Comparing the substrate specificities of cytochrome c
biogenesis Systems I and II
Bioenergetics
Alan D. Goddard*, Julie M. Stevens*, Arnaud Rondelet, Elena Nomerotskaia, James W. A. Allen
and Stuart J. Ferguson
Department of Biochemistry, University of Oxford, UK
Introduction
Nature employs at least five distinct systems for the
biogenesis of c-type cytochromes [1–3]; this post-trans-
lational modification process covalently links the heme
cofactor to, normally, two cysteines in a CXXCH
motif. System I is found in many Gram-negative bacte-
ria and various mitochondria, including from plants
[4,5]; System II appears in Gram-positive and some
Gram-negative bacteria, and chloroplasts [6]; System
III occurs in many non-plant mitochondria [5]; System
IV is specific for the unusual cytochrome b
6
involved
in photosynthesis [7], and a fifth system, which remains
to be characterized, exists in trypanosomatids [8]. Very
unusually, some thermophilic cytochromes c are able
to form spontaneously in vitro or in the cytoplasm of
Escherichia coli [9], although it is believed that they are
naturally matured by one of the biogenesis systems
above.
The experimental amenability of E. coli has allowed
the heterologous replacement of its own cytochrome
c maturation (Ccm) machinery (encoded by the
ccmABCDEFGH operon, called System I, Fig. 1) with
systems from other organisms to facilitate their analy-

from Hydrogenobacter thermo-
philus, both normally matured by System I. The production of holocyto-
chrome is enhanced by the addition of exogenous reductant. Single-cysteine
variants of these cytochromes were not efficiently matured by System II,
but System I was able to produce detectable amounts of AXXCH variants;
this adds to evidence that there is no obligate requirement for a disulfide-
bonded intermediate for the latter c-type cytochrome biogenesis system. In
addition, System II was able to mature an AXXAH-containing variant into
a b-type cytochrome, with implications for both heme supply to the peri-
plasm and substrate recognition by System II.
Abbreviations
Ccm, cytochrome c maturation; IPTG, isopropyl thio-b-
D-galactoside; MESA, 2-mercaptoethane sulfonate.
726 FEBS Journal 277 (2010) 726–737 ª 2009 The Authors Journal compilation ª 2009 FEBS
produce mitochondrial holocytochrome c. System II
from Helicobacter pylori has been substituted for the
E. coli Ccm machinery, enabling a comparison of the
heme delivery activities of the two systems towards a
diheme cytochrome c [11]. In E. coli, natural cyto-
chrome c biogenesis requires at least 10 proteins, con-
trasting with the single protein constituting System III.
System II is of intermediate complexity and comprises
four proteins in, for example, Bacillus subtilis and
Bordetella pertussis, namely ResA, ResB, ResC and
CcdA [12,13] (Fig. 1). Notably, the genomes of some
Bordetella species, e.g. B. parapertussis, encode both
Systems I and II [1].
In common with System I, it is not clear whether
heme is transported across the membrane by System II
itself or by some other process. Heme must then be

mitochondria of Euglenozoan organisms, such as Cri-
thidia fasciculata, and it has been demonstrated that
the overall structure of cytochrome c from this organ-
ism bears remarkable similarity to yeast cytochrome c
[21]. It is believed that organisms which possess such
single-cysteine c-type cytochromes exhibit an as yet
unidentified, novel biogenesis system. All fully
sequenced genomes of organisms expressing such sin-
gle-cysteine cytochromes lack identifiable homologues
of known c-type cytochrome biogenesis proteins. The
ability (or otherwise) of System I or II to mature such
cytochromes may shed light on the mechanism of
heme attachment in these systems. To date, System I
has not been clearly observed to attach heme to such
single-cysteine variants [22,23]. Contrastingly, System
II has been proposed to attach heme to an SXXCH
motif in NrfH [24].
In this work, we have cloned the ResBC-encoding
gene from H. pylori (ycf5) into the backbone plasmid
(pACYC184) of pEC86 which contains the E. coli ccm
operon [25] and which has been very widely used for
heterologous cytochrome c production. Heterologous
expression of H. pylori ResBC from this new plasmid
(pHP86) in E. coli allowed us to explore the substrate
Heme handling/ligation
System I
System II
CcmE
CcmF
ResA

specificity of System II in direct comparison with
System I.
Results
Various c-type cytochrome proteins were used to probe
different aspects of the specificity of the expressed
cytochrome c biogenesis systems. Experiments were
conducted in a strain of E. coli lacking all known
endogenous cytochrome c biogenesis proteins, EC06.
In each case, control experiments were performed for
spontaneous heme attachment to exogenous cyto-
chromes [using the biogenesis system plasmid back-
bone containing no cytochrome c biogenesis genes
(AD377)]. In addition, correction was performed for
the formation of any endogenous E. coli cytochromes
catalyzed by the products of the different biogenesis
plasmids in the absence of a gene for an exogenous
cytochrome.
System II can mature monoheme c-type
cytochromes in E. coli
Cytochrome c
550
from Paracoccus denitrificans is well
characterized as a heterologous holocytochrome pro-
duced by the E. coli Ccm system [26]. The ability of
System II to attach heme to this monoheme bacterial
cytochrome in the periplasm of EC06 cells was tested.
Holocytochrome c
550
was detected in periplasmic
extracts of cells containing pHP86 (H. pylori ResBC)

genobacter thermophilus cytochrome c
552
. This System
0.035
0.030
0.025
Absorbance
14
550 560
570540530
0.020
MI II
6
Δ Absorbance
400 450 500 550 600 650
Wavelen
g
th (nm)
Fig. 2. Maturation of P. denitrificans cytochrome c
550
by System II.
Visible absorption spectra reflecting the formation of P. denitrificans
cytochrome c
550
and variants in periplasmic extracts of E. coli EC06
catalyzed by H. pylori ResBC: wild-type cytochrome c
550
(full line),
AXXCH-containing variant (broken ⁄ dotted line) and CXXAH-contain-
ing variant (broken line). The vertical scale bar represents 0.01

System I System II
Cytochrome c
550
CXXCH 4.07 (0.55) 0.024 (0.009)
AXXCH 0.045 (0.002) ND
CXXAH 0.023 (0.005) ND
Cytochrome c
552
CXXCH 0.99 (0.42) 0.16 (0.054)
AXXCH 0.030 (0.004) 0.009 (0.002)
CXXAH ND ND
Specificity of cytochrome c biogenesis System II A. D. Goddard et al.
728 FEBS Journal 277 (2010) 726–737 ª 2009 The Authors Journal compilation ª 2009 FEBS
I-matured thermophilic cytochrome has also been used
as a substrate to test the properties of the E. coli cyto-
chrome c biogenesis system [22]. System I is able to
produce large quantities of the c
552
holocytochrome
(Table 1 and Fig. 3). Co-expression of cytochrome c
552
with the System II plasmid resulted in approximately
16% of the level produced by System I, a much higher
proportion than that observed with the mesophilic
cytochrome c
550
. The spectroscopic features and mobil-
ity on SDS-PAGE of the System II-produced cyto-
chrome c
552

bonds [21,28]. The determination of whether the pres-
ence of two cysteine thiols is essential could also
address the requirement for an intramolecular disulfide
bond, known to occur within apocytochromes [29], in
the heme attachment reaction. It has been argued that
System II can attach heme to one SXXCH motif gen-
erated by site-directed mutagenesis in the tetraheme
cytochrome NrfH from Wolinella succinogenes [24].
However, this is an important point requiring further
investigation.
Cytochrome c
550
containing an AXXCH motif
(C35A) acquired approximately 1.1% of the level of
heme attachment observed for the wild-type CXXCH
protein when expressed with System I (Table 1). The
values in Table 1 are based on the absorption values
at single wavelengths. However, they are only taken to
indicate the presence of the specific holocytochrome
under investigation if the features of the spectrum, in
terms of wavelength maxima, and the position and
intensity of heme-staining bands on SDS-PAGE gels,
also appropriately demonstrate holocytochrome forma-
tion. The AXXCH variant produced by System I has
spectroscopic features indicative of single-cysteine holo-
cytochrome formation, and a band of the expected
mass is observed on heme-stained gels (data not
shown). The values in Table 1 imply that a small
amount of the C38A (CXXAH) variant may have
undergone heme attachment by System I. However,

addition of a few grains of disodium dithionite. The absorbance
maxima are at 417, 521 and 552 nm. The inset gel illustrates the
detection of c-type cytochromes via SDS-PAGE analysis of periplas-
mic fractions from cells expressing H. thermophilus cytochrome
c
552
and the indicated biogenesis system (I or II), and subsequent
heme-staining of the gel. Loading was normalized for total protein
content. The left-most lane (M) contains See-Blue Plus 2 protein
markers of the indicated molecular weights (kDa).
A. D. Goddard et al. Specificity of cytochrome c biogenesis System II
FEBS Journal 277 (2010) 726–737 ª 2009 The Authors Journal compilation ª 2009 FEBS 729
c
550
serum, whereas the CXXAH variant was not
(Fig. S1, see Supporting Information).
Although it is possible that the CXXAH variant of
P. denitrificans cytochrome c
550
is unstable and cannot
be made, the two single-cysteine-containing (and
AXXAH) variants of H. thermophilus cytochrome c
552
can form stably in the E. coli cytoplasm [30,31]. Sys-
tem I was unable to produce the CXXAH variant
cytochrome c
552
, but some System I-dependent forma-
tion of the AXXCH variant (approximately 3% rela-
tive to CXXCH) was detected (this is the value after

spectroscopically detectable AXXCH above the level
of spontaneous cytochrome formation in two of the
cultures. These data are responsible for the apparent
formation of AXXCH by System II when compared
with AD377 (reported as mean values in Table 1). It is
possible that in the majority of our observations
single-cysteine cytochromes were formed by System II
at such low levels that they were undetectable either by
spectroscopic analysis of periplasmic fractions or heme
staining of appropriate SDS-PAGE gels.
System II mediates the formation of a b-type
cytochrome
Unexpectedly, the spectra of periplasmic extracts of
cells containing the System II plasmid and the double-
alanine cytochrome c
550
(AXXAH motif, C35A ⁄ C38A)
indicated the presence of small amounts of a typical
low-spin, b-type cytochrome (Fig. 5). The Soret band
is red shifted by 4 nm and the a-band by 5 nm com-
pared with the wild-type cytochrome c
550
, as would be
expected for noncovalently bound heme (saturation of
each heme vinyl group on formation of a c-type cyto-
chrome causes a blue shift of 2–3 nm in the a-band of
the absorption spectrum). To confirm the presence of
variant cytochrome c
550
, we performed a western blot

550
AXXAH variant (full line) and no cytochrome (pKK223-
3) (dotted line). The vertical scale bar represents 0.005 absorbance
units. Samples were reduced by the addition of a few grains of
disodium dithionite. The Soret and a-band absorbance maxima are
at 415 and 550 nm, respectively, for wild-type cytochrome c
550
,
and at around 419 and 555 nm for the AXXAH-containing variant.
The spectrum of the wild-type cytochrome c
550
has been reduced
by a factor of seven for clarity, and the spectra are vertically offset.
The vertical line indicates 550 nm.
Specificity of cytochrome c biogenesis System II A. D. Goddard et al.
730 FEBS Journal 277 (2010) 726–737 ª 2009 The Authors Journal compilation ª 2009 FEBS
cytochrome c
550
was evident in the strain expressing
AXXAH-containing cytochrome c
550
, but not in the
control strain containing pKK223-3 (Fig. S1, see Sup-
porting Information). It was not possible to detect low
levels of b-type complexes (if they exist) made with
System I because of the relatively high levels of endog-
enous cytochromes that are produced (see above)
would mask the spectroscopic features of the b-type
cytochrome. However, we were unable to detect the
formation of a b-type AXXAH variant by System I

550
C35A
variant did not result in the formation of detectable
holocytochrome, implying that the augmentation in
wild-type holocytochrome maturation with the addi-
tion of reductant is a result of the reduction of a disul-
fide in the apocytochrome.
Production of endogenous E. coli cytochromes
Escherichia coli contains a number of endogenous
c-type cytochromes that it expresses under different
anaerobic growth conditions. Some of these are
observed at low levels in periplasmic extracts when the
Ccm deletion strain EC06 is complemented with System
I (pEC86), but not with System II (pHP86), as shown in
Fig. 6. The two bands observed correspond to the
masses of the soluble cytochromes NapB (around
15 kDa) and NrfA (around 50 kDa). However, given the
relative maturation levels of exogenous cytochromes c
(see above), it may be that any endogenous cytochrome
matured by System II would be present below the lower
limit of detection in our experiments. We have
determined that the limit of detection for heme on a
heme-stained SDS-PAGE gel is 1 nmol per lane (A. D.
Goddard & S. J. Ferguson, unpublished observations).
E. coli NapB has two hemes and NrfA five. Therefore,
we would expect to detect 0.5 and 0.2 nmol of these
cytochromes, respectively.
Discussion
The complex and somewhat unpredictable natural
distribution of cytochrome c biogenesis systems does

lane (M) contains See-Blue Plus 2 protein markers of the indicated
molecular weights (kDa). Equal amounts of total protein were
loaded in each lane.
A. D. Goddard et al. Specificity of cytochrome c biogenesis System II
FEBS Journal 277 (2010) 726–737 ª 2009 The Authors Journal compilation ª 2009 FEBS 731
contain a heme lyase for each mitochondrial cyto-
chrome c (e.g. yeast cytochromes c and c
1
), whereas
others (e.g. animals) contain a single such enzyme that
apparently catalyzes heme attachment to both cyto-
chromes [37]. No study has examined the specificity of
System II, which in nature produces an array of
mono- and multiheme cytochromes.
System II produces monoheme bacterial
cytochromes
In this work, we have shown that coexpression of the
plasmid pHP86 (expressing System II) in E. coli with
the mesophilic cytochrome c
550
from P. denitrificans
and the thermophilic cytochrome c
552
from H. thermo-
philus, both naturally matured by System I, results in
heme attachment to these cytochromes, yielding prod-
ucts that are indistinguishable from those produced by
System I. This suggests that System II, in common
with System I and in contrast with System III [38], has
a broad substrate specificity and is able to mature c-

System II, the oxidoreductase that would normally
reduce such a disulfide bond, ResA, is absent; the oxi-
dation would also slow heme attachment. Our observa-
tion that added reductant results in a substantial
increase in cytochrome c
550
production indicates that
oxidation of the heme-binding motif can reduce the
efficiency of heme attachment by System II. Neverthe-
less, it is becoming increasingly clear from this work
and others [20] that dithiol ⁄ disulfide oxidoreductases
are not strictly necessary for cytochrome c maturation
in the periplasm of E. coli.
Maturation of single-cysteine-attached
cytochromes c
We found no detectable heme attachment to the sin-
gle-cysteine-containing variants of P. denitrificans c
550
with coexpression of the System II plasmid. A very
low, variable level of heme attachment was observed
with the AXXCH variant of cytochrome c
552
, but none
with the CXXAH form. If there is a capability to
attach heme to a single-cysteine cytochrome then, in
common with System I, it is to a very low extent com-
pared with heme attachment to CXXCH, below the
level of detection of the analysis conducted in this
study. It is notable that, in the work of Simon et al.
[24], evidence was found for heme attachment to only

background cytochrome c matured (producing no
detectable c-type cytochromes in the absence of a plas-
mid-borne biogenesis system). The sensitivity of the
analytical methods used (e.g. the former work did not
use heme-stained gels) may also contribute to the dif-
ferences. That System I can attach some heme to sin-
gle-cysteine-containing cytochromes is significant,
particularly in the context of a possible relationship
between heme attachment and a disulfide bond in the
CXXCH motif. The fact that two cysteines are not
absolutely essential for the heme attachment reaction
Specificity of cytochrome c biogenesis System II A. D. Goddard et al.
732 FEBS Journal 277 (2010) 726–737 ª 2009 The Authors Journal compilation ª 2009 FEBS
demonstrates that the chemistry of such a reaction is
not necessarily via an obligate disulfide-containing
intermediate. Breaking a disulfide bond could be envis-
aged as providing the driving force for formation of
the thioether bonds to heme. However, successful
in vitro thioether bond formation using a phosphine
(in the absence of thiol reagents) to reduce the apocy-
tochrome disulfide also indicated that disulfide-linked
chemistry is not involved in the heme attachment reac-
tion [39]. The present work implies that the formation of
the two thioether bonds is not thermodynamically neces-
sary to release heme from the covalent heme-binding
chaperone CcmE. We observe, as might be anticipated
[21,31,40], that the single-cysteine variant in which the
heme-binding cysteine is directly adjacent to the heme
iron-ligating histidine (i.e. XXXCH) is the more likely to
be recognized by the system and to undergo heme attach-

lent intermediate (CcmE–heme) [42], System I is
unable to pass heme to an AXXAH variant apocyto-
chrome; System II (ResBC) from Helicobacter hepati-
cus does not appear to covalently bind heme [20].
However, a recent study with Bacillus subtilis ResB
and ResC (unfused proteins in that organism) revealed
covalent binding of heme to the cytoplasmic side of
ResB [43]. It is therefore possible that an initial cova-
lent attachment of heme to ResB occurs, followed by
trafficking through ResC, before insertion of heme into
the periplasmic cytochrome. However, the residue
covalently bound to the heme of ResB was found to
be nonessential for cytochrome c biogenesis. The func-
tion, if any, of System II proteins covalently binding
heme therefore remains to be resolved.
That expression of the System II protein in E. coli
allows the formation of a b-type cytochrome suggests
that heme provision from the cytoplasm to the peri-
plasm might be performed by ResBC, concurrent with
recent observations [20]. The study by Frawley &
Kranz [20] also demonstrated the essentiality of H858
of H. hepaticus ResBC in holocytochrome formation,
and proposed that this residue, together with H77, is
involved in supplying heme to the periplasm. We note
that a H857E mutant in H. pylori ResBC (equivalent
to H858 in the H. hepaticus protein) is unable to
mature the b-type cytochrome described above (A. D.
Goddard & S. J. Ferguson, unpublished observations).
This is consistent with H857 playing a role in heme
transport. It is not known how heme is transported

from pEC86 [25]. To create a comparable plasmid lacking
any biogenesis system, inverse PCR was performed on
pEC86 using the primers AG234 and AG235, and the prod-
uct was self-ligated. This removed the entire ccm operon,
and the plasmid created is AD377 (no biogenesis system).
To create a suitable plasmid for the expression of other bio-
genesis systems, a XhoI site was introduced immediately
after the initiating ATG of ccmA in pEC86 via Quikchange
mutagenesis using the primers WC1 and WC2. The resul-
tant construct is pEC86x, in which the entire ccm operon
can be excised by digestion with XhoI and StuI. The ycf5
gene was amplified from H. pylori (strain 26695) genomic
DNA using oligonucleotides HelF and HelR. The PCR
product was cloned into XhoI ⁄ StuI-digested pEC86x. The
resultant plasmid for the expression of H. pylori ResBC is
pHP86 (System II).
Cytochrome c plasmids
P. denitrificans cytochrome c
550
was expressed from the iso-
propyl thio-b-d-galactoside (IPTG)-inducible promoter of
pKPD1 [26]. Mutations within the CXXCH heme-binding
motif of c
550
were created by Quikchange using the follow-
ing: c
550
C35A, C35AF and C35AR; c
550
C38A, C38AF

)1
and chlorampheni-
col at 34 lg Æ mL
)1
.
Analysis of cytochrome production
Periplasmic extractions were performed as described previ-
ously [22]. Extracts were analyzed by SDS-PAGE (Invitro-
gen pre-cast 10% Bis-Tris gels), followed by heme staining
[48], which stains proteins with covalently bound heme.
Samples were normalized for wet cell weight, and equal
amounts of protein were loaded per lane (5–10 lg).
Western blots to detect P. denitrificans cytochrome c
550
were performed according to the manufacturer’s instruc-
tions using anti-cytochrome c
550
rabbit serum and a
commercial alkaline-phosphatase-conjugated anti-rabbit
secondary IgG raised in goat. The marker used was See-
Blue Plus 2 (Invitrogen).
UV–visible spectroscopy was performed using a Perkin-
Elmer (Waltham, MA, USA) Lambda 2 spectrophotometer;
samples were reduced by the addition of a few grains of
disodium dithionite (Sigma-Aldrich Company Ltd, Poole,
UK). Pyridine hemochrome spectra were determined
according to the method described by Bartsch [49].
The normalized cytochrome content of each extract is pre-
sented as the number of milligrams of holocytochrome c
per gram of cell pellet. The data are averages of at least five

c
550
wild-type and variants, 140 mm
)1
Æcm
)1
at 415 nm [22].
The extinction coefficients for the cytochrome c
550
variants
are unknown; the wild-type value was therefore used. Cor-
rections of the average normalized values for each dataset
were performed by subtracting the value observed when no
biogenesis genes were expressed (i.e. with plasmid AD377
and the relevant cytochrome plasmid, to correct for sponta-
neous holocytochrome production) and subtracting the
value observed when no heterologous cytochrome gene was
expressed (i.e. with plasmid pKK223-3 and the relevant bio-
genesis plasmid, to correct for the production of endoge-
nous E. coli cytochromes). Finally, the values for cells
expressing the two empty vectors AD377 and pKK223-3
were added back, so that any corrections were for endoge-
nous or spontaneous cytochrome c production only.
Cultures for the corrections were grown and analyzed at
least three times, and the mean values were used for the
corrections.
Acknowledgements
This work was supported by the Biotechnology and
Biological Sciences Research Council (BBSRC; grant
numbers BB ⁄ D523019 ⁄ 1, BB ⁄ E004865 ⁄ 1 and BB ⁄

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