.

    Poulos, T. L. Heme enzyme structure and function. Chem. Rev. 114, 3919–3962 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Weichsel, A., Andersen, J. F., Roberts, S. A. & Montfort, W. R. Nitric oxide binding to nitrophorin 4 induces complete distal pocket burial. Nat. Struct. Biol. 7, 551–554 (2000).

    CAS  PubMed  Google Scholar 

  10. 10.

    Perutz, M. F., Kendrew, J. C. & Watson, H. C. Structure and function of hemeoglobin: II. Some relations between polypeptide chain configuration and amino acid sequence. J. Mol. Biol. 13, 669–678 (1965).

    CAS  Google Scholar 

  11. 11.

    Barker, P. D. & Ferguson, S. J. Still a puzzle: why is heme covalently attached in c-type cytochromes? Structure 7, R281–R290 (1999).

    CAS  PubMed  Google Scholar 

  12. 12.

    Moore, G. R. & Pettigrew, G. W. Cytochromes c. Evolutionary, Structural and Physicochemical Aspects. (Springer, 1990).

  13. 13.

    Keilin, D. On cytochrome, a respiratory pigment, common to animals, yeast, and higher plants. Proc. R. Soc. Lond. B 98, 312–339 (1925).

  14. 14.

    Thöny-Meyer, L. Biogenesis of respiratory cytochromes in bacteria. Microbiol. Mol. Biol. Rev. 61, 337–376 (1997).

    PubMed  PubMed Central  Google Scholar 

  15. 15.

    Kranz, R., Lill, R., Goldman, B., Bonnard, G. & Merchant, S. Molecular mechanisms of cytochrome c biogenesis: three distinct systems. Mol. Microbiol. 29, 383–396 (1998).

    CAS  PubMed  Google Scholar 

  16. 16.

    Methé, B. A. et al. Genome of Geobacter sulfurreducens: metal reduction in subsurface environments. Science 302, 1967–1969 (2003).

    PubMed  Google Scholar 

  17. 17.

    Babbitt, S. E., Sutherland, M. C., Francisco, B. S., Mendez, D. L. & Kranz, R. G. Mitochondrial cytochrome c biogenesis: no longer an enigma. Trends Biochem. Sci. 40, 446–455 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Babbitt, S. E. et al. Mechanisms of mitochondrial holocytochrome c synthase and the key roles played by cysteines and histidine of the heme attachment site, Cys-XX-Cys-His. J. Biol. Chem. 289, 28795–28807 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Stevens, J. M., Uchida, T., Daltrop, O. & Ferguson, S. J. Covalent cofactor attachment to proteins: cytochrome c biogenesis. Biochem. Soc. Trans. 33, 792–795 (2005).

    CAS  PubMed  Google Scholar 

  20. 20.

    Sanders, C., Turkarslan, S., Lee, D. W. & Daldal, F. Cytochrome c biogenesis: the Ccm system. Trends Microbiol. 18, 266–274 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Thöny-Meyer, L. Cytochrome c maturation: a complex pathway for a simple task? Biochem. Soc. Trans. 30, 633–638 (2002).

    PubMed  Google Scholar 

  22. 22.

    Schulz, H., Fabianek, R. A., Pellicioli, E. C., Hennecke, H. & Thöny-Meyer, L. Heme transfer to the heme chaperone CcmE during cytochrome c maturation requires the CcmC protein, which may function independently of the ABC-transporter CcmAB. Proc. Natl Acad. Sci. USA 96, 6462–6467 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Baysse, C. et al. Co-ordination of iron acquisition, iron porphyrin chelation and iron–protoporphyrin export via the cytochrome c biogenesis protein CcmC in Pseudomonas fluorescens. Microbiology 149, 3543–3552 (2003).

    CAS  PubMed  Google Scholar 

  24. 24.

    Page, M. D. & Ferguson, S. J. Mutational analysis of the Paracoccus denitrificans c-type cytochrome biosynthetic genes ccmABCDG: disruption of ccmC has distinct effects suggesting a role for CcmC independent of CcmAB. Microbiology 145, 3047–3057 (1999).

    CAS  PubMed  Google Scholar 

  25. 25.

    Richard-Fogal, C. L., Frawley, E. R. & Kranz, R. G. Topology and function of CcmD in cytochrome c maturation. J. Bacteriol. 190, 3489–3493 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Sanders, C. et al. The cytochrome c maturation components CcmF, CcmH, and CcmI form a membrane-integral multisubunit heme ligation complex. J. Biol. Chem. 283, 29715–29722 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Ahuja, U., Rozhkova, A., Glockshuber, R., Thöny-Meyer, L. & Einsle, O. Helix swapping leads to dimerization of the N-terminal domain of the c-type cytochrome maturation protein CcmH from Escherichia coli. FEBS Lett. 582, 2779–2786 (2008).

    CAS  PubMed  Google Scholar 

  28. 28.

    Fabianek, R. A., Hofer, T. & Thöny-Meyer, L. Characterization of the Escherichia coli CcmH protein reveals new insights into the redox pathway required for cytochrome c maturation. Arch. Microbiol. 171, 92–100 (1999).

    CAS  PubMed  Google Scholar 

  29. 29.

    San Francisco, B., Sutherland, M. C. & Kranz, R. G. The CcmFH complex is the system I holocytochrome c synthetase: engineering cytochrome c maturation independent of CcmABCDE. Mol. Microbiol. 91, 996–1008 (2014).

    CAS  PubMed  Google Scholar 

  30. 30.

    Lee, J. H., Harvat, E. M., Stevens, J. M., Ferguson, S. J. & Saier, M. H. Evolutionary origins of members of a superfamily of integral membrane cytochrome c biogenesis proteins. Biochim. Biophys. Acta Biomembr. 1768, 2164–2181 (2007).

    CAS  Google Scholar 

  31. 31.

    Richard-Fogal, C. L. et al. A conserved heme redox and trafficking pathway for cofactor attachment. EMBO J. 28, 2349–2359 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    San Francisco, B., Bretsnyder, E. C., Rodgers, K. R. & Kranz, R. G. Heme ligand identification and redox properties of the cytochrome c synthetase, CcmF. Biochemistry 50, 10974–10985 (2011).

    PubMed  Google Scholar 

  33. 33.

    San Francisco, B. & Kranz, R. G. Interaction of holoCcmE with CcmF in heme trafficking and cytochrome c biosynthesis. J. Mol. Biol. 426, 570–585 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Shoemaker, K. R., Kim, P. S., York, E. J., Stewart, J. M. & Baldwin, R. L. Tests of the helix dipole model for stabilization of α-helices. Nature 326, 563–567 (1987).

    CAS  PubMed  Google Scholar 

  35. 35.

    Lomize, M. A., Pogozheva, I. D., Joo, H., Mosberg, H. I. & Lomize, A. L. OPM database and PPM web server: resources for positioning of proteins in membranes. Nucleic Acids Res. 40, D370–D376 (2012).

    CAS  PubMed  Google Scholar 

  36. 36.

    Schulz, H., Hennecke, H. & Thöny-Meyer, L. Prototype of a heme chaperone essential for cytochrome c maturation. Science 281, 1197–1200 (1998).

    CAS  PubMed  Google Scholar 

  37. 37.

    Enggist, E., Thöny-Meyer, L., Guntert, P. & Pervushin, K. NMR structure of the heme chaperone CcmE reveals a novel functional motif. Structure 10, 1551–1557 (2002).

    CAS  PubMed  Google Scholar 

  38. 38.

    Arnesano, F. et al. Solution structure and characterization of the heme chaperone CcmE. Biochemistry 41, 13587–13594 (2002).

    CAS  PubMed  Google Scholar 

  39. 39.

    Ferreira, G. C. et al. Structure and function of ferrochelatase. J. Bioenerg. Biomembr. 27, 221–229 (1995).

    CAS  PubMed  Google Scholar 

  40. 40.

    Dailey, T. A. & Dailey, H. A. Identification of [2Fe-2S] clusters in microbial ferrochelatases. J. Bacteriol. 184, 2460–2464 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Richard-Fogal, C. & Kranz, R. G. The CcmC:heme:CcmE complex in heme trafficking and cytochrome c biosynthesis. J. Mol. Biol. 401, 350–362 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Ahuja, U. & Thöny-Meyer, L. CcmD is involved in complex formation between CcmC and the heme chaperone CcmE during cytochrome c maturation. J. Biol. Chem. 280, 236–243 (2005).

    CAS  PubMed  Google Scholar 

  43. 43.

    Verissimo, A. F., Mohtar, M. A. & Daldal, F. The heme chaperone apoCcmE forms a ternary complex with CcmI and apocytochrome c. J. Biol. Chem. 288, 6272–6283 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Verissimo, A. F. & Daldal, F. Cytochrome c biogenesis system I: an intricate process catalyzed by a maturase supercomplex? Biochim. Biophys. Acta 1837, 989–998 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Verissimo, A. F. et al. The thioreduction component CcmG confers efficiency and the heme ligation component CcmH ensures stereo-specificity during cytochrome c maturation. J. Biol. Chem. 292, 13154–13167 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Frawley, E. R. & Kranz, R. G. CcsBA is a cytochrome c synthetase that also functions in heme transport. Proc. Natl Acad. Sci. USA 106, 10201–10206 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

    CAS  PubMed  Google Scholar 

  48. 48.

    Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Vonrhein, C. et al. Data processing and analysis with the autoPROC toolbox. Acta Crystallogr. D Biol. Crystallogr. 67, 293–302 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Terwilliger, T. C. et al. Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol wizard. Acta Crystallogr. D Biol. Crystallogr. 65, 582–601 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    BUSTER v. 2.10.3 (Global Phasing Ltd., 2010).

  54. 54.

    Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    CAS  PubMed  Google Scholar 

Download references


We thank F. Kersten and S. Andrade for support and helpful discussions. This work was supported by the European Research Council (grant 310656) and Deutsche Forschungsgemeinschaft (CRC 1381, project ID 403222702, and RTG 2202, project ID 46710898). We thank the beam line staff at the Swiss Light Source, Villigen, Switzerland, for excellent assistance with data collection.

Author information




A.B. and O.E. designed the experiments. A.B. and L.I. produced protein and generated crystals. A.B. solved the crystal structure. A.B. and L.Z. built and refined the crystal structure. A.B. and O.E. analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to Oliver Einsle.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Chemical Biology thanks T. Iverson and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 The heme cofactor and c-type cytochromes.

a, Fe-protoporphyrin IX, the widely used tetrapyrrole cofactor heme, with IUPAC numbering of the carbon atoms in the aromatic ligand. Two vinyl side chains are located at position 3 and 8, and the negatively charged propionate side chains at positions 13 and 17 are relevant for the translocation of the cofactor across a lipid bilayer. b, in cytochromes of type c, the heme cofactor is covalently linked to two cysteine residues of the protein chain via thioether bonds. This linkage is catalyzed by heme lyases and allows for a high cofactor/protein ratio. c, heme cofactors are bound to signature CXXCH motifs in the protein sequence, with the two cysteine residues of the motif forming the thioether linkages, and the subsequent histidine acting as a proximal axial ligand to the iron ion of the cofactor. Figure made from cytochrome c nitrite reductase (PDB ID 1FS7)1.

Extended Data Fig. 2 The tryptophan-rich signature motif (WXWD).

In all classes of heme lyases, a tryptophan-rich motif is suggested to be directly involved in the handling of the heme cofactor. It is found in human cytochrome c synthase (HCCS), as well as in the lyase CcsA of system II and the components CcmC and CcmF of system I.

Extended Data Fig. 3 B-factor distribution in TtCcmF.

Elevated B-factors provide a measure of structural flexibility within the structure of CcmF. The cytoplasmic face of the protein shows high B-factors only in the loop connecting helices h8 and h14 near the C-terminus. In contrast, the periplasmic face of the heme lyase features multiple regions with increased flexibility, notably including the periplasmic domain in the loop connection helices h14 and h15.

Extended Data Fig. 4 Surface representations of TtCcmF.

a, Stereo representation of a low-resolution molecular surface of CcmF and its orientation within the membrane. The membrane is represented by a red disc for the periplasmic boundary and a blue disc for the cytoplasmic boundary. b, Three different views of the surface of CcmF with bound lipids in stick representation, highlighting the extensive periplasmic protrusion that is made up predominantly by the C-terminal periplasmic domain.

Extended Data Fig. 5 Environment of the accessory heme group in CcmF.

The stereo figure shows the b-type heme group liganded by residues H259 in helix h7 and H493 in helix h14. The open space above the accessory heme is the vestibule suggested to accommodate the substrate heme group for attachment to an apocytochrome chain. The refined 2Fo–Fc electron density map is contoured at the 1 σ level.

Extended Data Fig. 6 Docking model for a substrate heme group in CcmF.

The stereo figure details the docking result for a second b-type heme cofactor as a substrate heme within the cavity located above the accessory heme, seen from the opening of this vestibule towards the inner leaflet of the membrane. In order to accommodate the substrate heme, the two tryptophane residues of the WXWD motif in loop 6, W238 and W240, had to relocate, interacting with the bound heme moiety via π-stacking interactions.

Extended Data Fig. 7 Orientation of b-type heme groups in membrane proteins.

Membrane-integral heme groups are most commonly canonical Fe-protoporphyrin IX (heme b). Even bound within the protein matrix, they consistently orient with their propionate sidechains towards the hydrophilic surface of the membrane, revealing this to be a preferred orientation in either leaflet of the membrane. Panels show the orientation of protein complexes in the membrane above a detail of the orientation of the membrane-integral heme b groups. In the respiratory complexes cytochrome bc1 (a) from oxidative phosphorylation and cytochrome b6f (b) from oxygenic photosynthesis, the low- and high-potential heme b moieties have both propionates facing the hydrophilic phase. c, in TtCcmF, the single heme b cofactor is located at the boundary of membrane and cytoplasm, with an approximate 40° rotation with respect to a and b, but in a highly similar arrangement as the heme group in the cytoplasmic leaflet of formate dehydrogenase (d) and nitrate reductase (e).

Supplementary information

Supplementary Information

Supplementary Figs. 1–3 and Table 1.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Brausemann, A., Zhang, L., Ilcu, L. et al. Architecture of the membrane-bound cytochrome c heme lyase CcmF. Nat Chem Biol 17, 800–805 (2021).

Download citation


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing