9.B.14 The Heme Handling Protein (HHP) Family

The proteins of the HHP family can be large with ~650 amino acids and as many as 15 or 16 putative TMSs. Parts of them are homologous to the E. coli CycZ putative heme exporter (9.B.14.2.1), the plant chloroplast cytochrome c biogenesis proteins such as CcsA of Chlamydomonas reinhardii (spP48269) and to HelC of Rhodobacter capsulatus (spP29961), also a 6 TMS protein thought to be involved in heme export. Ccl1 of R. capsulatus has been experimentally shown to have eleven TMSs, while CcsA of Mycobacterium leprae has been shown to have 6 TMSs. UniProt puts these homologues in the CcsA/CcmF/CycK/Cel1 family. This family is distantly related to the UniProt CcmC/CycZ/HelC family (Lee et al., 2007). The functions of most of these proteins are not established, but evidence suggests that at least some of them are heme exporters (Baysse et al., 2003; Sutherland et al. 2018). CcmC of E. coli binds heme and interacts with CcmE, a heme chaperone protein that inserts heme into apocytochrome c (Ren and Thöny-Meyer, 2001). Kranz et al. (2009) have reviewed aspects of cytochrome c biogenesis including the mechanisms for covalent modifications and trafficking of heme, and for heme-iron redox control. CryoEM studies on CcsBA is both a heme transporter and an insertase (see TC# 9.A.14.3.1).

 Three members of the HHP family (CcmC, CcmF and CcsBA), involved in cytochrome c biosynthesis, possess a conserved tryptophan-rich region (called the WWD domain) in an external loop at the inner membrane surface. The WWD domain binds heme to present it to an acceptor protein (apoCcmE for CcmC or apocytochrome c for CcmF and CcsBA) such that the heme vinyl group covalently attaches to the acceptor. CcmE only interacts stably with CcmC when heme is present. Endogenously synthesized heme enters the external WWD domain of CcmC either via a channel within this six-transmembrane-spanning protein or from the membrane (Richard-Fogal and Kranz, 2010).

Frawley and Kranz, (2009) showed that CcsBA exports and protects heme from oxidation. CcsBA has 10 apparent TMSs and reconstitutes cytochrome c synthesis in the E. coli periplasm; thus, CcsBA is a heme exporter and cytochrome c synthetase. Purified CcsBA contains heme in an 'external heme binding domain' for which two external histidines are shown to serve as axial ligands that protect the heme iron from oxidation. There is also a heme binding site in the membrane domain of CcsBA (Sutherland et al. 2018). The former site may be the active site of the synthetase, while the latter site may be involved in transport. Furthermore, two conserved histidines in TMSs are required for heme to travel to the external heme binding domain. Thus, CcsBA is a heme channel or carrier with a heme binding site within the bilayer.

Organisms employ one of several different enzyme systems to mature cytochromes c (Simon and Hederstedt 2011). The biosynthetic process involves the periplasmic reduction of cysteine residues in the heme c attachment motif of the apocytochrome, transmembrane transport of heme b and stereospecific covalent heme attachment via thioether bonds. The biogenesis System II (or Ccs system) is employed by β-, δ- and ε-proteobacteria, Gram-positive bacteria, Aquificales and cyanobacteria, as well as by algal and plant chloroplasts. System II comprises four (sometimes only three) membrane-bound proteins: CcsA (or ResC) and CcsB (ResB) are the components of the cytochrome c synthase, whereas CcdA and CcsX (ResA) function in the generation of a reduced heme c attachment motif. Some ε-proteobacteria contain CcsBA fusion proteins constituting single polypeptide cytochrome c synthases especially amenable for functional studies (Simon and Hederstedt 2011).



Baert, B., C. Baysse, S. Matthijs, and P. Cornelis. (2008). Multiple phenotypic alterations caused by a c-type cytochrome maturation ccmC gene mutation in Pseudomonas aeruginosa. Microbiology 154: 127-138.

Baysse, C., S. Matthijs, M. Schobert, G. Layer, D. Jahn, and P. Cornelis. (2003). 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.

Bhattacharya, S., J.D. Choudhury, R. Gachhui, and J. Mukherjee. (2017). A new collagenase enzyme of the marine sponge pathogen Pseudoalteromonas agarivorans NW4327 is uniquely linked with a TonB dependent receptor. Int J Biol Macromol. [Epub: Ahead of Print]

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

Goldman, B.S., D.L. Beck, E.M. Monika, and R.G. Kranz. (1998). Transmembrane heme delivery systems. Proc. Natl. Acad. Sci USA 95: 5003-5008.

Hoel, C.M., L. Zhang, and S.G. Brohawn. (2022). Structure of the GOLD-domain seven-transmembrane helix protein family member TMEM87A. Elife 11:.

Inoue, K., B.W. Dreyfuss, K.L. Kindle, D.B. Stern, S. Merchant, and O.A. Sodeinde. (1997). Ccs1, a nuclear gene required for the post-translational assembly of chloroplast c-type cytochromes. J. Biol. Chem. 272: 31747-31754.

Kranz, R.G., C. Richard-Fogal, J.S. Taylor, and E.R. Frawley. (2009). Cytochrome c biogenesis: mechanisms for covalent modifications and trafficking of heme and for heme-iron redox control. Microbiol. Mol. Biol. Rev. 73: 510-28, Table of Contents.

Krumm, B.E. and R. Grisshammer. (2015). Peptide ligand recognition by G protein-coupled receptors. Front Pharmacol 6: 48.

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

Mendez, D.L., E.P. Lowder, D.E. Tillman, M.C. Sutherland, A.L. Collier, M.J. Rau, J.A.J. Fitzpatrick, and R.G. Kranz. (2022). Cryo-EM of CcsBA reveals the basis for cytochrome c biogenesis and heme transport. Nat Chem Biol 18: 101-108.

Page, M.D. and S.J. Ferguson. (1999). 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.

Page, M.D., Y. Sambongi, and S.J. Ferguson. (1998). Contrasting routes of c-type cytochrome assembly in mitochondria, chloroplasts and bacteria. Trends Biochem. Sci. 23: 103-108.

Pearce, D.A., M.D. Page, H.A.C. Norris, E.J. Tomlinson, and S.J. Ferguson. (1998). Identification of the contiguous Paracoccus denitrificans ccmF and ccmH genes: disruption of ccmF, encoding a putative transporter, results in formation of an unstable apocytochrome c and deficiency in siderophore production. Microbiology 144: 467-477.

Peleg, A., Y. Shifrin, O. Ilan, C. Nadler-Yona, S. Nov, S. Koby, K. Baruch, S. Altuvia, M. Elgrably-Weiss, C.M. Abe, S. Knutton, M.A. Saper, and I. Rosenshine. (2005). Identification of an Escherichia coli operon required for formation of the O-antigen capsule. J. Bacteriol. 187: 5259-5266.

Ren, Q. and L. Thony-Meyer. (2001). Physical interaction of CcmC with heme and the heme chaperone CcmE during cytochrome c maturation. J. Biol. Chem. 276: 32591-32596.

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

Schulz, H., E.C. Pellicioli, and L. Thöny-Meyer. (2000). New insights into the role of CcmC, CcmD and CcmE in the haem delivery pathway during cytochrome c maturation by a complete mutational analysis of the conserved tryptophan-rich motif of CcmC. Mol. Microbiol. 37: 1379-1388.

Schulz, H., R.A. Fabianek, E.C. Pellicioli, H. Hennecke, and L. Thöny-Meyer. (1999). 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.

Simon, J. and L. Hederstedt. (2011). Composition and function of cytochrome c biogenesis System II. FEBS J. 278: 4179-4188.

Sutherland, M.C., N.L. Tran, D.E. Tillman, J.M. Jarodsky, J. Yuan, and R.G. Kranz. (2018). Structure-Function Analysis of the Bifunctional CcsBA Heme Exporter and Cytochrome Synthetase. MBio 9:.

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TC#NameOrganismal TypeExample
9.B.14.1.1The cytochrome c biogenesis protein, CcmF Bacteria CcmF (Ccl1) of Rhodobacter capsulatus

Uncharacterized protein of 796 aas and 19 TMSs


UP of Leptospira interrogans


Heme maturase of 518 aas and 13 TMSs, YejR


YejR of Tetrahymena thermophila


Uncharacterized protein of 285 aas and 6 TMSs


UP of Candida albicans


AgrC of 429 aas and 6 TMSs


AgrC of Stahpylococcus epidermidis


Uncharacterized protein of 225 aas and 6 TMSs.


UP of Petrotoga mobilis


Uncharacterized protein of 263 aas and 7 TMSs

UP of Fusobacterium nucleatum


Uncharacterized protein of 257 aas and 6 TMSs

UP of Caldicellulosiruptor lactoaceticus


Uncharacterized protein of 434 aas and 7 TMSs.

UP of Roseburia intestinalis


Uncharacterized GHKL domain-containing protein of 445 aas and 6 TMSs.

UP of Clostridium hungatei


UP of 747 aas and 19 TMSs. Possibly related to 9.B.14.

UP of Lokiarchaeum sp GC14-75

9.B.14.1.2The cytochrome c biogenesis protein, CycK Bacteria CycK of Bradyrhizobium japonicum

Uncharacteerized ATP-binding protein of 424 aas and 7 N-terminal TMSs and a C-terminal hydrophilic domain.

UP of Ruminococcus flavefaciens

9.B.14.1.3The cytochrome c biogenesis protein, CcmFBacteriaCcmF (CycK) of E. coli (P33927)

C-type biosynthesis protein, CcmF (620 aas; 17 TMSs) (resembles 9.B.67.3.3, and possibly APC family (2.A.3) members)


CcmF of Cyanidioschyzon merolae (Q9ZZP7)


Cytochrome C-type biogenesis protein, CcmF 


CcmF of Haloarcula marismortui (Q5V2F3)


Mitochondrial cytochrome C-type biogenesis protein, CcmF of 442 aas and 6 - 8 TMSs.


CcmF of Arabidopsis thaliana (P93286)


Cytochrome biogenesis protein, CcmF (involved also in pyoverdine maturation) (Baert et al. 2008). It is 657 aas long with 15 TMSs in a 14 + 1 TMS arrangement.


CcmF of Pseudomonas aeruginosa


MURF1 protein of 443 aas and 16 TMSs


MURF1 of Leishmania tarentolae (Sauroleishmania tarentolae)


Putative XkR8 (TC family 2.A.112) homologue of 235 aas and 6 TMSs.


XkR8 homologue of Campylobacter jejuni


TC#NameOrganismal TypeExample

TC#NameOrganismal TypeExample
9.B.14.2.1The putative heme exporter, HelCBacteriaHelC of Rhodobacter capsulatus (P29961)
9.B.14.2.2The putative heme exporter, CcmC (CycZ). This protein is also listed under TC# 3.A.1.107.1 as part of an ABC transporterBacteriaCcmC (CycZ) of Bradirhizobium japonicum (P30962)
9.B.14.2.3The putative heme exporter, CycZ (CcmC)BacteriaCycZ (CcmC) of E. coli (P0ABM1)

Putative heme exporter, CcmC; also involved in pyoverdine maturation (Baert et al. 2008).


CcmC of Pseudomonas aeruginosa

ABC-type cytochrome c biogenesis putative heme transport system permease component C of 234 aas


Putative ABC-type cytochrome c biogenesis transport system permease component of Melioribacter roseus

TC#NameOrganismal TypeExample

Cytochrome c biogenesis protein, CcsBA (936 aas; 10 TMSs; 14 TMSs predicted by the WHAT program (Frawley and Kranz, 2009).  May be a bifunctional protein with heme transport (export from the cytoplasm to the periplasm) and attachment to apocytochrome c (cytochrome c synthetase) activities.  Two heme binding sites have been identified in a homologue, one in the membrane and one on the external surface (on the periplasmic side of the membrane) (Sutherland et al. 2018). Mendez et al. 2022 described cryo-EM structures of CcsBA, which, as noted above, is a bifunctional heme transporter and cytochrome c (cyt c) synthase. Models built from the cryo-EM densities show that CcsBA is trapped with heme in two conformations, closed and open states. The closed state has heme located solely at a transmembrane (TM) site, with a large periplasmic domain oriented such that access of heme to the cytochrome acceptor is prevented. The open conformation contains two heme moieties, one in the TM-heme site and another in an external site (P-heme site). The presence of heme in the periplasmic site at the base of a chamber induces a large conformational shift that exposes the heme for reaction with apocytochrome c (apocyt c). Consistent with these structures, in vivo and in vitro cyt c synthase studies suggest a mechanism for transfer of the periplasmic heme to cytochrome (Mendez et al. 2022).


CcsBA of Helicobacter hepaticus (Q7VHG9)


Uncharacterized protein of 271 aas and 8 TMSs.

UP of Gemmataceae bacterium (freshwater metagenome)

9.B.14.3.2Cytochrome c biogenesis protein CcsA


ccsA of Prochlorococcus marinus

Cytochrome c biogenesis protein CcsA or Ccs1 of 353 aas.  Required during biogenesis of c-type cytochromes (cytochrome c6 and cytochrome f) at the step of heme attachment (Inoue et al. 1997).


CcsA of Chlamydomonas reinhardtii


Cytochrome c biogenesis protein of 327 aas and 6 established TMSs, CcsA.


CcsA of Mycobacterium leprae

Membrane protein of 266 aas and 8 putative TMSs.


Membrane protein of Pseudomonas aeruginosa


Putative cytochrome c assembly protein, YpjD of 263 aas and 8 TMSs.  In an operon with YfjD, a putative Mg2+uptake protein (TC# 9.B.20.1.4).

YpjD of E. coli


Cytochrome c biogenesis protein, CcsA, of 309 aas and 8 TMSs.  May be a bifunctional protein with heme transport (export from the cytoplasm to the periplasm) and attachment to apocytochrome c (cytochrome c synthetase) activities.  Two heme binding sites have been identified in a homologue, one in the membrane and one on the external surface (on the periplasmic side of the membrane) (Sutherland et al. 2018).

CcsA of Prochlorococcus marinus


Cytochrome c biogenesis protein, CcsA, of 309 aas and 8 TMSs.  May be a bifunctional protein with heme transport (export from the cytoplasm to the periplasm) and attachment to apocytochrome c (cytochrome c synthetase) activities.  Two heme binding sites have been identified in a homologue, one in the membrane and one on the external surface (on the periplasmic side of the membrane) (Sutherland et al. 2018).

CcsB of Helicobacter pylori


Cytochrome c biogenesis protein, CcsB, of 1041 aas and 14 TMSs.  May be a bifunctional protein with heme transport (export from the cytoplasm to the periplasm) and attachment to apocytochrome c (cytochrome c synthetase) activities.  Two heme binding sites have been identified in a homologue, one in the membrane and one on the external surface (on the periplasmic side of the membrane) (Sutherland et al. 2018).

CcsB of Flavobacterium psychrophilum


TC#NameOrganismal TypeExample

XkR8 homologue of 383 aas and 11 TMSs.


XkR8 homologue of Brachyspira murdochii


Putative XkR8 homologue of 404 aas and 11 TMSs.


XkR8 homologue of Brachyspira pilosicoli


Putative XkR8 homologue of 587 aas and 12 TMSs.


Putative XkR8 homologue of Mycoplasma mycoides


Uncharacterized protein of 431 aas and 5 TMSs in a 1 (N-terminal) + 4 (C-terminal) TMS arrangement.

UP of Plasmodium yoelii


O-antigen polymerase of 439 aas and 10 TMSs.

Polymerase of Candidatus Pelagibacter


Uncharacterized protein of 497 aas and 12 TMSs.

UP of Mycoplasma collis


TC#NameOrganismal TypeExample

Putative bacteriocin immunity protein of 572 aas and 14 putative TMSs.


Imm protein of Bacillus cereus


Uncharacterized protein of 588 aas and about 15 TMSs.


UP of Bacillus cereus


TC#NameOrganismal TypeExample

TC#NameOrganismal TypeExample

TC#NameOrganismal TypeExample