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.