Heme Attachment To Cytochrome C

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b1243 Handbook of Porphyrin Science
93 Heme Attachment
to Cytochromes c
Julie M. Stevens and Stuart J. Ferguson
Department of Biochemistry, University of Oxford, South Parks Road,

by MASSACHUSETTS INSTITUTE OF TECHNOLOGY on 05/23/13. For personal use only.
Oxford, OX1 3QU, UK

Handbook of Porphyrin Science Downloaded from www.worldscientific.com
List of Abbreviations 372

I. Cytochromes c 372
A. Structures and Functions 372
B. Why is Heme Covalently Attached to c-Type Cytochromes? 375
C. Insights from Spontaneous Heme Attachment to
Cytochromes 376
II. The Diversity of Heme Attachment Pathways 379
III. System I 379
A. Distribution 379
B. Heme Handling Components 381
C. The Heme Chaperone CcmE 381
D. The Heme Attachment Reaction to Cytochromes c 384
E. Redox Control and Chaperoning of the Apocytochrome 385
F. Substrate Specificity 386
G. Variations of the System I Pathway 387
IV. System II 388
A. Distribution 388
B. Redox Control 388
C. Heme Transport and Attachment 389
D. Substrate Specificity 390
E. Variations of the System II Pathway 390
V. System III 391
A. Distribution 391
B. HCCS and Mitochondrial Cytochrome c Import 391
C. HCCS Function 392
D. Substrate Specificity 393
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372 Stevens and Ferguson
VI. System IV 394

A. Distribution 394
B. Components and Interactions 395
VII. Conclusion and Perspectives 395
VIII. Acknowledgments 396
IX. References 397
List of Abbreviations
ABC ATP-binding cassette

CCB cofactor assembly on complex C subunit B
Ccm cytochrome c maturation

HCCS holocytochrome c synthase
IMS intermembrane space
Nrf periplasmic nitrite reductase with ammonium as reaction product
TPR tetratricopeptide repeat
I. Cytochromes c
A. Structures and Functions
The protein commonly called cytochrome c refers to the heme-containing mito-
chondrial protein that has been studied for over 70 years1 and has a vital role in
respiration. The importance of the protein in the electron transport chain, its
relative abundance in some tissues and its stability and simplicity have made it a
model protein for a myriad of studies. The fields of protein folding, electron
transfer, hemoprotein spectroscopies, NMR and crystallography have focused
on studying the properties and function of this protein. There is renewal of interest
in the protein,2 particularly in the context of apoptosis, human disease and aging.3
The defining feature of cytochrome c is the covalent attachment of the vinyl
groups of heme to two cysteine thiols in a CXXCH motif in the apocytochrome
protein. In some unusual cases the heme is linked to the protein via a single
covalent bond.4–6 Unmodified b-type heme and the characteristic thioether bonds
of c-type heme are shown in Figure 1. The α-carbons of the heme vinyl groups
form bonds with the cysteine sulfurs. The histidine side chain of the CXXCH motif
provides one of the heme iron axial ligands as indicated in the early crystal struc-
ture of a monoheme cytochrome c from the bacterium Paracoccus denitrificans
(Figure 2A),7 which has a similar fold to mitochondrial cytochrome c. In this case
the other iron ligand is a methionine side chain. Mitochondrial cytochrome c is a
low molecular weight soluble protein with a single heme molecule. It has an
93/Heme Attachment to Cytochromes c 373

Figure 1. The structures of (A) b-heme (Fe-protoporphyrin IX) as it occurs in b-type

cytochromes and (B) c-heme, indicating the two thioether bonds formed between the 2- and
4-vinyl groups (Fischer nomenclature; 3- and 8-vinyl groups using IUPAC nomenclature) of
the heme and the two cysteine thiols on the protein (indicated by the line between the
cysteines). The 2-vinyl position is always bound to the N-terminal of the two cysteines in the
classical CXXCH cytochrome c motif, and the 4-vinyl to the C-terminal cysteine.

Figure 2. (A) A monoheme cytochrome c (cytochrome c550 from Paracoccus denitrificans,7

PDB accession 155C) that has a typical mitochondrial cytochrome c fold. The heme is shown
in red, the protein in green and the two ligands to the heme iron (a histidine from the CXXCH
motif, and a methionine residue) are shown in blue. (B) A multiheme cytochrome c. MtrF
(PDB accession 3PMQ10) is a decaheme c-type cytochrome functioning at a bacterial cell
surface as an electron transfer conduit. The 10 covalently bound hemes are shown in red.
(C) The nitrite reductase NrfA (the monomer is shown, PDB 1QDB)100 with the five hemes
indicated in red. The unusual active-site heme, that has a lysine coordinating the heme iron,
is shown in blue. All structures were rendered in Pymol (DeLano, W.L. The Pymol Molecular
Graphics System (2002) http://www.pymol.org).
essential function in the cell to shuttle electrons, in the mitochondrial intermem-

brane space, between the cytochrome bc1 complex (Complex III) and cytochrome
c oxidase as part of the respiratory chain. Cytochrome c1 is the second of the two
c-type cytochromes that function in the mitochondrial electron transfer pathway;
its globular domain has a similar fold to cytochrome c itself but c1 has an anchor-
ing transmembrane helix. Cytochrome c1 has an integral function in Complex III
in transferring electrons from the Rieske FeS protein to cytochrome c. This essen-
tial life-sustaining process of respiration contrasts with the role of cytochrome c
in apoptosis, where, on release from the mitochondrion, it has a role in triggering
formation of the apoptosome and activation of the signaling cascade leading to

cell death.8
Cytochromes c similar to the mitochondrial protein occur in many species of
bacteria, many of which contain the cytochrome bc1 complex. Besides the previ-
ously mentioned Paracoccus denitrificans, a well-known example is the photosyn-
thetic organism Rhodobacter sphaeroides in which cytochrome c2 (the subscript 2
is a legacy of a time when only a few c-type cytochromes where known) acts as an
electron shuttle analogous to mitochondrial cytochrome c. However, bacteria
employ cytochromes c in a diverse set of roles compared with the two mitochon-
drial proteins. The greater diversity of function is partly related to the ability of bac-
teria to attach multiple hemes to a single polypeptide chain, often resulting in a very
high density of the cofactor relative to the protein. Some bacteria contain very large
numbers of c-type cytochromes, many of which contain surprisingly large numbers
of heme molecules, and in which the polypeptide fold is very different from that in
mitochondrial cytochromes c. Interest in the genomes of bacteria that reduce metals
(in particular radionuclides) has revealed genes for many multiheme c-type
cytochromes. Their role in Geobacter, for example,9 has been suggested to provide
redundancy and flexibility in respiratory electron transfer chains that allow the
organisms to live in varied environmental conditions. The structure of a multiheme
cytochrome c from Shewanella oneidensis with 10 covalently bound heme groups
is shown in Figure 2B.10 The function of many hemes on a polypeptide appears to
be to optimize electron transfer and in some cases to harvest electrons11 or shuttle
electrons out of the cell as “nanowires.”10 There are a variety of other protein folds
found amongst the c-type cytochromes in bacteria. The majority of cytochromes c
function in electron transfer although a variety of other functions in catalysis and
sensing of gases (NO and CO) are known.12 A c-type cytochrome also functions in
plant photosynthesis; this is cytochrome f which is actually a c-type cytochrome,
albeit one with a unique, for a c-type cytochrome, β-sheet structure.13
B. Why is Heme Covalently Attached to c-Type Cytochromes?

The question of why heme is covalently attached in c-type cytochromes has been
considered extensively.12,14 Some change in heme reduction potential in the pres-
ence of the two covalent bonds has been measured, as well as increased thermo-
dynamic stability of the protein.12,15 Heme retention may have been an
evolutionary driving force in the prokaryotic mitochondrial ancestors, where com-
petition for iron in heme meant that covalent heme-binding conferred an advan-
tage. The most compelling reason for the appearance and conservation of covalent
porphyrin attachment is the packing of multiple heme molecules into bacterial
multiheme cytochromes. The high density of heme packing would be difficult to
achieve if the hemes were non-covalently bound as the overall protein fold is
dependent on the presence of the hemes; in other words the apoprotein could not
fold by itself to provide nascent pockets for heme. The covalent attachment also
allows strict control of heme stereochemistry and orientation, which is seemingly
vital to the function of the cytochromes c.
It has long been known that the mitochondrial c-type cytochromes of
trypanosomes and related organisms have only one thioether bond.4 The crystal
structure of one such cytochrome, from Crithidia fasciculata, has now been deter-
mined.6 It is clear that the absence of one thioether bond has very little conse-
quence for the structure, which is consistent with earlier observations that the
oxidation and reduction potential, along with stability, were not markedly affected
by the loss of a thioether bond. The same conclusion can be drawn from studies of
a c-type cytochrome that assembles spontaneously in the cytoplasm of E. coli. In
this case both single-cysteine variants, AXXCH and CXXAH could be made,16
whereas in the trypanosomes only the former variant is known. It is far from clear
why the single bond attachment has emerged in trypanosomes; there are no anal-
ogous proteins yet identified in bacteria. In the case of monoheme c-type
cytochromes, the question can be reversed to ask why two thioether bonds are
required. It is hard to give an answer but in the many multiheme c-type
cytochrome found in bacteria two thioether bonds may be advantageous. As noted
above, in many of these cytochromes there is a high ratio of heme to polypeptide
chain and the precise packing of heme, and stereochemical control of binding,
may not be possible with only one thioether bond per heme. It is presumably
sensible to have only one biogenesis system per cell type for making c-type
cytochromes and so it can be argued that all c-type cytochromes of any cell type
therefore have two thioether bonds (chloroplasts are an exception to this,
see below).
C. Insights from Spontaneous Heme Attachment to Cytochromes

As discussed below, physiological c-type cytochrome formation in cells is a cat-
alyzed process. However, there are examples (both in vivo and in vitro) of non-
catalyzed c-type cytochrome formation. Some examples are described here.
Expression of the apoform of Hydrogenobacter thermophilus cytochrome c552 in
the cytoplasm of E. coli surprisingly produced a holocytochrome c with the cor-
rect attachment of heme.17,18 When the two cysteines were converted into alanine,
a b-type cytochrome was obtained. This system then allowed a comparison of
stabilities and other properties between cytochrome with covalently and non-
covalently bound heme.15,19 The observations were interpreted as reflecting the
formation of an apoform with sufficient structure to bind heme stereospecifically
such that a proximity effect would ensure the formation of the thioether bonds.
NMR experiments have demonstrated that the apocytochrome form of the b-type
variant of H. thermophilus cytochrome c552 has a compact fold that is able to bind
heme, in contrast with mitochondrial cytochromes c which in their apoforms are
random coils.20 This view was subsequently supported by the finding that the wild-
type apocytochrome c552 protein could bind heme in vitro to give a spectrum
initially characteristic of a b-type cytochrome and then, over time, to a c-type pro-
viding the reaction conditions were reducing.21 Variation of pH conditions for
in vitro reaction of heme with apocytochrome c gave modest support for a reac-
tion scheme in which the vinyl group might be protonated before attack by the
cysteine sulfur. Subsequently, it was shown that the apoform of cytochrome c555
from Aquifex aeolicus (with its cysteines replaced with alanines) adopts a similar
folded structure as the holoprotein and was able to bind heme.22 The interpretation
of the results showing spontaneous heme attachment to thermostable cytochromes
is that in the reducing environment of E. coli cytoplasm the heme either enters the pre-
formed heme binding site (Aquifex) performed heme binding site or H. thermophilus
induces the formation of the site from a precursor conformation. The binding is
stereospecific such that a proximity effect is sufficient to result in the formation of
the thioether bonds. If only one cysteine is present then a single thioether bonded
variant is obtained.16 However, it must be stressed that this is exceptional behav-
ior and in general spontaneous correct attachment of heme will not occur. Thus,
for example the cytochrome c552 from Thermus thermophilus does not acquire
homogeneous and correct heme attachment when produced spontaneously in the
E. coli cytoplasm. Oxidation of the heme 2-vinyl to a formyl group was observed
in one product23 and rotation of the heme group by 180° with covalent bond for-
mation to the incorrect vinyl group has also been described.24 Advantage has been
taken of the failure of heme to attach properly to the Thermus protein to obtain
apoprotein with a single cysteine.25
In the cases of certain other c-type cytochromes, including the horse heart
mitochondrial protein, some formation of the physiological product was detected
in vitro following incubation of apoprotein with heme.26 In vitro H. thermophilus
cytochrome c reconstitution with heme derivatives lacking either of the vinyl
groups (2-vinyldeuteroheme and 4-vinyldeuteroheme) and the single-cysteine
containing variants of the apocytochrome proteins showed that the correct prod-
ucts were formed.27 The N-terminal of the two cysteines reacted predominantly
with the heme 2-vinyl group and the C-terminal cysteine with the 4-vinyl group.
It is the case that the apoproteins of cytochromes c can be converted by muta-
genesis to more stable forms that are largely folded.28 Presumably this might be
done for any mono heme c-type cytochrome protein which might then react spon-
taneously with heme. Evolution has not followed either this route or that of
making a very stable b-type cytochrome to perform the roles of mitochondrial

cytochromes c and c1. The shift of Eo′ in the H. thermophilus cytochrome c552
(245 mV) to a value of 170 mV in the b-type analog is significant, but it is diffi-
cult to imagine that the tuning of the polypeptide chain could not, in principle,
restore the latter value to the former.
Experiments with metalloporphyrins other than heme have also given insight
into the requirements for thioether bond formation in c-type cytochromes. In vitro
formation of cytochrome c using the Hydrogenobacter c552 wild-type apocy-
tochrome demonstrated that Zn-protoporphyrin IX was readily incorporated.29
This added valuable support for the idea that it is the 2+ oxidation state of Fe in
heme that is needed for thioether bond formation. On the other hand, other transi-
tion metals, irrespective of oxidation state did not result in homogeneous products.
It has been possible to incorporate various metal-substituted protoporphyrins (e.g.
Cu and Co) into c-type cytochromes for various applications.30,31
It is also possible to form thioether bonds in proteins that are not themselves
c-type cytochromes. The earliest study of this kind was with a single-cysteine vari-
ant of cytochrome b5 but a variety of products was obtained.32 Later a double cys-
teine variation of cytochrome b5 was studied.33 The original work led to the
conversion of the four-helix bundle E. coil protein cytochrome b562 into a c-type
cytochrome by introduction of two cysteine residues in appropriate positions for
attachment to the heme vinyl groups.34 Mixed products have been obtained includ-
ing a verdoheme derivative.35 When the c-type variant of b562 is directed to the
periplasm of E. coli it is acted upon by the biogenesis system producing a bona fide
c-type cytochrome.36 On the other hand, it has been reported that a natural c-type
cytochrome with a four helix bundle structure assembles spontaneously in the
periplasm and intriguingly its formation is inhibited by the co-expression of the
Ccm (see below) biogenesis system.37 A similar effect has been noted in studies of
cysteine-containing variants of cytochrome b562.36
Another example of reductive formation of a thioether bond occurs in an
engineered variant of ascorbate peroxidase.38 Normally this protein has non-
covalently bound heme at its active site. However, it could be predicted from the
crystal structure that if a serine residue was changed to cysteine then the
proximity of the side chain sulfur to the vinyl group might result in spontaneous
formation of a thioether bond. Such a prediction was realized, provided incuba-
tion was under reducing conditions.38 This was important because if the serine
was changed to methionine then oxidative conditions, but not reductive condi-
tions, resulted in the formation of a covalent bond to heme, a type of bond that
occurs naturally in some peroxidases.39 These observations show that proximity
itself is not sufficient for covalent bond formation; the correct redox conditions
are also needed.
Overall the study of spontaneous formation of c-type cytochromes, whether

spontaneous in the cytoplasm, or in vitro, suggests that formation in vivo requires
heme to be in the ferrous state. This is in agreement with earlier studies that sug-
gested that cytochrome c biogenesis catalyzed by mitochondrial extracts also
required reductant. However, the rate of spontaneous thioether bond is rather slow
and thus it is unlikely that the need for a catalyst can ever be avoided in vivo, espe-
cially as the vinyl groups of heme are not intrinsically very reactive.
II. The Diversity of Heme Attachment Pathways

In cells the attachment of heme to the apoforms of cytochromes c is a protein-

catalyzed process. The reaction follows the transport of the apocytochrome to the
compartment where the holocytochrome functions (the mitochondrial intermem-
brane space, the extracytoplasmic space of bacteria (the periplasm in Gram nega-
tive bacteria) and the chloroplast lumen and stroma). The transport of heme to
these compartments also needs to occur, as heme biosynthesis is completed else-
where in each case (with one exception in this regard; see below). Unexpectedly,
very different post-translational modification systems, of varying complexity,
occur in these different compartments for the heme attachment reactions. The var-
ious pathways have come to be known as Systems I, II, III and IV with an as yet
unidentified system in the trypanosomatids which, as mentioned earlier, have an
unusual heme attachment via a single thioether bond to the cytochrome.40–43 The
latter putative cytochrome c biogenesis apparatus is known as System V.44
What is known about the composition and function of these systems will be
described below in more detail. The complexity of the distribution of cytochrome
c biogenesis systems is exemplified in plants, where System I occurs in mito-
chondria and Systems II and IV both function in plastids, on opposite sides of the
membrane. The evolutionary origins of the different systems are not clear, espe-
cially given their distribution pattern45 and very varied complexity. All the systems
perform the same chemical reaction and yield products with highly conserved, in
stereochemical terms, attachments of the porphyrin to the protein.
III. System I
A. Distribution
The most complex cytochrome c biogenesis pathway is also known as the Ccm sys-
tem (cytochrome c maturation). In Escherichia coli the system comprises the pro-
teins CcmABCDEFGH that are located in the cytoplasmic membrane and perform
the heme attachment reaction in the periplasm, as shown in Figure 3. System I is

Figure 3. Cytochrome c maturation (Ccm) System I. The Ccm proteins (illustrated here as found
in E. coli) produce c-type cytochromes in many Gram-negative bacteria and in the mitochondria
of plants and some protozoa. CcmA hydrolyses ATP on the cytoplasmic side of the cytoplasmic
membrane (in grey) and along with the membrane proteins CcmB, C and D assists in the func-
tion of the heme chaperone CcmE in binding and releasing heme in the periplasm. The heme
transfer reaction from CcmE to apocytochromes (shown in red) depends on the protein CcmF.
Provision of the apocytochrome in a form suitable for heme attachment (with the cysteine thiols
reduced) is generally believed (uncertanities remain) to be performed by the thiol oxidoreductases
CcmG and the N-terminal domain of CcmH (N-CcmH); their cysteine residues are shown in
yellow. The C-terminal domain of CcmH (C-CcmH) occurs as a separate polypeptide in many
organisms (called CcmI) and is suggested to function as a chaperone for apocytochrome proteins.
The structure shown here for C-CcmH is that of the protein NrfG, which is expected to be similar
in structure. The reductant needed to reduce the cysteines in the apocytochrome is transported
from the cytoplasm by the transmembrane protein DsbD (not shown). It is not known how heme
is transported from its cytoplasmic site of synthesis to the periplasm, as indicated by the ques-
tion mark. The PDB accession codes for the structures of the soluble domains are: CcmE, 1LIZ;59
N-CcmH, 2HL7;77 NrfG (as paralog of C-CcmH), 2E2E;87 CcmG, 2B1K.154
found in many species of Gram-negative bacteria, plant mitochondria and some

protozoal mitochondria.45–47 The proteins occur in complexes and function in heme
provision and preparation of the apocytochrome for heme attachment, which can
involve reversing by reduction any disulfide bond formation between the cysteine
residues in the CXXCH heme-binding motif. The mechanism of the final step of
heme attachment is not known. The majority of what is known about the Ccm pro-
teins comes from studies in E. coli and Rhodobacter capsulatus; some complemen-
tary information comes from studies on the plant Arabidopsis thaliana. The Ccm
system is the most intensively studied of the cytochrome c biogenesis pathways.
B. Heme Handling Components

The proteins CcmA and CcmB form a complex in the cytoplasmic membrane,
with CcmA on the cytoplasmic side, forming an ABC-protein (ATP-binding
cassette).48 Different stoichiometries of the ABC protein formed by the Ccm com-
ponents have been suggested, specifically CcmA2B2 and CcmA2BC.48,49 Evidence
has been presented showing that CcmC functions independently of CcmAB,50,51
suggesting that CcmC, although it interacts with CcmAB, does not form an ABC
protein with them. Although proteins in the ABC family are usually transporters,
there is no evidence for a transport function in this case. ATP hydrolysis activity
has been shown48,52 but there is evidence that CcmAB is not involved in
heme transport.53 It appears that energy from ATP hydrolysis is somehow used in
the function of the heme chaperone CcmE.48,49 CcmE binds heme in the periplasm
via an unusual covalent bond between a histidine residue in the protein and a vinyl
group on the porphyrin.54 Importantly, heme is still attached to CcmE when the
ATPase activity of CcmAB is blocked,49 an observation that provides good evi-
dence against the notion of a heme transport function for CcmAB. The heme is
provided by the membrane protein CcmC50 which is associated with CcmAB.49
CcmC has been shown to bind heme; it has a pair of conserved histidine residues
that are essential for its function of heme transfer to CcmE50 and are thought to lig-
ate heme on the periplasmic face of the cytoplasmic membrane.55 CcmC also dis-
plays a tryptophan-rich motif (WWD motif) that is also found in other
heme-handling proteins in both Systems I and II, and is thought to interact directly
with the heme.55 Heme biosynthesis occurs in the cytoplasm and it is not known
how the heme is transported across the cytoplasmic membrane to the periplasm.
There is no evidence to support the otherwise appealing idea that CcmC might be
involved in the transport. Following transfer of heme from CcmC to CcmE, the
heme is passed on to an apocytochrome c in an ATP-dependent process; the latter
dependency is deduced from the failure of this step when the ATPase activity of
CcmAB has been blocked by mutation.49 CcmD, a low molecular weight mem-
brane protein, is essential for unknown reasons for the interaction of CcmABC
and CcmE; the details of its function have not been agreed upon.56,57
C. The Heme Chaperone CcmE

The structures of apoCcmE proteins (i.e. without heme bound) from E. coli and
Shewanella putrefaciens have been solved by NMR spectroscopy.58,59 Attempts to
solve the structure of the holoprotein have failed. ApoCcmE has a so-called oligo-
binding (OB) fold with a compact core consisting of β-strands with both termini
observed to be largely unstructured. The structure of the protein from E. coli is
shown in Figure 4B. The structure does not show an obvious heme-binding
pocket. It has been possible to model a heme-binding site on to the surface of the
protein guided by mutagenesis experiments.59,60 The binding site consists of a
hydrophobic patch on the core of the protein and places a heme vinyl group close
to the histidine known to form the covalent bond to heme. The histidine is located
near the unstructured C-terminal domain which has prompted studies of this
region of the protein.61,62 While removal of the C-terminal domain affected the
in vitro heme ligation properties of the protein, the truncated protein retained some
ability to bind and transfer heme in vivo. Resonance Raman studies of CcmE with
covalently and non-covalently bound heme showed that the ligands to the heme
vary depending on the oxidation state of the iron,63,64 suggesting that ligand
switching may form part of the function of the protein in the cell. Further experi-
ments designed to characterize the heme-binding site of CcmE involved insertion
of alanine residues around the histidine residue; these had a dramatic effect on
heme-binding, in contrast with amino acid replacements in the same positions.65
These results suggest the existence of a well-defined heme-binding site where the
relative position of the histidine is crucial. A full description of how heme is bound
to CcmE will depend on structure determination of the holoprotein.
The heme-histidine bond in CcmE has been investigated by NMR analysis of a
peptide derived from the holoprotein produced in vivo.66 The nature of the bond,
involving the Nδ1 of the histidine and the β-carbon of a heme vinyl group (shown in
Figure 4A), does not obviously suggest how this bond would be broken and allow
onward heme transfer to an apocytochrome c. It is hard to see how an attack on the
α-carbon by a cysteine could be linked to the loss of a hydride from that carbon and
effective transfer of that hydride to the β-carbon with concomitant displacement of
the histidine. As an alternative it has been suggested that the histidine departs via a
reverse of the Michael-type addition reaction through which it was presumably
formed.47 Why a heme chaperone forms a transient covalent bond in the first place
is not clear, though stereochemical control would seem an obvious possible justifi-
cation. Although the heme-protein bond in CcmE is unique, another example of a
histidine-heme bond has been described. Cyanobacterial hemoglobins have been
found that form covalent bonds between the 2-vinyl group of heme and a histidine
side chain.67 The bond is different from that seen in CcmE because the α-carbon of
the heme vinyl group is involved and the Nε 2 of the histidine side chain. Experiments
to examine the mechanism of heme attachment to the hemoglobins support an
electrophilic addition reaction with protonation of the heme vinyl group being rate-
limiting,68 which may be a similarity to in vitro cytochrome c formation.21
Spectroscopic experiments have implicated Y134 (in the E. coli protein) in
ligation of the heme iron in purified holoCcmE.63,69 However, replacement of the
tyrosine residue does not appear to affect the function of the protein in cytochrome

Figure 4. (A) The covalent attachment of heme to the heme chaperone CcmE. The Nδ1 of the
side chain of histidine 130 (E. coli numbering) is attached to the β-carbon of the 2-vinyl group
of heme.66 The NMR structure did not confirm which of the two heme vinyl groups were
modified, though spectroscopic data have implicated the 2-vinyl group.63 (B) The solution
structure of E. coli apoCcmE (PDB 1LIZ)59 showing the location of heme-binding histidine His
130. (C) The solution structure of Desulfovibrio vulgaris apoCcmE (PDB 2KCT) showing the
cysteine residue that covalently binds heme in System I*.73 The structures were rendered in
Pymol (DeLano, W.L. The Pymol Molecular Graphics System (2002) http://www.pymol.org).
c maturation in either the heme binding or transfer reactions.65 The characterization

of a complex of CcmC, heme and CcmE, in which the heme is ligated by histidine
residues on CcmC,55 could indicate that the physiological CcmE heme-binding
site is completed by CcmC and explain why Y134 is not needed in vivo. It has,
however, been shown that purified holoCcmE is able to transfer heme to an apocy-
tochrome c spontaneously in vitro under certain conditions.70 Both the heme and
the apocytochrome need to be reduced for the heme transfer reaction to occur. The
mechanism of heme binding and release by CcmE remain to be fully elucidated.
It has been possible to attach heme to apoCcmE in vitro provided reducing condi-
tions are used.70 However, the exact nature of the heme-histidine bond formed in
this case has not been established.
A further development has indicated that an intermediate step in c-type
cytochrome synthesis is the formation of a species in which a nascent c-type
cytochrome has formed one thioether bond to heme that is still covalently attached
via histidine to CcmE.71 This putative intermediate was detected by expressing a
form of cytochrome b562 that has one cysteine in the heme-binding pocket. Recall
that it has been shown that variants of cytochrome b562 with a CXXCH motif can
become a c-type cytochrome with heme attached by the Ccm apparatus.36 The Ccm
system makes at best very small amounts of variant c-type cytochromes with only
one cysteine.72,73 Cytochrome b562 and its c-type variants are expressed at relatively
higher levels than most bona fide c-type cytochromes in E. coli; additionally the
apoforms of cytochrome b562 and its variants are stable in the periplasm whereas
most apocytochromes are not. Expression of a single-cysteine variant of b562, along-
side elevated expression of the Ccm proteins, allowed detection and characteriza-
tion of a species that contained covalently linked heme and had a mass consistent
with the sum of the cytochrome plus CcmE. Peptide mapping of this complex
showed that the two protein molecules were joined together by heme linked to his-
tidine 130 of CcmE and the introduced cysteine in the cytochrome b562. Formation
of this species was attenuated in a predictable way if inactive variants of CcmA or
CcmC were present. Thus loss of ATPase activity in CcmAB or of key histidines,
believed to be important in heme handling, in CcmC abolished formation of the
complex. On the other hand, loss of CcmF enhanced the levels of the CcmE-heme-
cytochrome complex. Loss of ATPase activity of CcmAB has no effect on heme
attachment to CcmE but stops its onward movement to apocytochromes c. Thus
this ATPase activity is needed to make the heme on CcmE available to the mono-
cysteine cytochrome. The fact that this complex can form without CcmF suggests
that any role, see later, for the latter in keeping heme reduced is apparently not
needed for the attachment of heme on CcmE to cysteine of a cytochrome. It follows
that any electron donation role for the heme in ubiquinone in CcmF (see below)
may be related to the breakage of the histidine to heme bond.
D. The Heme Attachment Reaction to Cytochromes c

The heme transfer reaction from CcmE to apocytochrome depends on the protein
CcmF, a large membrane protein (with many transmembrane helices) that has
been found to bind heme itself.74 It also possesses some tryptophan residues in a
predicted periplasmic loop that could be involved in assisting heme transfer to the
apocytochrome c. The heme bound to CcmF may function in providing electrons,
possibly derived from ubiquinol in the membrane, to maintain the heme on CcmE
that is to be transferred to apocytochromes in the reduced form. The heme bound
to CcmF has been reported to be a hexacoordinated low-spin heme with a mid-
point reduction potential of −147 mV.75 Heme is synthesized in the ferrous form in
the cytoplasm but the oxidizing environment of the periplasm could make it nec-
essary to have a source of heme reductant. There is general acceptance of a require-
ment of ferrous heme for the formation of the thioether bonds, as demonstrated by
the uncatalyzed formation of a c-type cytochrome in vitro (see earlier).21
E. Redox Control and Chaperoning of the Apocytochrome

CcmG and CcmH are involved in thiol-disulfide oxidoreductase functions and

reverse the oxidation of the heme-binding cysteines in the apocytochrome
CXXCH to disulfides, a reaction that is known to occur in the oxidizing environ-
ment of the periplasm. The protein DsbA is a powerful periplasmic oxidase that
functions in introducing disulfide bonds into a range of extracytoplasmic
proteins.76 The role of CcmH, which has two domains and occurs as two separate
proteins (called CcmH and CcmI) in most organisms, appears to be more complex
than disulfide handling and may have a chaperone-like function.77,78 The reducing
power required for reduction of the CXXCH motif in the apocytochrome is pro-
vided by the proteins DsbD or CcdA, which transport reductant from thioredoxin
in the cytoplasm across the cytoplasmic membrane via disulfide cascades.79 DsbD
is a membrane protein with two soluble periplasmic domains. The C-terminal
domain has a thioredoxin fold and transfers reductant from the transmembrane
domain to the N-terminal domain, which in turn continues the disulfide cascade
by interacting with various periplasmic partners.80 The N-terminal domain of
DsbD interacts with CcmG to provide reductant to the Ccm system.81
Structural information for the Ccm proteins is entirely lacking for the membrane-
associated components (see Figure 3). Structures of the soluble domains of CcmG,82
CcmH77 and DsbD83 have given insight into how these proteins are involved in the
processes of handling the apocytochrome and transfer of reducing power. CcmH
from Pseudomonas aeruginosa (corresponding to N-CcmH in E. coli) has a three-
helix bundle structure77 contrasting with the thioredoxin fold usually found for thiol-
disulfide oxidoreductases. The structure of the E. coli homolog of this domain
showed a dimeric structure in which the solvent exposure of the cysteine residues was
affected; the physiological significance of the dimerization is not yet clear.84 The role
of N-CcmH as a thiol-disulfide oxidoreductase is not straightforward as there is evi-
dence that the cysteine residues are not essential for function under anaerobic condi-
tions,85,86 whereas the protein itself is essential. CcmG, however, has a typical
thioredoxin fold with an acidic active-site that is thought to contribute to its substrate
specificity.82 Whether CcmG or CcmH transfers reductant to oxidized apocy-

tochromes remains to be established but two-hybrid studies with the A. thaliana
proteins46 support interaction of N–CcmH with the apocytochrome.
The CcmI protein (the C-terminal region of CcmH in E. coli and some other
organisms) can be considered as comprising two parts: an N-terminal region
which has two transmembrane helices with a linking cytoplasmic loop which con-
tains a leucine-zipper motif and a C-terminal region which has three tetratri-
copeptide (TPR) repeats. A structure of a CcmI analog, NrfG, has confirmed the
presence of the characteristic TPR structure.87 TPR motifs are widely employed as
facilitators of protein–protein interactions.88 CcmI is expected to have a similar

structure; the NrfG structure is shown in Figure 3. There have been mixed reports
about the requirement for CcmI. A knock-out of the ccmI gene (at the time con-
fusingly called cycH) of P. denitrificans resulted in no detectable soluble c-type
cytochromes but some 5–10% residual levels of certain membrane-bound
cytochromes.89 It was suggested that CcmI might be an assembly factor, as
opposed to being directly involved in heme attachment to polypeptide. In R. cap-
sulatus, the absence of the entire CcmI resulted in no c-type cytochromes but the
presence of the N-terminal region did allow the formation of a c-type
cytochrome.90 On the other hand, in E. coli the loss of the entire CcmI-like part of
CcmH does not abolish formation of soluble heterologous c-type cytochromes.85
It has been shown that apocytochrome c2 from R. capsulatus can interact with
CcmI whereas the holoform of the cytochrome does not.78 This is consistent with
chaperone-type role. Overall, it is reasonable to conclude at this stage of under-
standing that CcmI aids, and thus provides optimal performance, but is not
absolutely essential for operation of the Ccm system in several organisms. The
necessity for CcmI could depend on the properties of the substrate cytochrome.
F. Substrate Specificity
System I has broad substrate specificity towards both diverse endogenous
cytochromes c and cytochromes from other organisms. The Ccm proteins are the
system of choice for heterologous cytochrome c overexpression, using a plasmid
encoding the E. coli ccm operon;91 the other systems are also used.92,93 There is
strong evidence that all that is recognized for heme attachment is the CXXCH motif,
conferring versatility on the system in terms of substrate recognition. It was demon-
strated that the Ccm proteins could covalently attach heme to a peptide of only 12
amino acids containing a CXXCH motif.94 The close spacing, in some cases, of the
CXXCH motifs in multiheme cytochromes c also suggests that no more than local
primary CXXCH sequence is needed for recognition and heme attachment. The
Ccm system does not, however, mature cytochromes c lacking the heme-ligating
histidine and those with single-cysteine heme-binding motifs are very marginally
processed.95 It has been found that modification of the CXXC motif of the soluble
C-terminal thioredoxin-like domain of DsbD to CXXCH (in other words incorpo-
rating a heme-binding motif into a non-cytochrome protein) resulted in low but
detectable levels of heme attachment.96 It could be that the redox properties of the
cysteine pair in the motif affect the level and rate of heme attachment in the
periplasm; rapid folding of a protein when it is delivered across the membrane could
protect the motif against heme attachment by the Ccm proteins. An effect of this
kind could explain why some CcmG proteins are not c-type cytochromes despite
possessing a CXXCH motif. Insight into substrate recognition by the Ccm system
has also come from examining the processing of heme-binding motifs with varied
numbers of residues between the cysteines. In variants of cytochrome b562 contain-
ing two heme-binding cysteines, but with five or six residues between them (instead
of the usual two), it was found that the Ccm system had attached heme covalently
to the motif but that an additional sulfur had been incorporated into the heme-protein
linkage.97 An understanding of the origin of these unexpected persulfide linkages
will potentially give insight into the heme attachment reaction.
G. Variations of the System I Pathway

Variants of the classical Ccm system have been identified and studied. These
include a set of proteins (called System I*) found in archaea and some bacteria
that have CcmE proteins employing a transient covalent bond from heme to cys-
teine, as opposed to the E. coli histidine.73 A CcmI-like protein associated with
System I* enhances, but is not required for, c-type cytochrome production, at least
when functioning heterologously in E. coli. System I* can function without a pro-
tein corresponding to the N-terminal domain of CcmH in E. coli, which is essen-
tial for cytochrome c maturation in E. coli.85 The implications of these surprising
variations remain to be determined. Of particular intrigue is the cysteine that cova-
lently binds to heme in the CcmE protein of System I* (CcmE*). This cysteine is
essential for formation of c-type cytochromes and cannot be substituted by the his-
tidine found in System I73; nor can CcmE of System I tolerate a cysteine in place of
histidine.60 Covalent bond formation between the cysteine of CcmE of System I*
and heme has been demonstrated, but it is not known whether the bond formed is a
thioether bond, and if so whether the bond involves reaction between the α- or β-
carbons of the vinyl group. In either case breakage of a thioether bond to be replaced
by the thioether bond of the c-type cytochrome would be a novel kind of reaction.
The structure of the apoform of a System I* CcmE, with the cysteine residue,
showed it to closely resemble the fold of the histidine-containing CcmE protein
(PDB 2KCT, unpublished), as shown in Figure 4C. It was speculated earlier that a
reductive function of CcmF may be related to cleavage of the bond between the
His of CcmE and heme. It is hard to understand how this could apply to CcmE*
as in this case it is presumably a thioether bond that has to be broken to release
heme from the cysteine-containing CcmE.
Additional biogenesis proteins, called NrfEFG, are required for the maturation
of an unusual c-type cytochrome, the nitrite reductase NrfA,98,99 which has a
CXXCK heme-binding motif at its active-site.100 The structure of NrfA is shown
in Figure 2C, highlighting the unusual lysine coordination of the heme iron.
NrfEFG are paralogs of the proteins CcmF and CcmH, suggesting that these are
key to recognition of the substrate cytochrome. The ligand to the iron provided by
the heme-binding motif appears to be key for the substrate recognition by the bio-
genesis proteins in a way that is not yet understood. A model has been proposed
for the interaction between NrfA and the TPR-containing NrfG.87 The physiologi-
cal relevance is hard to appreciate since a chaperone would be expected to stabi-
lize the apoform of NrfA as is suggested by the finding mentioned above that
CcmI of R. capsulatus only interacts with apocytochrome c2.
IV. System II
A. Distribution
In Gram-positive bacteria, some Gram-negative bacteria (β, δ and ε-proteobacteria),
cyanobacteria and chloroplasts, a simpler biogenesis machinery occurs, referred to
as System II. The proteins comprising this system are referred to as Ccs and Res in
different organisms, and are shown in Figure 5 (as reviewed101). These proteins are
less well understood than those of System I. There appear to be no more than four
proteins for cytochrome c biogenesis in the organisms/organelles in which System II
occurs. Some components were identified by characterizing Chlamydomonas
reinhardtii mutants with cytochrome deficiencies.102,103 Experiments in Bacillus
subtilis and Bordetella pertussis revealed the System II elements in bacteria.104,105
The proteins are integral to or associated with the cytoplasmic membrane, and
perform the heme attachment reaction outside that membrane in bacteria, or in the
chloroplast lumen. Their functions can be separated into apocytochrome disulfide
reduction and heme provision/attachment (heme is synthesized in a different
compartment from the site of holocytochrome synthesis).
B. Redox Control
ResA (also called CcsX) is the most well-studied of the System II proteins and is a
thiol-oxidoreductase with a well-defined role in providing the reductant needed to
reverse oxidation of the cysteine thiols in the apocytochrome c.106 In Bacillus,

Figure 5. Cytochrome c biogenesis System II. The Res proteins as found in Bacillus subtilis
are illustrated (the cytoplasmic membrane is shown in grey). System II occurs in Gram-positive
bacteria, some Gram-negative bacteria as well as chloroplasts. ResA provides reductant to the
apocytochrome protein (shown in red) via a pair of cysteines (shown in yellow). It has been
suggested that heme is transported through a channel in ResBC,113 which also catalyzes the
heme attachment reaction.
for example, the protein BdbD is a powerful oxidase that introduces disulfide bonds
into apocytochromes,107 a process that needs to be reversed before the heme attach-
ment reaction can occur. The ResA structure has given some insight into how it rec-
ognizes the apocytochrome cysteines to specifically reduce the disulfide bond in the
heme-binding motif.108 A redox-dependent conformational change causes the
appearance of a cavity that recognizes the substrate cytochrome. The function of
ResA has been confirmed by experiments showing that its absence can be comple-
mented by the addition of disulfide reductants to growth media, or by the lack of oxi-
dizing proteins (BdbD).106,109 ResA acquires reductant from the membrane proteins
CcdA/DsbD, which in turn transfer electrons from thioredoxin in the cytoplasm in a
manner analogous to DsbD in System I.110,111 CcdA displays homology to the trans-
membrane domain of DsbD and has been demonstrated to be a functional homolog
(both are able to transfer reductant from the cytoplasm across the membrane).112
C. Heme Transport and Attachment

ResB and ResC function in the heme attachment reaction to apocytochromes c.113
Both are integral membrane proteins with some soluble regions; ResB has a
substantial predicted extracytoplasmic domain. The predicted number of trans-

membrane domains in the two proteins vary.101 They occur as a fusion
(ResBC/CcsBA) in some ε-proteobacteria. The fused protein from Helicobacter
species functions heterologously in E. coli lacking its endogenous biogenesis
operon, which has facilitated its study. Purified CcsBA was found to contain
heme; histidine residues were identified that are thought to enable heme transfer
across the membrane by forming a heme channel.113 Studies of ResB and ResC
from Bacillus subtilis have also demonstrated heme binding.114 Purified ResB had
heme covalently bound at a cysteine residue. The significance of this observation

is not yet clear because the cysteine residue is not fully conserved and its absence
does not abolish cytochrome c production. Further evidence has been presented
suggesting a heme transport function by the fused proteins in Wolinella
succinogenes,115 a bacterium that has three CcsBA fusions with different substrate
specificities. CcsA/ResC has an evolutionary relationship with the proteins CcmC
and CcmF of System I116 and all contain tryptophan-rich regions (WWD motifs)
on the p-side of the membrane that are suggested to be involved in heme han-
dling.47 Mutations to remove the tryptophan residues have demonstrated their
functional importance.117
D. Substrate Specificity
The substrate specificity of System II has been examined in E. coli; 118 it is able to
attach heme to exogenous monoheme cytochromes c, but not to single-cysteine
forms of these proteins. The presence of CcsBA facilitated the production of a
b-type cytochrome (in contrast with System I), consistent with a possible role for
the System II proteins in heme provision to the periplasm. The System II proteins
can be used for large-scale heterologous expression of multiheme cytochromes c in
W. succinogenes.93 The latter organism contains 23 c-type cytochromes (according
to genome analysis) including mono- and multi-heme cytochromes, indicating that
its cytochrome maturation system has naturally broad substrate specificity.
E. Variations of the System II Pathway

Variants of the System II proteins have been identified that have specificities for
substrates other than the classical CXXCH heme-binding motif. These include the
protein NrfI in W. succinogenes that is involved in heme attachment to the
CXXCK motif of the nitrite reductase NrfA119 and CcsA1 that handles an unusual
CX15CH heme-binding site in an octaheme cytochrome c.120 Although the
evidence is not as developed as for System I, it seems reasonable to conclude that
System II recognizes little more than the CXXCH motif (or CXXCK and
CX15CH). There is no evidence so far that there are any apocytochromes c that are
specifically recognized by only one of these two systems. In this context it is
notable that System II does not appear to involve any ATP-dependent process, a
TPR-containing chaperone or a covalent heme-protein intermediate analogous to
that seen for CcmE.
V. System III
A. Distribution
The simplest, at least in terms of polypeptide composition, cytochrome c biogen-
esis system is found in the mitochondria of fungi, invertebrates, and vertebrates44

where a single type of protein is required for heme attachment to cytochromes c;
it has been called heme lyase as well as holocytochrome c synthase (HCCS). The
latter nomenclature is preferred here. The evolutionary origins of System III are
unclear as there is no ancestral prokaryotic protein resembling HCCS (α-pro-
teobacteria use System I for producing cytochromes c). It is notable that many
bacterial species make multiple cytochromes c, often containing multiple hemes
per protein, whereas mitochondria contain only two cytochromes c (c and c1) and
these are both monoheme cytochromes. As noted above, monoheme c-type
cytochromes seem to have the same properties with just one (rather than the nor-
mal two) thioether bond. Nevertheless System III, which presumably appeared
later in evolution than Systems I and II evolved to make c-type cytochromes with
two thioether bonds.
B. HCCS and Mitochondrial Cytochrome c Import

HCCS is a nuclear-encoded protein that is transported to the mitochondrial inter-
membrane space (IMS), by a non-conservative mechanism,121 where it is essential
for the mitochondrial import of apocytochrome c and for the covalent attachment
of heme to the CXXCH motif (as indicated in Figure 6).122,123 In yeast, for exam-
ple, there are two separate HCCS proteins specific for cytochrome c and
cytochrome c1 (with only 30% identity between them).124,125 The proteins are called
HCCS and HCC1S, respectively. In animals (and some other organisms including
Dictyostelium discoideum) a single protein is thought to process both types of mito-
chondrial cytochrome c, i.e. humans produce a single HCCS protein that is thought
to be able to produce both cytochromes c and c1.126 Specificity studies of the
different forms of HCCS towards the different cytochromes have been performed
in yeast.127 While there appears to be some cross-reactivity (corroborated by the
fact that some organisms contain one biogenesis protein for both substrates), it

Figure 6. Cytochrome c biogenesis System III. The protein holocytochrome c synthase

functions in heme attachment to cytochromes c in the intermembrane space of the mito-
chondria of fungi, invertebrates, and vertebrates. The two membranes are shown in grey. The
apocytochrome proteins (shown in red) are transported across the outer membrane without a
classical mitochondrial targeting sequence and it is not known how heme is transported from
its site of synthesis in the matrix to the IMS, as indicated by the question mark. The protein
HCC1S attached heme in some organisms to cytochrome c1, which is membrane anchored
and has a different mitochondrial import mechanism than cytochrome c.
might be expected that the different import mechanisms for the two types of c-type
cytochrome make the mechanisms of heme attachment different. Cytochrome c
does not have a classical mitochondrial targeting sequence but relies on HCCS for
its import.128 It has been suggested that the attachment of heme causes the
cytochrome to fold and therefore traps the product holocytochrome c in the IMS,
but HCCS binding to the apocytochrome could also keep the apocytochrome in the
IMS. Cytochrome c1 has a bipartite signal sequence that leads to its insertion into
the inner mitochondrial membrane before the heme cofactor is attached.129
C. HCCS Function
It has been suggested that HCCS binds heme,130 as would be expected as one of its
substrates. Heme biosynthesis is completed in the mitochondrial matrix and it is
currently unknown how the heme is transported across the inner membrane to be
used for holocytochrome c synthesis in the IMS. CP motifs are common in HCCS
sequences and these have been implicated in the heme-binding function of the pro-
tein in mitochondria.130 Experiments studying HCCS function heterologously in
E. coli have shown that these motifs are not required for the actual heme attachment
reaction;131 they may possibly be required for a heme acquisition function in mito-
chondria. CP motifs are known as heme-regulatory motifs in an array of other pro-
teins, where heme binding controls a variety of protein functions.132 These include
the bacterial iron response regulator Irr where heme binding causes degradation,133
the activity of the transcription factor Bach1134 and in inhibition of mitochondrial
import of δ-aminolevulinate synthase.135 HCCS has been shown to produce
holocytochromes in both the E. coli cytoplasm92 (where holocytochromes are not

normally produced) and periplasm (in the absence of the endogenous cytochrome
c biogenesis machinery that naturally functions in this compartment).136
Understanding the mechanism of heme attachment to cytochromes by HCCS
has been hampered by the lack of structural information and because the protein
appears to occur at low levels in mitochondria.44 HCCS association with the mito-
chondrial inner membrane has been demonstrated123 although analysis of the
hydrophobicity of the protein does not indicate the presence of transmembrane
helices. Studies of HCCS activity in purified mitochondria have given some
insight;137 evidence for classical Michaelis–Menten kinetics was shown, in addi-
tion to inhibition by holocytochrome c. However, whether HCCS binds the apoc-
ytochrome in a suitable heme-binding conformation, or if it has a classical
catalytic role, is currently unclear and remains to be established.
The protein Cyc2p has been implicated in holocytochrome c production in
yeast, where its role is suggested to be in redox control.138 Its precise function
remains to be determined.
Disruption of HCCS function by mutation has been found to cause human dis-
ease. Microphthalmia with linear skin defects syndrome (MLS) is an X-linked
dominant male-lethal disorder characterized by abnormalities of the eyes and skin.
Mutations in the HCCS gene have been identified in patients suffering from MLS
and the variant proteins are unable to complement for the absence of HCCS in
yeast.139,140 It is thought that the HCCS deficiency disrupts cytochrome c-mediated
apoptosis and causes necrosis in the affected cells. The corresponding yeast HCCS
variants have been found to be inactive in cytochrome c biogenesis in E. coli.131
The functional importance of the amino acids involved will become clear only
with structural data on the HCCS protein.
D. Substrate Specificity
The substrate specificity of HCCS has been examined. Early studies in yeast tested
activity towards cytochromes c from different species (e.g. yeast HCCS towards
animal cytochromes c127), as well as a series of variant cytochromes c. Some level

of heme attachment has been observed with single-cysteine containing
cytochromes141 (as occur naturally in some organisms), as well as when the heme
ligands are replaced (e.g. CXXCR 142). The key role of the N-terminal region of the
cytochrome c protein in recognition by HCCS has been demonstrated, with a con-
served phenylalanine residue appearing to be central to the substrate recogni-
tion.143–145 Fusion of the N-terminal region of yeast cytochrome c to a bacterial
cytochrome (but with retention of the CXXCH sequence of the latter) that is not
itself recognized by HCCS, and lacks its natural N-terminus, resulted in heme
attachment to the chimeric protein by HCCS.144 It has also been shown that HCCS
(like System I) can mature a cytochrome with more than two residues between the
heme-binding cysteines (i.e. a CXXXCH motif).145 It appears that recognition of the
cytochrome by the HCCS involves at least some unfolded conformation and primary
sequence elements, as demonstrated by barely detectable maturation by HCCS of a
trypanosome cytochrome c that has a virtually identical final fold as other mito-
chondrial cytochromes c.6 However, HCCS will not process bacterial cytochromes
c with very similar folds to mitochondrial cytochrome c.146 Hence in contrast to
Systems I and II, and not surprisingly in view of its evolution, HCCS and its variant
HCC1S appear to be capable of only handling mitochondrial proteins.
VI. System IV
A. Distribution
System IV is the most recently identified of the cytochrome c biogenesis systems
and consists of the CCB proteins (cofactor assembly on complex C subunit B).147
It occurs in all organisms that perform oxygenic photosynthesis and is completely
distinct from the other known systems, presumably because its substrate
cytochrome c is different in several respects from other cytochromes c. The b6 f
complex functions in the process of photosynthesis in a manner analogous to the
bc1 complex of respiratory electron transport chains.148 B6 f has been found to con-
tain an additional heme that is covalently bound but only via a single thioether
bond (to the 2-vinyl group of the porphyrin).5,149 This heme is also unusual in that
there are no ligands to the Fe from protein side chains; the heme is 5-coordinate
with a hydroxyl/water acting as a ligand. Unlike the other biogenesis systems dis-
cussed (all of which act on the p-side of the energy transducing membrane),
System IV functions on the n-side (i.e. the plastid stroma or the bacterial cyto-
plasm). The implication of this is that the components for cytochrome c biogene-
sis are produced in the same compartment as the location for heme attachment
(heme and apocytochrome are both produced on the n-side).
B. Components and Interactions

Genetic studies in Chlamydomonas reinhardtii (a green alga) identified four genes
that were essential for attaching the unusual heme to the b6 f complex (the need for
other components has not been excluded).150,151 All encode transmembrane proteins
located in the chloroplast in Arabidopsis.152 CCB1 has three transmembrane domains,
CCB2 and CCB4 are low similarity paralogs with two transmembrane domains with
their C-terminal domains in the stroma; the proteins have been shown to interact with
each other.153 CCB3 has two transmembrane domains with its termini located in the
lumen. Other features found in proteins of the other biogenesis systems do not occur:
there are no conserved histidine residues, no covalently bound heme has been
detected, and there are no conserved cysteine residues characteristic of the thio-redox
components of the other systems. The different subcellular environment in which the
CCB proteins function could account for these observations.
Interaction studies of the CCB proteins have suggested a model in which
CCB1 interacts with b6 (in a chaperone complex), CCB3 then binds, followed by
the CCB2/4 heterodimer, resulting in the attachment reaction of the porphyrin to
the cytochrome protein.147
VII. Conclusion and Perspectives

There is still a common belief that there are only two c-type cytochromes, the mito-
chondrial c sand c1. As discussed at the outset in this chapter, there is a wide range of
distinct types of c-type cytochromes in bacteria and even one in disguise, the
cytochrome f, of the plant photosynthesis system. In fact, the cytochrome c2 of photo-
synthetic bacteria and the cytochrome c3 (ironically a tetraheme protein) in sulfate-
reducing organisms were discovered in the early 1950s, very unexpected at the time,
but the optimistic numbering of what were expected to be a very limited number of
such cytochromes has proved inadequate as ever more examples have been identified
in the bacterial world, largely in recent times through genome sequences. The CXXCH
motif is a valuable search criterion in this regard. Naturally occurring cytochromes c
with different numbers of residues between the cysteines do, however, exist.
It was first realized in the 1980s that the formation of the two thioether bonds of
mitochondrial c-type cytochromes is not spontaneous. The biogenesis system
known as heme lyase or holocytochrome c synthase was identified and for a while
it was assumed that a similar system functioned in other organisms. This proved to
be a false assumption and indeed it has emerged that not all mitochondria use this
system for cytochrome c production. There are other systems at work in
mitochondria, the System I or Ccm system that is also found in many species of
bacteria, and the yet to be identified system (called System V in anticipation) that
must operate in those organisms (e.g. trypanosomes) that attach heme by a single
thioether bond (we assume that for reasons laid out in this chapter that heme attach-
ment is not spontaneous in this case).
System I is clearly very complex and there is still much to be understood about it.
For example, why is it so important to have heme covalently bonded to the CcmE pro-
tein as an intermediate step in attaching heme to polypeptide? The usual covalent bond
in CcmE is heme to histidine but in the variant System I* this seems to be thioether
bond. In the former case (System I) it is clear that the CcmC protein is important for
this heme attachment and that the ATPase activity of CcmAB is not. But how exactly
is the CcmE-heme bond formed? In the case of System I* at the time of writing coun-
terpart information about roles of CcmAB and CcmC is not available, but it is hard to
understand how a thioether bond between heme and CcmE* can be formed or broken.
In the case of CcmE the breaking of the CcmE-histidine bond needs energy from ATP
hydrolysis. It is uncertain whether this involves the formation of free heme itself before
the two thioether bonds are formed although very recent work suggests that one
thioether bond is formed before the bond between heme and CcmE is broken.71 The
full role of CcmF in the maturation has yet to be established. Presumably this protein
provides a template for the binding of the CXXCH motif and provides an environment
for heme transfer from CcmE and reaction with the two cysteines of an apocytochrome
c. In general terms, the roles of the remaining Ccm proteins can be seen more simply
as being to keep the cysteine side chains reduced.
In view of the complexity of System I, it is perhaps not surprising that this sys-
tem has been supplanted in some eukaryotic cells by the, superficially at least,
simpler System III. It is challenging to explain though why all eukaryotes have not
made this switch. Although System III is apparently simpler it is far from under-
stood. Key points of contrast are that System III seems far more specific for its
substrate, with certain features of the N-terminal region of the apocytochrome c
being very important for recognition of mitochondrial, but not other c-type
cytochromes. There is no evidence for a protein-heme covalent complex in
System III nor of any need for a chaperone protein as is found in System I (CcmI).
The absence of a covalent complex analogous to CcmE or of a chaperone-type
protein is also a feature of System II, which unlike System III, has a broad substrate
specificity, again draws attention as to why these features are needed in System I.
Overall, it is not only the mechanisms of the different biogenesis systems that
demand our attention, but why all these systems have persisted during evolution.
VIII. Acknowledgments
The authors acknowledge funding from the Biotechnology and Biological
Sciences Research Council (BBSRC) of the United Kingdom.
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Heme Attachment To Cytochrome C

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b1243_Vol-19_Ch-93.

qxd 3/19/2012 9:23 AM Page 371

b1243 Handbook of Porphyrin Science

Julie M. Stevens and Stuart J. Ferguson

Department of Biochemistry, University of Oxford, South Parks Road,

Oxford, OX1 3QU, UK

List of Abbreviations 372

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372 Stevens and Ferguson

VI. System IV 394

ABC ATP-binding cassette

Ccm cytochrome c maturation

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93/Heme Attachment to Cytochromes c 373

Figure 1. The structures of (A) b-heme (Fe-protoporphyrin IX) as it occurs in b-type

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374 Stevens and Ferguson

Figure 2. (A) A monoheme cytochrome c (cytochrome c550 from Paracoccus denitrificans,7

essential function in the cell to shuttle electrons, in the mitochondrial intermem-

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93/Heme Attachment to Cytochromes c 375

formation of the apoptosome and activation of the signaling cascade leading to

B. Why is Heme Covalently Attached to c-Type Cytochromes?

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376 Stevens and Ferguson

C. Insights from Spontaneous Heme Attachment to Cytochromes

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making a very stable b-type cytochrome to perform the roles of mitochondrial

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93/Heme Attachment to Cytochromes c 379

Overall the study of spontaneous formation of c-type cytochromes, whether

II. The Diversity of Heme Attachment Pathways

In cells the attachment of heme to the apoforms of cytochromes c is a protein-

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found in many species of Gram-negative bacteria, plant mitochondria and some

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93/Heme Attachment to Cytochromes c 381

B. Heme Handling Components

C. The Heme Chaperone CcmE

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382 Stevens and Ferguson

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93/Heme Attachment to Cytochromes c 383

c maturation in either the heme binding or transfer reactions.65 The characterization

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384 Stevens and Ferguson

D. The Heme Attachment Reaction to Cytochromes c

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93/Heme Attachment to Cytochromes c 385

E. Redox Control and Chaperoning of the Apocytochrome

CcmG and CcmH are involved in thiol-disulfide oxidoreductase functions and

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386 Stevens and Ferguson

specificity.82 Whether CcmG or CcmH transfers reductant to oxidized apocy-

facilitators of protein–protein interactions.88 CcmI is expected to have a similar

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93/Heme Attachment to Cytochromes c 387

G. Variations of the System I Pathway

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