Getting iron inside

Introduction

Many proteins require helper molecules for full biological activity, which are known as cofactors. These are typically organic molecules with unique chemical properties. Incorporation of a cofactor therefore dramatically extends the chemical versatility of a protein relative to the limited versatility that is achievable with only amino acids. Heme represents a well-known cofactor that has been used for over 3 billion years in a variety of biological processes such as electron transfer, binding and sensing of gases, catalysis and gene regulation (1). Heme has a characteristic red color and chemically, heme is composed of an aromatic ring-like protoporphyrin IX scaffold with a covalently bound iron atom in its center. 

The core of the scaffold is made up of four pyrroles that form a large ring structure that is known as a porphyrin (2). The iron atom represents the business end of a heme molecule because it is responsible for the binding of gasses and its redox properties ensure efficient electron transfer within electron transport chains or catalysis. The heme iron is coordinated to four nitrogen atoms of the porphyrin moiety, which point towards the inside of the ring (figure 1 adapted from 2). The most common types of heme found in Nature are heme b and heme c (figure 1), which from a chemical perspective are very similar. However, heme b associates non-covalently to the polypeptide backbone, whereas heme c is covalently bound to the protein typically through thioether bonds (2). The precise reason why some proteins contain covalently bound heme is not clear yet, although it increases their overall stability and prevents the loss of the heme cofactor in extracytoplasmic environments (1). Proteins that contain heme c are known as c-type cytochromes or cytochromes c for short. These cytochromes contain a varying number of heme groups ranging from one up to 36 in Geobacter sulfurreducens (3) and typically function as electron carriers in a variety of photosynthetic and respiratory electron transport chains. C-type cytochromes are defined by the presence of a characteristic heme-binding motif (CXXCH) (4,5). Within this pentapeptide, histidine serves as axial ligand to the heme iron, while the two cysteine residues form thioether bonds to vinyl groups 2 and 4 of the porphyrin’s tetrapyrrole ring (figure 1). Currently, five different systems have been described in various organisms that enable the post-translational assembly of cytochrome c proteins. Of these, the cytochrome maturation (Ccm) system (or system I) represents a complex cytochrome c assembly machinery that is present in Gram-negative bacteria, archaea and plant mitochondria (4,5). Much of what we know about the bacterial Ccm system has been learned from E. coli. Following export of the apocytochrome c through the SecYEG translocon into the E. coli periplasm, heme assembly is completed by the cytochrome maturation machinery. This is composed of eight membrane-embedded proteins (CcmABCDEFGH) and proceeds via four consecutive steps, namely: (i) translocation of heme into the periplasm that is facilitated by CcmA, CcmB, CcmC and CcmD, (ii) CcmE-dependent chaperoning of heme through the periplasm towards the CcmHF complex, (iii) reduction of unwanted disulfides in the CXXCH motif by CcmG, CcmH and CcmI and (iv) finally covalent attachment of heme into the apocytochrome c catalyzed by CcmF (4,5). A series of elegant genetic and biochemical studies have revealed the basics of bacterial cytochrome c assembly (4,5). However, the precise mechanistic details of this process are as yet not clear owing to a lack of structural information on the different components of the Ccm machinery. With regards to this, it is important to note that recently the atomic structure of CcmF from Thermus thermophilus was determined, providing significant insight into how heme is delivered from within the membrane and transferred onto the apocytochrome (6). Here, I will discuss this structure as well as its functional understanding. 

Overview of Ccm-mediated cytochrome c assembly

 

Genetic studies performed with different bacteria in the late 80’s and early 90’s revealed a complex pathway for the maturation of cytochrome c proteins. It is now well established that this process occurs in the periplasm and requires the proteins of the Ccm operon (CcmABCDEFGH) that are located in the cytoplasmic membrane (4,5). The absence of any of these proteins blocks the ability of bacterial cells to produce cytochrome c proteins. The cast of players is presented below, starting with CcmA. Two copies of this protein associate with two molecules of CcmB to form an ABC transporter. CcmA is peripheral subunit exposed to the cytoplasm and displays ATPase activity, while CcmB is a membrane-embedded protein with six transmembrane helices (TMHs) that firmly anchors the ABC transporter into the membrane (5). Considering the role of CcmAB as ABC transporter it seemed likely that it facilitated the export of heme into the periplasm (5). However, it is now clear that this ABC transporter is required for releasing heme from CcmE (1). CcmC is an integral membrane protein with six TMHs that is probably involved in the translocation of heme and the subsequent transfer and ligation of heme to CcmE (4). CcmD is a poorly conserved, small integral membrane protein with one TMH that is thought to assist the heme ligation activity of CcmC as well as the release of heme-bound CcmE from the CcmABCDE complex (5). CcmE is the heme chaperone of the Ccm system and is embedded into the membrane with one TMH (5). CcmF contains eleven TMHs and binds heme-bound CcmE. It functions as lyase and is responsible for catalyzing the covalent attachment of heme onto the apocytochrome (1). CcmG is a small membrane-embedded protein with one TMH and is equipped with a periplasmic thioredoxin motif. It is responsible for reducing the cysteine residues in the CXXCH motif, thereby enabling the formation of thioether bonds with the vinyl groups of the porphyrins tetrapyrrole ring (5). Like CcmG, CcmH is a single-spanning membrane protein with a periplasmic thioredoxin-like motif. It forms a complex with the heme lyase CcmF and probably binds the apocytochrome, while its periplasmic domain is involved in reducing the cysteine residues in the CXXCH motif (4,5). In addition to the Ccm proteins described above, several other proteins play an important role in the maturation of cytochrome c proteins, namely: CcmI and DsbD. CcmI is a membrane protein with two TMHs and is part of the CcmFH complex. Functionally, CcmI probably binds the apocytochrome (5). DsbD is anchored into the membrane through its 6 TMHs and contains two periplasmic domains. It is involved in the transfer of electrons from cytoplasmic thioredoxin to CcmG in the periplasm, thereby keeping the latter in a reduced state and ensuring that CcmG is able to reduce the cysteines in the CXXCH motif (5).  

The maturation of cytochrome c proteins by the bacterial Ccm system is schematically shown in figure 2 (adapted from 6), revealing that this process comprises four basics steps. These are: (i) translocation of the apocytochrome across the cytoplasmic membrane into the periplasm, (ii) transport of heme from the cytoplasm towards the periplasm, (iii) reduction and chaperoning of the apocytochrome and finally (iv) formation of thioether bonds between the heme and the cysteine residues of the CXXCH motif (7). Apocytochrome proteins are synthesized in the cytoplasm and are translocated post-translationally across the cytoplasmic membrane into the periplasm through the SecYEG translocon. Membrane translocation requires that the apocytochrome is unfolded and is kept in this state following translocation in the periplasm. Folding only occurs after covalent attachment of heme, yielding holocytochrome c proteins (1,5). Heme is also produced in the cytoplasm and needs to be exported to the periplasm as well (1). It was initially thought the ABC transporter made up of CcmA and CcmB was responsible for this. However, it is now clear that this ABC transporter is required for the release of heme from the heme chaperone CcmE (1). This raises the question how heme is transferred across the cytoplasmic membrane into the periplasm. With regards to this question, it should be noted that CcmC is required for heme attachment to CcmE and it is therefore also conceivable that it plays a role in the periplasmic export of heme (4). Moreover, immunobiochemical data showed that CcmC, CcmB and CcmD interact (1). This indicates that these proteins may form a complex that is required for heme translocation and subsequent transfer to CcmE (figure 2) (4,5). Following its translocation into the periplasm, heme has to be delivered to CcmE. This is a heme chaperone that covalently binds the heme group through a histidine residue (4,5). How heme is loaded onto CcmE is not well understood. It has, moreover, been shown that ATP hydrolysis by CcmA induces the release of heme from holoCcmE, thereby supplying heme for cytochrome c assembly (figure 2) (1). While the functional details of CcmE remain poorly understood, it is clear that it is a crucial part of the Ccm machinery. It is well established that the Ccm system recognizes the CXXCH motif present in apocytochrome proteins (1,4,5). In order to keep the cysteines of this motif competent for heme ligation, it is essential that the formation of disulfides, which is catalyzed by the periplasmic DsbA-DsbB system, is prevented through reduction of the cysteines in the CXXCH motif. The latter is achieved by CcmG and CcmH that contain thioredoxin-like motifs, enabling participation in dithiol-disulfide oxidoreduction reactions (4,5). The electrons that are required for this are provided by thioredoxin in the cytosol which are subsequently transferred across the plasma membrane to CcmG located at the periplasmic face of the plasma membrane (5). In vitro experiments with truncated Ccm components suggested that electrons are passed on from CcmG to CcmH and from there to apocytochrome c to reduce the cysteines of the CXXCH motif (5). However, several lines of biochemical evidence challenged a direct role of CcmH in the reduction of apocytochrome c. Conceivably, the oxidized apocytochrome c is a direct partner of CcmG instead of CcmH. This means that CcmG functions as a thioredoxin to reduce the cysteines of the heme-binding motif as well as a chaperone that binds the apocytochrome c (5). In most bacteria, CcmH and CcmF form a complex together with CcmI. In R. capsulatis, for example, a complex of about 800 kDa embedded in the plasma membrane has been identified that comprises CcmI, CcmH and CcmF (8). This CcmFHI complex scans the apocytochrome to locate heme-binding motifs and catalyze heme ligation. CcmH and CcmI probably serve as a chaperone by binding the apocytochrome and present it to CcmF for heme attachment. CcmF functions as heme lyase and, therefore, represents the functional core of the CcmFHI complex (4,5,6). Several reaction mechanisms that describe covalent heme attachment to the cysteines of the heme-binding motif have been proposed (1) but in principle CcmF catalyzes an addition reaction of the cysteine thiols of heme-binding motif to the vinyl side chains of the heme molecule. The precise details of this reaction are unknown but it is clear that in vivo this reaction and its stereospecificity are controlled by the Ccm system. 

Biological and structural features of CcmF - the cytochrome c heme lyase

 

E. coli CcmF is protein of about 71 kDa that contains four conserved histidine residues exposed to the periplasm as well as a functionally essential tryptophan-rich region called the WXWD motif that is probably involved in heme binding. Based on the presence of this motif, CcmF is a member of the family of putative heme handling proteins (HHP). CcmF is tightly anchored into the cytoplasmic membrane by virtue of its 13 TMHs. Its N-terminus is located in the cytoplasm, while its C-terminus resides in the periplasm according to an experimentally derived topological model employing CcmF-PhoA fusions (9). Moreover, this study also revealed that CcmF contains heme b that is located between its fourth and fifth TMH in an external heme-binding site that is part of the WXWD motif with His261 in TMH5 as axial heme ligand. This heme is not delivered by CcmE but represents a tightly bound cofactor of CcmF in a 1:1 stoichiometry. It was, moreover, proposed that CcmF represents a heme lyase that transfers heme from CcmE to apocytochrome c (10). Bacterial heme lyases are relatively unspecific because they are able to attach heme groups to every CXXCH motif present in a polypeptide chain. Furthermore, pull-down experiments showed that in R. capsulatis and E. coli CcmF is associated with CcmH (9, 11). This finding points towards a CcmF/H complex, which scans apocytochrome proteins to locate the heme-binding motif and initiate heme attachment. 

 

The mechanistic understanding of how heme transport is coupled to the recognition of the CXXCH motif in apocytochrome proteins and subsequent attachment of the heme group is hampered by the lack of detailed structural information on CcmF. It is therefore important to note that recently the structure of CcmF was reported (6). To elucidate this structure, CcmF from Thermus thermophilus was recombinantly produced and subjected to X-ray crystallography. This resulted in the 3D structure of CcmF at 2.7 Å resolution, which is shown in figure 3 in surface (left panel) and ribbon representation (right panel). 

T. thermophilus CcmF is a polytopic membrane protein with 15 TMHs, which is in contrast to earlier predictions that proposed 11-13 TMHs. The N-terminus is located in the periplasm, while the N-terminal half contains different periplasmic loop regions that connect TMH2 and 3 as well as TMH4 and 5. The C-terminal part of the protein contains a large periplasmic domain (shown in purple) that is made up of two antiparallel four stranded ß-sheets. This domain is located in the loop that links TMH14 and 15. 

The TMHs of CcmF for three distinct segments, namely: TMH1-4 are grouped in a four- helix bundle at the front of the protein, whereas TMH5-10 and 11-15 make up a central channel that spans the lipid bilayer but is blocked by a heme group. This is shown in the left and bottom panel of figure 4 with heme in yellow and residues that comprise the heme-binding site shown as blue ball and sticks. This heme group is non-covalently bound to the protein through interactions of the iron ion with histidine 259 of TMH7 (not resolved) and histidine 493 in TMH4 (right panel). Importantly, this heme is not transferred to apocytochrome c but plays a role in electron transfer. Conceivably, this heme might reduce the second heme group that is obtained from CcmE, which is transferred to the apocytochrome c protein. The channel that is formed by TMH5-10 and 11-15 is partly capped at the periplasmic side by loop 6. This contains the WXWD motif (figure 4 right panel) of which both tryptophan residues (238 and 240) face the lumen of the channel, while aspartate 241 is oriented away from the channel. Moreover, TMHs 6, 8 and 14 create a cleft that runs from the channel’s center to the outer leaflet of the membrane, which is large enough to accommodate a second heme group that is delivered by CcmE and is ultimately transferred to apocytochrome c. The role of this cleft in accommodating heme delivered by CcmE is supported by molecular docking simulations.

Summarizing conclusion

 

Heme is one of the most widely used protein cofactors in Nature. It is utilized by all organisms for a variety of biological processes such as electron transfer, binding and sensing of gases, catalysis and gene regulation. Chemically, heme is composed of an aromatic ring-like protoporphyrin IX scaffold with a covalently bound iron atom in its center. The core of the scaffold is made up of four pyrroles that form a large ring structure that is known as a porphyrin (2). The heme iron is coordinated to four nitrogen atoms of the porphyrin moiety. The heme structure contains a proximal and distal position for interaction with amino acid residues of the polypeptide chain. Heme b and heme c are commonly present in proteins but despite their striking similarity, heme b associates non-covalently to the polypeptide backbone, whereas heme c is covalently bound to the protein most often through thioether bonds (2). Proteins that contain heme c are known as c-type cytochromes or cytochromes c and typically function as electron carriers in a variety of photosynthetic and respiratory electron transport chains (1). These proteins contain the characteristic CXXCH heme-binding motif. Five different systems are known that enable the post-translational assembly of cytochrome c proteins in various organism. Of these, the cytochrome maturation (Ccm) system (or system I) represents a complex cytochrome c assembly machinery that is present in Gram-negative bacteria, archaea and plant mitochondria. In Gram-negative bacteria, cytochrome c assembly occurs in the periplasm and requires the proteins of the Ccm operon (CcmABCDEFGH) that are localized to the cytoplasmic membrane (4,5). This process comprises four basics steps. These are: (i) translocation of the apocytochrome across the cytoplasmic membrane into the periplasm, (ii) transport of heme from the cytoplasm towards the periplasm, (iii) reduction and chaperoning of the apocytochrome and finally (iv) formation of thioether bonds between the heme and the cysteine residues of the CXXCH motif (7).). Despite the basic biochemical understanding of bacterial cytochrome c assembly, the precise mechanistic details of how heme transport is coupled to the recognition of the CXXCH motif in apocytochrome proteins and subsequent attachment of the heme group is hampered by the lack of detailed structural information on CcmF. It is therefore important to note that recently the structure of CcmF from Thermus thermophilus was reported (6).  

 

Based on this structure, a model was proposed (figure 5 adapted from 6) explaining how heme is delivered from within the membrane and transferred onto the apocytochrome. This model revolves around the membrane partitioning of heme owing to its hydrophobic porphyrin ring, while the charged propionate side chains will prefer the polar lipid head groups. It has been established that the CcmA/B ABC transporter does not transport heme into the periplasm, although its precise role is not well understood. Possibly, it functions as a flippase to enrich heme in the outer leaflet of the cytoplasmic membrane. The presence of a lateral cleft that runs from the channel to the outer leaflet of the lipid bilayer and is able to accommodate a heme group suggests that heme is inserted into CcmF from within the membrane. CcmF arranges the heme group in such a way that its vinyl groups are accessible for an attack by the cysteine thiols of the heme-binding motif of the apocytochrome c protein. Specifically, CcmF catalyzes first attachment at the second cysteine of the heme-binding motif of the 8-vinyl group. This implies that release from CcmE occurs after this step, meaning that the heme group remains bound to either CcmE or the cytochrome polypeptide chain.

 


References

1.     Kranz RG, Richard-Fogal C, Taylor JS, Frawley ER. 2009. Cytochrome c biogenesis: mechanisms for covalent modifications and trafficking of heme and for heme-iron redox control. Microbiol Mol Biol Rev. 73: 510-528.

2.     Bowman SEJ, Bren KL. 2008. The chemistry and biochemistry of heme c: functional bases for covalent attachment. Nat Prod Rep. 25: 1118-1130.

3.     Methé BA, Nelson KE, Eisen JA, Paulsen IT. et al. 2003. Genome of Geobacter sulfurreducens: metal reduction in subsurface environments. Science. 302: 1967-1969.

4.     Stevens JM, Mavridou DAI, Hamer R, Kritsiligkou P, Goddard AD, Ferguson SJ. 2011. Cytochrome c biogenesis System I. FEBS J. 278: 4170-4178.

5.     Sanders C, Turkarslan S, Lee CW, Daldal F. 2010. Cytochrome c biogenesis: the Ccm system. Trends Microbiol. 18: 266-274.

6.     Brausemann A, Zhang L Ilcu L, Einsle O. 2021. Architecture of the membrane-bound cytochrome c heme lyase CcmF. Nat Chem Biol. 17: 800-805.

7.     Mavridou DA, Ferguson SJ, Stevens JM. 2013. Cytochrome c assembly. IUBMB Life. 65(3): 209-216.

8.     Sanders C, Deshmukh M, Astor D, Kranz RG, Daldal F. 2005. Overproduction of CcmG and CcmFH (Rc) fully suppresses the c-type cytochrome biogenesis defect of Rhodobacter capsulatus CcmI-null mutants. J Bacteriol. 187(12):4245-4256.

9.     Richard-Fogal CL, Frawley ER, Bonner ER, Zhu H, San Francisco B, Kranz RG. 2009. A conserved haem redox and trafficking pathway for cofactor attachment. EMBO J. 28(16):2349-2359.

10.  Ren Q, Ahuja U, Thöny-Meyer L. 2002. A bacterial cytochrome c heme lyase. CcmF forms a complex with the heme chaperone CcmE and CcmH but not with apocytochrome c. J Biol Chem. 277(10):7657-7663.

 

11.  Sanders C, Turkarslan S, Lee DW, Onder O, Kranz RG, Daldal F. 2008. The cytochrome c maturation components CcmF, CcmH, and CcmI form a membrane-integral multisubunit heme ligation complex. J Biol Chem. 283(44):29715-29722.

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