Báo cáo khoa học: Structure of a trypanosomatid mitochondrial cytochrome c with heme attached via only one thioether bond and implications for the substrate recognition requirements of heme lyase potx

Structure of a trypanosomatid mitochondrial
cytochrome c with heme attached via only one thioether
bond and implications for the substrate recognition
requirements of heme lyase
Vilmos Fu
¨
lo
¨
p
1
, Katharine A. Sam
2
, Stuart J. Ferguson
2
, Michael L. Ginger
3
and James W. A. Allen
2
1 Department of Biological Sciences, University of Warwick, Coventry, UK
2 Department of Biochemistry, University of Oxford, UK
3 School of Health and Medicine, Division of Biomedical and Life Sciences, Lancaster University, UK
The principal physiological role of mitochondrial cyto-
chrome c is electron transfer from the cytochrome bc
1
complex to cytochrome aa
3
oxidase during oxidative
phosphorylation. c-Type cytochromes form a large
family in bacteria, archaea, mitochondria and chlorop-
lasts, in which the iron cofactor heme is covalently
bound to the polypeptide chain. Such cytochromes

have many distinct folds (often unrelated to that of
mitochondrial cytochrome c); bacterial c-type cyto-
chromes frequently have many hemes [1]. Despite this
variety, the covalent heme attachment to protein is
highly stereospecific and regiospecific, and is almost
Keywords
Cytochrome c; heme lyase; intermembrane
space; thioether bond; trypanosome
Correspondence
J. W. A. Allen, Department of Biochemistry,
University of Oxford, South Parks Road,
Oxford, OX1 3QU, UK
Fax: +44 0 1865 613201
Tel: +44 0 1865 613330
E-mail:
Database
X-ray structure coordinates for Crithidia
fasciculata cytochrome c have been
deposited in the Protein Data Bank under
the accession code 2w9k
(Received 23 December 2008, revised 19
February 2009, accepted 13 March 2009)
doi:10.1111/j.1742-4658.2009.07005.x
The principal physiological role of mitochondrial cytochrome c is electron
transfer during oxidative phosphorylation. c-Type cytochromes are almost
always characterized by covalent attachment of heme to protein through
two thioether bonds between the heme vinyl groups and the thiols of cyste-
ine residues in a Cys-Xxx-Xxx-Cys-His motif. Uniquely, however, members
of the evolutionarily divergent protist phylum Euglenozoa, which includes
Trypanosoma and Leishmania species, have mitochondrial cytochromes c

with heme attached through only one thioether bond [to an (A ⁄ F)XXCH
motif]; the implications of this for the cytochrome structures are unclear.
Here we present the 1.55 A
˚
resolution X-ray crystal structure of cyto-
chrome c from the trypanosomatid Crithidia fasciculata. Despite the funda-
mental difference in heme attachment and in the cytochrome c biogenesis
machinery of the Euglenozoa, the structure is remarkably similar to that of
typical (CXXCH) mitochondrial cytochromes c, both in overall fold and,
other than the missing thioether bond, in the details of the heme attach-
ment. Notably, this similarity includes the stereochemistry of the covalent
heme attachment to the protein. The structure has implications for the
maturation of c-type cytochromes in the Euglenozoa; it also hints at a dis-
tinctive redox environment in the mitochondrial intermembrane space of
trypanosomes. Surprisingly, Saccharomyces cerevisiae cytochrome c heme
lyase (the yeast cytochrome c biogenesis system) cannot efficiently mature
Trypanosoma brucei cytochrome c or a CXXCH variant when expressed in
the cytoplasm of Escherichia coli, despite their great structural similarity to
yeast cytochrome c, suggesting that heme lyase requires specific recognition
features in the apocytochrome.
Abbreviations
Ccm, cytochrome c maturation; IMS, intermembrane space; NCS, noncrystallographic symmetry; SHAM, salicylhydroxamic acid.
2822 FEBS Journal 276 (2009) 2822–2832 ª 2009 The Authors Journal compilation ª 2009 FEBS
always through two thioether bonds between the vinyl
groups of heme and the thiols of cysteine residues that
occur in a CXXCH amino acid motif; the histidine
serves as the proximal ligand to the heme iron atom
[2–4]. However, in one group of eukaryotes, the mem-
bers of the protist phylum Euglenozoa, biochemical,
spectroscopic and genetic evidence suggests that,

uniquely, the mitochondrial c-type cytochromes (c and
c
1
) both have heme covalently bound to the polypeptide
chain through only a single thioether bond [to an
(F ⁄ A)XXCH motif] [5–10]. The Euglenozoa include
ubiquitous, free-living phagotrophic flagellates (e.g.
Bodo saltans), photosynthetic algae (e.g. Euglena graci-
lis), and parasitic trypanosomatids [e.g. the causal
agents of the tropical diseases African sleeping sickness
(Trypanosoma brucei), Chagas disease (Trypanosoma
cruzi), and leishmaniasis (Leishmania species)]. Note
that some euglenozoans, such as E. gracilis, also contain
a chloroplast with typical CXXCH cytochromes c [11].
Covalent attachment of heme to cytochromes c is a
catalyzed post-translational modification. Remarkably,
at least five completely distinct biogenesis systems have
evolved to achieve this heme attachment in different
organisms and organelles. Some eukaryotes (land
plants and various protists) use the multicomponent
System I [also called the cytochrome c maturation
(Ccm) system], and others (animals, fungi, and many
evolutionarily diverse protozoa and algae) use System
III, the enzyme heme lyase, for maturation of their
mitochondrial cytochromes c [10]. Surprisingly little is
known about the mechanism and substrate recognition
features of heme lyase-dependent heme attachment to
apocytochrome c, and the origins of this critical
eukaryote-specific enzyme are obscure [10]. Strikingly,
none of the known cytochrome c biogenesis proteins

are present in the several trypanosomatid species for
which complete genome sequences are available
[9,10,12]. The presence throughout the Euglenozoa of
unique single cysteine mitochondrial cytochromes c,
coupled with the absence from trypanosomatids of any
known cytochrome c biogenesis proteins, points
towards a novel maturation apparatus for all eugleno-
zoan mitochondrial cytochromes c [9,10].
The reason for the loss of one thioether bond from
the mitochondrial cytochromes c of euglenozoans is a
longstanding puzzle. It could be a means of altering
the structure of these cytochromes c and ⁄ or a conse-
quence of some biosynthetic demand. A high-resolu-
tion structural comparison between a euglenozoan
mitochondrial cytochrome c and a typical cyto-
chrome c (with heme bound by two thioether bonds) is
therefore important. Moreover, in all the available,
diverse structures of c-type cytochromes, there is an
invariant stereospecific arrangement of the heme [1,13];
there is no a priori reason to expect this to be the same
in the single thioether bond euglenozoan cytochromes.
Finally, it is likely that (at least for cytochrome c bio-
genesis Systems I and II) folding of holocytochromes c
mainly takes place after covalent attachment of
heme to the polypeptide chain. Thus, it is not axiom-
atic that anchoring the heme to protein through only a
single thioether bond would result in the same local
structure that is characteristic of heme attachment to a
CXXCH motif. Therefore, we have determined the
X-ray crystal structure of mitochondrial cytochrome c

from the trypanosomatid C. fasciculata; we have also
investigated the maturation of trypanosome cyto-
chrome c by the poorly understood yeast cytochrome c
heme lyase.
Results
Cytochrome-dependent respiration in
C. fasciculata
For determination of a euglenozoan cytochrome c
structure, we isolated holocytochrome from C. fascicu-
lata, a monogenetic insect parasite that is not patho-
genic to humans. Some trypanosomatids (e.g.
Leishmania spp. and T. cruzi) possess a classic mito-
chondrial respiratory chain [14–16]. In others (e.g. the
life cycle stage of T. brucei found in the midgut of the
tsetse fly), the mitochondrial respiratory chain is
branched, and electrons can be transferred from ubiqu-
inol to either the cytochrome bc
1
complex, or to the
enzyme alternative oxidase, which reduces oxygen to
water but is not coupled to ATP production by oxida-
tive phosphorylation because proton translocation is
absent [17,18]. In other cases, such as pathogenic
forms of T. brucei found in the mammalian blood-
stream, cytochrome-dependent respiration is repressed,
and alternative oxidase is the essential sole terminal
oxidase in mitochondrial electron transport [16,19].
Thus, before solving the X-ray structure of C. fascicu-
lata cytochrome c, we first confirmed its importance in
mitochondrial electron transport in that organism. The

presence of 200 lm salicylhydroxamic acid (SHAM), a
specific inhibitor of alternative oxidase, had little effect
on the growth rate of C. fasciculata when compared
with control cultures. Addition of 2 lgÆmL
)1
antimycin
A (a cytochrome bc
1
complex inhibitor), however,
resulted in no growth over 72 h (from a starting inocu-
lum of 10
5
cellsÆmL
)1
). Similarly, addition of SHAM
to a concentration of 3 mm exerted no effect on
oxygen consumption by C. fasciculata as measured
using a Clark oxygen electrode, whereas following
V. Fu
¨
lo
¨
p et al. Structure of Crithidia fasciculata cytochrome c
FEBS Journal 276 (2009) 2822–2832 ª 2009 The Authors Journal compilation ª 2009 FEBS 2823
addition of antimycin to 2 lgÆmL
)1
, oxygen consump-
tion by C. fasciculata effectively ceased within a few
seconds (Fig. 1). A very similar result was obtained if
2mm KCN was added instead of antimycin A; cyanide

inhibits the cytochrome aa
3
oxidase of the classic
respiratory chain, but not alternative oxidase. Thus,
cytochrome-dependent respiration is essential in
C. fasciculata.
The structure of C. fasciculata mitochondrial
cytochrome c
The overall structure of oxidized C. fasciculata cyto-
chrome c, determined by X-ray crystallography to a
resolution of 1.55 A
˚
, is shown in Fig. 2. The asymmet-
ric unit is a trimer. The structure is ordered for resi-
dues 5–114 of the 114 amino acid polypeptide chain.
The fold is typical for a class I c-type cytochrome (e.g.
mitochondrial cytochrome c and bacterial cyto-
chromes c
2
) [20]. The structure unequivocally confirms
the earlier conclusion that trypanosomatid cyto-
chromes c contain only one thioether bond between
heme and protein; there are no other compensatory
covalent bonds to the heme cofactor. The heme is (as
expected [21]) covalently attached to the protein
through its (original) 4-vinyl group (also called the C
8
vinyl), as is observed for the C-terminal cysteine of the
typical CXXCH c-type cytochrome heme-binding
motif [1,13]. The heme iron is coordinated by the N

e
of the histidine of the ‘AXXCH’ (actually AAQCH)
heme-binding motif, and the sulfur of Met91. The
heme iron–ligand distances were restrained to 2 A
˚
(his-
tidine) and 2.3 A
˚
(methionine), and the model fits well
with these values. The vinyl a-carbon–Cys28 distances
were restrained to 1.8 A
˚
, but these refined to consider-
ably longer bond lengths: 2.25, 1.98 and 2.00 A
˚
,
respectively, for the three subunits of the asymmetric
unit. Residue 83 is a trimethyllysine [5]; a moderate fit
to the electron density suggests that the trimethyl
group is flexible.
The structure of C. fasciculata cytochrome c (in red)
is overlaid with that of S. cerevisiae iso-1-cytochrome c
(in blue) in Fig. 3. The two cytochromes have 48%
amino acid identity and 72% similarity. The structures
are remarkably similar overall, and in the details of
the heme attachment and heme position. The rmsd
between the structure of C. fasciculata cytochrome c
and S. cerevisiae iso-1-cytochrome c is 0.94 A
˚
for the

104 a-carbon atoms fitted. The positions and stereo-
chemistries of both axial heme ligands are essentially
Time (s)
0 100 200 300 400 500
+ SHAM
+ Antimycin
55 nmol O
2
Fig. 1. Oxygen consumption by C. fasciculata in the presence of
respiratory inhibitors. C. fasciculata cells (3–7 · 10
7
cells in 0.66 mL
of culture medium taken directly from a growing culture and diluted
to 2 mL with water) were placed in the chamber of a Clark oxygen
electrode, and the oxygen consumption of the cells was measured
at room temperature. Inhibitors of alternative oxidase (SHAM) and
the cytochrome bc
1
complex (antimycin A) were added at the
points indicated to final concentrations of 3 m
M and 2 lgÆmL
)1
,
respectively. The electrode was calibrated using a saturated solu-
tion of air in water, and water treated with disodium dithionite to
remove all of the oxygen. One representative experiment (of
seven) is shown.
Fig. 2. The X-ray crystal structure of C. fasciculata cytochrome c to
1.55 A
˚

resolution. The molecule is shown rainbow colored, from
the N-terminus (residue 5) in blue to the C-terminus (residue 114)
in red. The heme cofactor is shown in ball and stick representation,
as are the methionine and histidine side chains that coordinate the
heme iron, and the cysteine side chain that forms a thioether bond
between heme and protein. Also shown is the methyl group of the
alanine of the AXXCH heme-binding motif, which is found in place
of the first cysteine of a typical c-type cytochrome CXXCH heme-
binding motif.
Structure of Crithidia fasciculata cytochrome c V. Fu
¨
lo
¨
p et al.
2824 FEBS Journal 276 (2009) 2822–2832 ª 2009 The Authors Journal compilation ª 2009 FEBS
identical between the two structures. These observa-
tions concur with those of Wuthrich et al., who used
proton NMR to investigate the heme environment in
cytochrome c from another trypanosomatid species in
the 1970s, and concluded that the heme crevice and
heme electronic structure were very similar to those in
mammalian (CXXCH) mitochondrial cytochrome c
[21,22]. (Notably, these authors also correctly predicted
the overall structure of the trypanosomatid cyto-
chrome.) The stereochemistry of heme attachment
through the thioether bond is conserved in C. fascicu-
lata cytochrome c (Figs 3 and 4); it therefore remains
the same (i.e. S-stereochemistry) as in all known c-type
cytochrome structures [1,13]. Strikingly, the methyl
group of Ala25 in C. fasciculata cytochrome c (the

equivalent residue of the first cysteine of the CXXCH
motif of S. cerevisiae cytochrome c) overlays almost
perfectly with the CH
2
group of the equivalent cyste-
ine. Thus, the position of the polypeptide chain around
the cysteine(s) is very similar in the two structures,
even though one has attachment through two thioether
bonds and the other through only a single bond. As
discussed in a recent review [23], ‘the backbone struc-
ture of the CXXCH motif [of typical c-type cyto-
chromes] shows little variation, even among proteins in
which the variable ‘‘XX’’ residues have very different
properties’. The observation that this remains true in
natural single thioether cytochrome c is a notable
finding.
Details of the heme attachment in C. fasciculata
cytochrome c are shown in Fig. 4. The methyl group
Fig. 3. Comparison between the structures of C. fasciculata cyto-
chrome c (protein main chain in red) and S. cerevisiae iso-1-cyto-
chrome c (Protein Data Bank entry: 1YCC) (in blue). Also shown are
the heme ligands (histidine and methionine in each case), the
cysteines that form thioether bonds to the heme, and the methyl
group of the alanine of the C. fasciculata AXXCH heme-binding
motif, which is found in place of the first cysteine of the S. cerevi-
siae CXXCH heme-binding motif. The rmsd between the structure
of C. fasciculata cytochrome c and S. cerevisiae iso-1-cytochrome c
is 0.94 A
˚
for the 104 a-carbon atoms fitted.

Fig. 4. Detail of the heme-binding site in C. fasciculata cyto-
chrome c from two angles. The
SIGMAA [50] weighted 2mF
o
)DF
c
electron density, using phases from the final model of the half-
reduced form, is contoured at the 1.5r level, where r represents the
rms electron density for the unit cell. Contours more than 1.5 A
˚
from
any of the displayed atoms have been removed for clarity. Thin lines
indicate heme axial ligand coordination and hydrogen bonds. Sulfur
atoms (in methionine and cysteine) are colored yellow, nitrogen
blue, oxygen red, and iron purple. The methyl group of the alanine of
the AXXCH heme-binding motif is green, and the unsaturated vinyl
group of heme is cyan. Pro41 is conserved in class I c-type cyto-
chromes, and its main chain carbonyl is hydrogen bonded to the N
d
atom of the heme axial histidine side chain; this interaction main-
tains the correct orientation of the histidine ring to the heme iron.
V. Fu
¨
lo
¨
p et al. Structure of Crithidia fasciculata cytochrome c
FEBS Journal 276 (2009) 2822–2832 ª 2009 The Authors Journal compilation ª 2009 FEBS 2825
of the alanine of the AXXCH motif (Ala25) (in
green) and the unsaturated vinyl group of the heme
(cyan) are separated by 3.41 A

˚
(as compared with a
typical thioether bond length of 1.8 A
˚
). As the posi-
tion of the polypeptide chains in this region is essen-
tially the same in the two structures (Fig. 3), this
means that in the C. fasciculata cytochrome the
heme moves away from the polypeptide relative to
its position in the S. cerevisiae protein. The unsatu-
rated 2-vinyl group of C. fasciculata cytochrome c
remains almost coplanar with the porphyrin ring.
The conserved residue Pro41 is hydrogen bonded
through its carbonyl group to the N
d
of the proxi-
mal heme histidine ligand [20]. The electron density
shows no additional modifications (e.g. oxidation) to
the sulfurs of the heme-binding methionine or cyste-
ine residues.
Maturation of trypanosome cytochrome c by
yeast cytochrome c heme lyase
Given the remarkable overall structural similarity
between the mitochondrial cytochromes c from C. fas-
ciculata and S. cerevisiae (Fig. 3), we also investigated
whether the poorly understood enzyme responsible for
heme attachment to cytochrome c in yeast, heme lyase,
can mature a trypanosomatid cytochrome c. Heme
lyases can mature AXXCH variants of yeast or human
cytochrome c, albeit at a lower level than the CXXCH

wild-type [24,25]. Thus, we coexpressed S. cerevisiae
cytochrome c heme lyase with either T. brucei cyto-
chrome c or a CXXCH variant in the cytoplasm of
E. coli (the cytochromes c from C. fasciculata and
T. brucei have 84% sequence identity and 92% simi-
larity, and both have an AAQCH heme-binding
motif). As a control, we also coexpressed heme lyase
with S. cerevisiae iso-1-cytochrome c. Cells expressing
the yeast cytochrome were bright red, and were shown
by absorption spectroscopy to have produced
$ 1.4 mg of cytochrome c per gram of wet cells
(assuming a reduced Soret band extinction coefficient
of 130 000 m
)1
Æcm
)1
[20]). However, neither wild-type
T. brucei cytochrome c nor the CXXCH variant was
matured at levels immediately detectable by spectros-
copy or by staining of SDS ⁄ PAGE gels for proteins
with covalently bound heme. Expression of the protein
was, however, readily confirmed by western blotting of
the E. coli cytoplasmic extracts using a polyclonal
antibody raised against recombinant T. brucei
CXXCH holocytochrome c (Fig. 5A), the latter
matured by the E. coli Ccm system [9]; this antibody
is sensitive to both T. brucei holocytochrome c and
T. brucei apocytochrome c (J. W. A. Allen, unpub-
lished observation). Following concentration of the
E. coli cytoplasmic extracts, both T. brucei wild-type

holocytochrome c and T. brucei CXXCH holocyto-
chrome c could be observed on heme-stained
SDS ⁄ PAGE gels, and ran at the same molecular
mass as purified recombinant T. brucei CXXCH
cytochrome c matured by the E. coli Ccm system
A
B
14 kDa
14 kDa
400 450 500 550 600
0.00
0.02
0.04
0.06
0.08
0.10
C
1 2 3 4
1 2 3 4 5 6 7 8 9 10
Normalised absorbance
Wavelength (nm)
Fig. 5. Maturation of T. brucei cytochrome c and a CXXCH variant
by S. cerevisiae cytochrome c heme lyase. (A) Western blot of
cytoplasmic extracts from E. coli coexpressing heme lyase and
cytochrome c, using a primary antibody raised against the CXXCH
variant of T. brucei holocytochrome c (the latter matured in the
periplasm of E. coli by the E. coli Ccm apparatus [9]). Lane 1:
molecular mass markers. Lanes 2–5: four independent cultures
expressing wild-type T. brucei cytochrome c. Lanes 6–9: four inde-
pendent cultures expressing the CXXCH variant of T. brucei cyto-

chrome c. Lane 10: as the positive control, purified CXXCH variant
T. brucei holocytochrome c matured in the periplasm of E. coli by
the E. coli Ccm apparatus. (B) SDS ⁄ PAGE gel of concentrated cyto-
plasmic extracts from E. coli coexpressing heme lyase and cyto-
chrome c, stained for proteins containing covalently bound heme.
Lane 1: molecular mass markers. Lane 2: wild-type T. brucei cyto-
chrome c. Lane 3: the CXXCH variant of T. brucei cytochrome c.
Lane 4: as the positive control, purified CXXCH variant T. brucei
holocytochrome c matured in the periplasm of E. coli by the E. coli
Ccm apparatus. (C) Absorption spectra of concentrated cytoplasmic
extracts from E. coli coexpressing heme lyase and cytochrome c.
Wild-type T. brucei cytochrome c (black line) and the CXXCH variant
(gray line). A few grains of disodium dithionite were added to the
samples to reduce the cytochromes. As no extinction coefficients
are available, the spectra were normalized by intensity of the Soret
band. The spectra were also corrected for light scattering by sub-
traction of a wavelength to the power four curve.
Structure of Crithidia fasciculata cytochrome c V. Fu
¨
lo
¨
p et al.
2826 FEBS Journal 276 (2009) 2822–2832 ª 2009 The Authors Journal compilation ª 2009 FEBS
(Fig. 5B). Yields of heme lyase-matured T. brucei
holocytochrome were calculated from absorption
spectra of the concentrated cytochromes (Fig. 5C) as
3.1 lg (wild-type) and 3.2 lg (CXXCH) of holocyto-
chrome c per gram of wet cells, assuming in each
case a reduced Soret band extinction coefficient of
130 000 m

)1
Æcm
)1
. This cytochrome maturation was
heme lyase-dependent, because virtually no holocyto-
chrome was observed if expressed in the absence of
heme lyase. We previously investigated maturation of
T. brucei cytochrome c in E. coli by the Ccm system,
which produced approximately 1.6 mg of CXXCH
variant holocytochrome per gram of wet cells [9];
hence, the low yields of heme lyase-matured holo-
cytochrome in the present work are not due to, for
example, poor expression of the apoprotein or
T. brucei codon usage. We conclude that both wild-
type and CXXCH T. brucei cytochrome c are
matured by heme lyase, to similar extents, but, sur-
prisingly, at only approximately 0.25% of the level
of maturation of S. cerevisiae iso-1-cytochrome c,
even though the various cytochromes are structurally
extremely similar.
Discussion
Euglenozoan cytochrome c – evolution and
maturation
We report here the first high-resolution structure of
mitochondrial cytochrome c from a euglenozoan
organism. The mitochondrial cytochromes c and c
1
from this evolutionarily divergent protist group are
unique because they contain heme covalently bound
through only one cysteine residue to an (A ⁄ F)XXCH

heme-binding motif, rather than through two thioether
bonds to CXXCH, as in all other eukaryotes. No
apparatus for the post-translational attachment of
heme to apocytochrome c has yet been identified in
any euglenozoan, and, in contrast to all other eukary-
otes possessing mitochondrial cytochromes c, no appa-
ratus is evident from the analysis of multiple
completely sequenced trypanosomatid genomes
[9,10,12]. Identification of the novel biogenesis system
for cytochrome c in trypanosomes is a demanding
task. Remarkably, despite these fundamental differ-
ences in heme attachment and cytochrome biogenesis,
the structure of C. fasciculata cytochrome c is very
similar to the structures of typical mitochondrial cyto-
chromes c, e.g. from S. cerevisiae (Fig. 3). This simi-
larity was also observed, other than the missing
thioether bond, in the details of the heme attachment
and around the heme-binding site (Figs 3 and 4) [21].
Different c-type cytochromes have very different folds
[1], but the structural arrangement of the heme-bind-
ing motif around the thioether linkages is absolutely
conserved. The present work extends this observation
to a cytochrome with a natural single cysteine heme-
binding motif. Moreover, as illustrated with E. gracilis
cytochrome c
558
, single thioether attachment of heme
does not significantly affect the reaction between the
cytochrome c and (mammalian) cytochrome bc
1

or
cytochrome aa
3
oxidase (when compared with horse
heart cytochrome c) [26]. The biophysical properties
of euglenozoan and typical mitochondrial cyto-
chromes c are also similar [8]. Priest and Hajduk [6]
speculated that single thioether heme attachment in
both cytochromes c and c
1
(rather than just in one)
might be mutually compensatory, allowing efficient
interaction between them for respiratory electron
transfer in spite of their distinctive mode of heme
binding. However, our data (Fig. 3) suggest that the
unique mode of heme attachment is probably unre-
lated to the interaction between cytochrome c and its
redox partners. There is no apparent structural com-
pensation for the presence of only one thioether bond
in C. fasciculata cytochrome c, although properties
such as the reduction potential may be subtly fine-
tuned by the protein.
There is no obvious reason from the protein struc-
ture or functional data why one group of protists has
evolved a unique type of cytochrome c and a corre-
sponding novel biogenesis pathway. However, it is
clear from our data that euglenozoan cytochrome c
could structurally accommodate two cysteines in a
typical CXXCH heme-binding motif (Fig. 3). So, how
might the occurrence of these single cysteine cyto-

chromes c be explained? Considerable evidence points
to catalyzed formation and subsequent reduction of
an intramolecular disulfide bond in the CXXCH
motif during cytochrome c biogenesis in bacteria [3];
this may also happen in yeast [27]. It therefore seems
plausible that evolution of the euglenozoan single cys-
teine heme-binding motif, while the protein structure
was otherwise retained (Fig. 3), relates to the redox
environment of the euglenozoan mitochondrial inter-
membrane space (IMS) (the location of the cyto-
chrome c). Loss of one cysteine from the
cytochrome c heme-binding motif could: (a) signifi-
cantly affect the interactions between the apocyto-
chrome and other thiol proteins in the IMS; and ⁄ or
(b) prevent the formation of an undesirable intramo-
lecular disulfide bond in the apocytochrome for
which no suitable reductant would be available in the
IMS; and ⁄ or (c) provide a selective advantage by alle-
viating a constraint on other IMS redox proteins.
V. Fu
¨
lo
¨
p et al. Structure of Crithidia fasciculata cytochrome c
FEBS Journal 276 (2009) 2822–2832 ª 2009 The Authors Journal compilation ª 2009 FEBS 2827
Our structure thus adds to other recent evidence [28]
hinting that the redox environment of the mitochon-
drial IMS in trypanosomatids may be different from
that in animals and yeast.
Notably, the stereochemistry of heme attachment

remains conserved in all cytochromes c, including
that from C. fasciculata (Figs 3 and 4). Heme is not
symmetrical; to date, it has always been observed to
be attached to cytochromes c with S-stereochemistry
[1], although the physiological advantage of such ste-
reospecific attachment is not known. It was therefore
not clear a priori whether euglenozoan cytochromes c
with heme attached through a single cysteine would
have the same attachment stereochemistry as cyto-
chromes c with two thioether linkages [13]. The fact
that they do must be reflected in stereospecific con-
trol of the heme attachment by the euglenozoan cyto-
chrome c biogenesis machinery. If heme is not
inserted into cytochromes in a controlled orientation,
it enters initially in a mixed, roughly equal popula-
tion, and equilibrates slowly until one orientation
dominates (as observed for b-type cytochromes and
globins) [29–31]. This implies that there may be a
heme-handling chaperone in the trypanosomatid
cytochrome c biogenesis machinery (as there is in
biogenesis System I [32]).
One surprising difference in the structure of C. fas-
ciculata cytochrome c relative to ‘normal’ CXXCH
c-type cytochromes is that the thioether bond between
heme and protein is longer than is typical (1.98–
2.25 A
˚
as compared with 1.8 A
˚
). Our structure was

refined with a nonredundant first part of the dataset,
which showed similar bond lengths, as did refinement
against a lower-resolution dataset collected at a much
less intense beamline (ESRF, BM16). Therefore, this
observation cannot be interpreted as a result of
X-ray-induced radiation damage; rather, it is an
intrinsic feature of the structure. In single thioether
cytochrome c, the heme is less constrained than in a
normal (CXXCH) c-type cytochrome, because it is
covalently anchored to the protein only once rather
than twice. This leads to greater conformational flexi-
bility of the heme, which is reflected, for example, in
broadening of the peaks in the absorption spectrum
[9,24]. Moreover, when heme is attached to a
CXXCH motif, the (quite significant) strain of con-
straining the heme position is spread over two thioe-
ther bonds plus the histidine ligand to the iron,
whereas in the euglenozoan mitochondrial cyto-
chromes, the load must be borne by only one thioe-
ther bond plus the histidine. Together, these factors
presumably lead to a weaker, and hence longer, thioe-
ther bond.
Cytochrome c maturation by other biogenesis
systems
The structure reported here is also informative in the
context of the failure of the E. coli Ccm apparatus to
effectively mature wild-type (AXXCH) T. brucei cyto-
chrome c [9]; the system can mature both a CXXCH
variant [9] and the structurally very similar (Fig. 3)
yeast (CXXCH) mitochondrial cytochrome c [33].

Hence, we can now conclude with a high degree of
confidence that the inability of the Ccm system to
mature the single cysteine trypanosomatid cyto-
chrome c was due to the Ccm apparatus itself, and not
to some (previously unidentified) structural feature of
the substrate cytochrome. It has been argued that cyto-
chrome c biogenesis System II can catalyze single
cysteine heme attachment within the four-heme c-type
cytochrome NrfH from Wolinella succinogenes which is
unrelated to mitochondrial cytochrome c [34]. The
present work suggests that such single cysteine attach-
ment could be structurally accommodated within that
protein as a variant of the wild-type double cysteine
attachment. The bioinformatic implications are clear;
an XXXCH sequence could be indicative of a c-type
cytochrome in genomes of bacterial species that use
System II [34]. (Note also that the cytochrome b
6
f
complex of cyanobacteria and chloroplasts contains a
heme covalently bound to protein via a single thioether
bond; this heme attachment is dependent on the
recently described biogenesis System IV [12].)
Our data further show, unexpectedly, that S. cerevi-
siae heme lyase (System III) matures a CXXCH vari-
ant of T. brucei cytochrome c very poorly, in spite of
the great structural homology between the trypanoso-
matid and yeast cytochromes. There are two extreme
possibilities for how the scarcely understood heme
lyase recognizes its target apocytochrome. The first, by

analogy with the Ccm system [35], is that it recognizes
little more than the CXXCH heme-binding motif. The
second is that it recognizes as yet undefined features of
the apoprotein, leading to a productive complex within
which heme is attached. Our results here suggest the
second possibility, and that the recognition features in
the apocytochrome are not related to the overall struc-
ture of the cytochrome. This complements the previous
observation that heme lyase is unable to mature a
bacterial class I c-type cytochrome, Paracoccus denitrif-
icans cytochrome c
550
[33]. Moreover, many taxa that
have heme lyase apparently have separate heme lyases
for the maturation of cytochromes c and c
1
[10]; this
has been demonstrated biochemically for S. cerevisiae,
where only very limited overlap of substrate specificity
was observed [36]. Again, ‘simple’ interaction between
Structure of Crithidia fasciculata cytochrome c V. Fu
¨
lo
¨
p et al.
2828 FEBS Journal 276 (2009) 2822–2832 ª 2009 The Authors Journal compilation ª 2009 FEBS
heme lyase and the apocytochrome CXXCH motif
would appear to be unlikely if separate heme lyases are
required to mature cytochromes c and c
1

. Bernard et al.
[36] identified point mutations in S. cerevisiae cyto-
chrome c
1
that enhanced the activity of cytochrome c
heme lyase towards the former protein. The identifica-
tion of such residues both within and upstream of the
CXXCH heme-binding motif supports our conclusion
that heme lyase recognizes specific features in its apocy-
tochrome substrate, rather than just the heme-binding
motif or the overall fold of the protein.
Experimental procedures
C. fasciculata choanomastigotes were cultured at 27 °Cin
media containing 37 gÆL
)1
heart–brain infusion, 10 mgÆL
)1
hemin, 10 mgÆL
)1
folic acid and 5% (v ⁄ v) heat-inactivated
fetal bovine serum. Cells inoculated at $ 10
4
cellsÆmL
)1
in
500 mL tissue culture flasks containing 100 mL of medium
were grown for 60–72 h. Harvested cells were washed twice
in NaCl ⁄ P
i
(8 gÆL

)1
NaCl, 0.2 gÆL
)1
KCl, 1.42 gÆL
)1
Na
2
HPO
4
and 0.27 gÆL
)1
KH
2
PO
4
, pH 7.2) and stored at
)80 °C until required. Growth assays for C. fasciculata
were conducted by the addition of respiratory inhibitors as
described in the text; growth was assessed either by counts
using a hemocytometer, or by measurement of D
600 nm
val-
ues. Respiration of C. fasciculata was also investigated
using an oxygen electrode (Rank Brothers, Bottisham,
UK), which was calibrated and used according to the man-
ufacturer’s directions. Cells were placed in the electrode
chamber in their growth medium, and respiratory inhibitors
were added as required.
Purification of cytochrome c
Extracts of C. fasciculata were prepared by disrupting the

cells from $ 20 L of culture twice in buffer containing
1.42 gÆL
)1
Na
2
HPO
4
, 0.27 gÆL
)1
KH
2
PO
4
,1mm
Na
2
EDTA, 2 mm EGTA, 5 lm 2-mercaptoethanol, 0.25
mm phenylmethanesulfonyl fluoride, four ‘Complete’ prote-
ase inhibitor tablets (Roche) for every 50 mL of buffer
(containing $ 2 · 10
11
cells), and 2% (v ⁄ v) Nonidet P40
Substitute detergent (Igepal CA-630; USB Corporation,
Cleveland, OH, USA). Cell debris was removed by centrifu-
gation at 17 000 g for 15 mins, and the soluble cell extracts
were diluted five-fold with 50 mm Tris ⁄ HCl buffer (pH
8.0), and then applied to an XK26 ⁄ 20 column containing
SP-Sepharose fast-flow resin (GE Healthcare, Amersham,
UK) at room temperature. The column was washed with
the same buffer, and then with 50 mL of 2 mm K

3
Fe(CN)
6
dissolved in the buffer to ensure that the C. fasciculata
cytochrome c (sometimes called cytochrome c
555
[5]) was all
oxidized. The protein was eluted from the column with a
500 mL gradient of 0–500 mm NaCl in 50 mm Tris ⁄ HCl
buffer (pH 8.0), with a flow rate of 10 mLÆmin
)1
;8mL
fractions were collected. Fractions were assessed by their
red color, and those with maximum Soret band absorbance
more than one-third that of the best fraction were retained.
The pooled fractions were diluted five-fold in 50 m m
Tris ⁄ HCl (pH 8.0), and applied to an XK26 ⁄ 20 column
containing CM-Sepharose fast-flow resin (GE Healthcare)
at room temperature. The protein was eluted as described
above. Retained fractions containing the purest cytochrome
were concentrated to a volume of $ 1.5 mL, and applied to
a Sephacryl S-200 column (2.6 cm diameter, 1 m length),
pre-equilibrated with 50 mm potassium phosphate buffer
(pH 7.0). This chromatography step was conducted at 4 °C.
The cytochrome was eluted in the same buffer at a flow
rate of 15 mLÆh
)1
; 5 mL fractions were collected and
assessed for purity by absorption spectroscopy and
SDS ⁄ PAGE. Those with A

Soret
(oxidized) ⁄ A
280 nm
(oxi-
dized) > 3.8 were regarded as pure, and were concentrated
to 16 mgÆmL
)1
($ 1.3 mm) for crystallography.
Crystallization, structure determination, and
model refinement
Redox homogeneity of the purified C. fasciculata cyto-
chrome c was ensured by the addition of 5 mm K
3
Fe(CN)
6
.
Crystals initially formed in sitting drops made by mixing
the protein 1 : 1 with a solution containing 2.7 m
(NH
4
)
2
SO
4
, 0.1 m Hepes (pH 6.5) and 0.1 m LiCl at 19 °C
(all crystallographic reagents purchased from Hampton
Research, Aliso Viejo, CA, USA). These crystals were of
poor quality, but they were used for seeding. Diffraction-
quality crystals were produced in hanging drops by mixing
the protein 1 : 1 with 2.45 m (NH

4
)
2
SO
4
and 0.1 m Hepes
(pH 6.5); these drops were seeded with microcrystals after
equilibration for $ 48 h. Crystals grew, and were harvested,
within 1 week. Crystals were then picked up from the
mother liquor containing 15% glycerol using a cryoloop,
placed in a nitrogen stream at 100 K, and stored in liquid
nitrogen until data collection. Initial diffraction data were
collected at beamline BM16 (European Synchrotron Radia-
tion Facility), but the final dataset used for structure deter-
mination and refinement was collected at the Diamond
Light Source, UK. Integration and scaling were performed
using denzo and scalepack [37]. Subsequent data handling
was carried out using the ccp4 software package [38].
Molecular replacement was carried out using the coordi-
nates of S. cerevisiae iso-1-cytochrome c (Protein Data
Bank code: 1YCC) as a search model with the phaser pro-
gram [39]. Refinement of the structure was carried out by
alternate cycles of refmac [40], using noncrystallographic
symmetry restraints and manual rebuilding in o [41]. Water
molecules were added to the atomic model automatically
by arp ⁄ warp [42], and in the last steps of refinement all
the noncrystallographic symmetry restraints were released.
V. Fu
¨
lo

¨
p et al. Structure of Crithidia fasciculata cytochrome c
FEBS Journal 276 (2009) 2822–2832 ª 2009 The Authors Journal compilation ª 2009 FEBS 2829
A summary of the data collection and refinement statistics
is given in Table 1. Figures were drawn using molscript
[43,44] and rendered with raster 3d [45].
Heme lyase maturation assays
Maturation of T. brucei cytochrome c by heme lyase was
investigated in the cytoplasm of E. coli BL21-DE3 cells.
Plasmids for T. brucei cytochrome c (pKK223–Tbcytc), its
CXXCH variant (pKK223–TbcytcCXXCH), S. cerevisiae
cytochrome c heme lyase (pACcyc3) and iso-1-cytochrome c
(pScyc1) were as previously described [9,33]. Cells were
cotransformed with the heme lyase plasmid and the plasmid
for each of the cytochromes, respectively. Cells were grown
overnight at 37 °C with vigorous shaking, in 50 mL of 2·
TY medium (16 gÆL
)1
peptone, 10 gÆL
)1
yeast extract,
5gÆL
)1
NaCl) supplemented with 100 lgÆmL
)1
ampicillin,
34 lgÆmL
)1
chloramphenicol and 1 mm isopropyl-thio-b-d-
galactoside. Nine separate cultures were grown for each

combination of heme lyase and cytochrome. The E. coli
periplasmic fraction was prepared as previously described
[46], and discarded. The spheroplast pellet was resuspended
by vigorous vortexing in 50 mm Tris ⁄ HCl plus 150 mm
NaCl (pH 7.3), and broken by six freeze–thaw cycles (at
)78 and 37 °C); this was followed by centrifugation at
25 000 g for 1 h to remove the cell debris. The soluble cyto-
plasmic fraction was initially assayed by running the pro-
teins on SDS ⁄ PAGE gels that were stained for proteins
containing covalently bound heme [47]. Subsequently, the
extracts from multiple cultures were pooled and applied to a
5 mL Hi-Trap column containing SP-Sepharose (GE
Healthcare). The bound protein was batch eluted using
500 mm NaCl, concentrated, and then assessed using
absorption spectroscopy and heme-stained SDS ⁄ PAGE gels.
Western blotting was performed using a polyclonal
primary antibody raised against purified, recombinant, Ccm
system-matured CXXCH variant T. brucei holocyto-
chrome c (protein as described in [9]; antibody raised by
Covalab, Villeurbanne, France). Unconcentrated E. coli
soluble cytoplasmic extracts were resolved by SDS ⁄ PAGE
and blotted onto Hybond-C Extra nitrocellulose membrane
(GE Healthcare). The membrane was blocked for 1 h in 5%
(w ⁄ v) milk powder dissolved in NaCl ⁄ Tris [50 mm Tris ⁄ HCl,
pH 7.5, 120 mm NaCl, 1% (v ⁄ v) Tween-20]. It was then
incubated for 1 h with primary antibody diluted 200-fold in
10 mL of 5% milk ⁄ NaCl ⁄ Tris solution; the primary anti-
body was used as crude (unpurified) serum. The membrane
was washed four times (1 · 15 min, 3 · 5 min) in 10 mL of
NaCl ⁄ Tris, and then incubated with the secondary antibody

for 1 h in 30 mL of 5% milk ⁄ NaCl ⁄ Tris; the secondary anti-
body was affinity purified anti-rabbit IgG whole molecule
alkaline phosphatase conjugate (purchased from Sigma,
Poole, UK), and was used at 6000-fold dilution. The mem-
brane was then washed three times in NaCl ⁄ Tris (each wash
for 5 min), and stained by incubation in 10 mL of H
2
O
containing a dissolved FAST 5-bromo-4-chloroindol-2-yl
phosphate ⁄ Nitro Blue tetrazolium tablet (Sigma).
Acknowledgements
This work was supported by the Biotechnology and
Biological Sciences Research Council [grant numbers
BB ⁄ C508118 ⁄ 1 and BB ⁄ D019753 ⁄ 1]. J. W. A. Allen is
a BBSRC David Phillips Fellow, and M. L. Ginger is
a Royal Society University Research Fellow. KAS is
the William R. Miller Junior Research Fellow,
Table 1. Summary of crystallographic data collection and refine-
ment statistics. Numbers in parentheses refer to values in the high-
est-resolution shell. R
sym
¼
P
j
P
h
I
h;j
ÀhI
h

ij


.
P
j
P
h
hI
h
i, where
I
h,j
is the jth observation of reflection h, and <I
h
> is the mean inten-
sity of that reflection. R
cryst
¼
P
F
obs
jj
À F
calc
jjjj=
P
F
obs
j

, where
F
obs
and F
calc
are the observed and calculated structure factor
amplitudes, respectively. R
free
is equivalent to R
cryst
for a 4% sub-
set of reflections not used in the refinement [48]. DPI, diffraction
component precision index [49].
Data collection
Synchrotron radiation,
detector and
wavelength (A
˚
)
Diamond, IO2, ADSC
Q315 CCD 0.9511
Unit cell (A
˚
) a = 85.93, b = 110.88,
c = 60.93, b = 131.2
Space group C2
Resolution (A
˚
) 56–1.55 (1.61–1.55)
Observations 392 320

Unique reflections 60 049
I ⁄ r(I) 14.8 (2.0)
R
sym
0.118 (0.693)
Completeness (%) 97.6 (100.0)
Refinement
Nonhydrogen atoms 3199 (including three c-type
hemes, seven sulfates and
563 waters)
R
cryst
0.210 (0.291)
Reflections used 57 638 (4079)
R
free
0.247 (0.320)
Reflections used 2411 (171)
R
cryst
(all data) 0.212
Average temperature
factor (A
˚
2
)
24.4
Protein 21.3
Hemes 15.4
Solvent 39.4

Wilson plot 21.6
Rmsds from ideal values
Bonds (A
˚
) 0.016
Angles (°) 1.7
DPI coordinate error (A
˚
) 0.09
Ramachandran plot
Most favored (%) 89.1
Additionally allowed (%) 10.9
Structure of Crithidia fasciculata cytochrome c V. Fu
¨
lo
¨
p et al.
2830 FEBS Journal 276 (2009) 2822–2832 ª 2009 The Authors Journal compilation ª 2009 FEBS
St Edmund Hall, Oxford. We thank N. Brown for very
helpful crystallographic discussions, A. Holehouse for
preliminary experiments, and H. Lill for the gift of
plasmids encoding the S. cerevisiae proteins used in
this work. Crystallographic data were collected at
beamline IO2 at Diamond Light Source, UK, and we
acknowledge the support of T. Sorensen at Diamond
and A. Labrador at the ESRF, France.
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