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Architecture of the membrane-bound cytochrome c heme lyase CcmF

Abstract

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.

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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 www.pdb.org under the accession number 6ZMQ.

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Acknowledgements

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.

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Contributions

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.

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The authors declare no competing interests.

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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).

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Supplementary Figs. 1–3 and Table 1.

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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). https://doi.org/10.1038/s41589-021-00793-8

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