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.
Huynh et al. 2023 evaluated cryoEM and crystal structures of two molecular machines that traffick heme and attach it to cytochrome c (cyt c), the second activity performed by a cyt c synthase. These integral membrane proteins, CcsBA and CcmF/H, both covalently attach heme to cyt c, but carry it out via different mechanisms. A CcsB-CcsA complex transports heme through a channel to its external active site, where it forms two thioethers between reduced (Fe(+2)) heme and CysXxxXxxCysHis in cyt c. The active site is formed by a periplasmic WWD sequence and two histidines (P-His1 and P-His2). They evaluated each proposed functional domain in CcsBA cryoEM densities, exploring their presence in other CcsB-CcsA proteins from a wide distribution of organisms (e.g. from Gram positive to Gram negative bacteria to chloroplasts.) Two conserved pockets, for the first and second cysteines of CXXCH, explain stereochemical heme attachment. In addition to other universal features, a conserved periplasmic beta stranded structure, called the Beta cap, protects the active site when external heme is not present. Analysis of CcmF/H, an oxidoreductase and cyt c synthase, addresses mechanisms of heme access and attachment. Huynh et al. 2023 provided evidence that CcmF/H receives Fe+3 heme from holoCcmE via a periplasmic entry point in CcmF, whereby heme is inserted directly into a conserved WWD /P-His domain from above. Evidence suggests that CcmF acts as a heme reductase, reducing holoCcmE (to Fe+2) through a transmembrane electron transfer conduit, which initiates a complicated series of events at the active site.
References:
Uncharacterized protein of 796 aas and 19 TMSs
Spirochaetes
UP of Leptospira interrogans
Heme maturase of 518 aas and 13 TMSs, YejR
Ciliates
YejR of Tetrahymena thermophila
Uncharacterized protein of 285 aas and 6 TMSs
Fungi
UP of Candida albicans
AgrC of 429 aas and 6 TMSs
Firmicutes
AgrC of Stahpylococcus epidermidis
Uncharacterized protein of 225 aas and 6 TMSs.
Thermatogae
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
Uncharacteerized ATP-binding protein of 424 aas and 7 N-terminal TMSs and a C-terminal hydrophilic domain.
UP of Ruminococcus flavefaciens
CcmA/CcmD proteins, probably involved in cytochorme c matureation (heme insertion). CcmA iis of 353 aas with 9 TMSs, while CcmD is of 240 aas with 6 TMSs. They may function with CcmB (see TC# 3.A.1.107.5 (Gupta et al. 2022).
CcmA/CcmD of Methanosarcina acetivorans
C-type biosynthesis protein, CcmF (620 aas; 17 TMSs) (resembles 9.B.67.3.3, and possibly APC family (2.A.3) members)
Eukaryotes
CcmF of Cyanidioschyzon merolae (Q9ZZP7)
Cytochrome C-type biogenesis protein, CcmF
Archaea
CcmF of Haloarcula marismortui (Q5V2F3)
Mitochondrial cytochrome C-type biogenesis protein, CcmF of 442 aas and 6 - 8 TMSs.
Plants
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.
γ-Proteobacteria
CcmF of Pseudomonas aeruginosa
MURF1 protein of 443 aas and 16 TMSs
Eukaryotes
MURF1 of Leishmania tarentolae (Sauroleishmania tarentolae)
Putative XkR8 (TC family 2.A.112) homologue of 235 aas and 6 TMSs.
Plants
XkR8 homologue of Campylobacter jejuni
Putative heme exporter, CcmC; also involved in pyoverdine maturation (Baert et al. 2008).
γ-Proteobacteria
CcmC of Pseudomonas aeruginosa
ABC-type cytochrome c biogenesis putative heme transport system permease component C of 234 aas
Ignavibacteriae
Putative ABC-type cytochrome c biogenesis transport system permease component of Melioribacter roseus
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).
Bacteria
CcsBA of Helicobacter hepaticus (Q7VHG9)
Uncharacterized protein of 271 aas and 8 TMSs.
UP of Gemmataceae bacterium (freshwater metagenome)
Bacteria
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).
Algae
CcsA of Chlamydomonas reinhardtii
Cytochrome c biogenesis protein of 327 aas and 6 established TMSs, CcsA.
Actinobacteria
CcsA of Mycobacterium leprae
Membrane protein of 266 aas and 8 putative TMSs.
Proteobacteria
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
XkR8 homologue of 383 aas and 11 TMSs.
Spirochaetes
XkR8 homologue of Brachyspira murdochii
Putative XkR8 homologue of 404 aas and 11 TMSs.
Spirochaetes
XkR8 homologue of Brachyspira pilosicoli
Putative XkR8 homologue of 587 aas and 12 TMSs.
Tenericutes
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
Putative bacteriocin immunity protein of 572 aas and 14 putative TMSs.
Firmicutes
Imm protein of Bacillus cereus
Uncharacterized protein of 588 aas and about 15 TMSs.
Firmicutes
UP of Bacillus cereus