Remarkably high activities of testicular cytochrome c
in destroying reactive oxygen species and in
Zhe Liu*†‡, Hao Lin*‡§, Sheng Ye†, Qin-ying Liu§, Zhaohui Meng†, Chuan-mao Zhang§, Yongjing Xia*,
Emanuel Margoliash¶?, Zihe Rao†?, and Xiang-jun Liu*?
*Institute of Biomedical Informatics, School of Medicine, and†MOE Laboratory of Protein Science and Laboratory of Structural Biology, Tsinghua University,
Beijing 100084, China;§State Key Laboratory of Biomembrane and Membrane Biotechnology, College of Life Sciences, Peking University, Beijing 100871,
China; and¶Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208
Contributed by Emanuel Margoliash, April 28, 2006
Hydrogen peroxide (H2O2) is the major reactive oxygen species
(ROS) produced in sperm. High concentrations of H2O2 in sperm
induce nuclear DNA fragmentation and lipid peroxidation and
result in cell death. The respiratory chain of the mitochondrion is
one of the most productive ROS generating systems in sperm, and
thus the destruction of ROS in mitochondria is critical for the cell.
It was recently reported that H2O2 generated by the respiratory
chain of the mitochondrion can be efficiently destroyed by the
cytochrome c-mediated electron-leak pathway where the electron
of ferrocytochrome c migrates directly to H2O2 instead of to
cytochrome c oxidase. In our studies, we found that mouse testis-
specific cytochrome c (T-Cc) can catalyze the reduction of H2O2
three times faster than its counterpart in somatic cells (S-Cc) and
that the T-Cc heme has the greater resistance to being degraded by
H2O2. Together, these findings strongly imply that T-Cc can protect
sperm from the damages caused by H2O2. Moreover, the apoptotic
activity of T-Cc is three to five times greater than that of S-Cc in a
well established apoptosis measurement system using Xenopus
egg extract. The dramatically stronger apoptotic activity of T-Cc
might be important for the suicide of male germ cells, considered
a physiological mechanism that regulates the number of sperm
produced and eliminates those with damaged DNA. Thus, it is very
likely that T-Cc has evolved to guarantee the biological integrity of
sperm produced in mammalian testis.
mouse testis ? antioxidation
duction of reactive oxygen species (ROS) in sperm, which can
induce nuclear DNA fragmentation, lipid peroxidation, and pro-
form in sperm is hydrogen peroxide (H2O2), the concentration of
which in sperm has not been measured (2–4), whereas in liver cells,
to 10,000-fold higher than that of superoxide (O2
liter) (6, 9, 10). O2
can be rapidly converted to H2O2by several pathways and does not
accumulate in the cell (10, 11). The downstream ROS product of
H2O2, namely hydroxyl radical (?OH), is highly reactive in directly
inducing DNA single strand break and lipid peroxidation (12, 13).
of the cell, making it the most prevalent ROS form (14), its
reduction becomes a major challenge for the normal functions of
the cell. Recently, it was demonstrated that the cytochrome c
(Cc)-mediated electron-leak pathway can strongly suppress the
formation of H2O2by the respiratory chain and efficiently destroy
preexisting H2O2(10, 15, 16). Cc is thus considered a significant
physiological contributor for H2O2detoxification in the cell (10,
oughly half of infertility cases are caused by male infertility (1).
One of the mechanisms of male infertility is the excess pro-
??, as the precursor of H2O2and a short-lived ion,
cells because of their high content of polyunsaturated fatty acids
within the plasma membrane and a low concentration of ROS
scavenging enzymes in the cytoplasm (4, 18). During the develop-
ment of male mammalian germ cells, a testis-specific form of Cc
(T-Cc) that differs from its counterpart in somatic cells (S-Cc) is
expressed. In the mouse, the genes coding for T-Cc and S-Cc are
located at chromosomes 2 and 6, respectively (19). Although the
primary structures of these two proteins are 86.5% identical, T-Cc
and S-Cc are different not only in the intron–exon genomic
gradually declines as the cells undergo meiosis, with pachytene
(21). Thus, the biochemical property changes of T-Cc would have
great impact on the Cc-mediated biological processes in male germ
cells rich of T-Cc, which include electron transfer chain operation,
antioxidation, and probably apoptosis.
Here, we compare the biological functions of T-Cc with those of
S-Cc from various aspects. First, measurements of the electron
transfer activities with Cc oxidase (CcO) in beef mitochondrial
particles show that T-Cc is the better substrate in an alkaline
environment. Secondly, the apoptotic activity of T-Cc is shown to
be three to five times larger than that of S-Cc in the Xenopus egg
extract apoptosis measurement system. Furthermore, we demon-
strate that T-Cc catalyzes the reduction of H2O2three times faster
than S-Cc and that the T-Cc heme has the greater resistance to
being degraded by H2O2. Finally, in attempting to illustrate these
differences of T-Cc from S-Cc, a 1.6-Å crystal structure of T-Cc is
resolved and provides a structural interpretation to the increased
H2O2detoxification activity of T-Cc.
For further details, see Supporting Text, Figs. 6–9, and Table 3,
which are published as supporting information on the PNAS web
Overall Structure of Mouse T-Cc. The final structure of T-Cc was
The (?, ?) angles of all residues were evaluated by Ramachandran
plot (data not shown) and, except for several glycines and for
Conflict of interest statement: No conflicts declared.
Abbreviations: ROS, reactive oxygen species; Cc, cytochrome c; T-Cc, testis-specific Cc; S-Cc,
somatic Cc; CcO, Cc oxidase; hh-Cc, horse heart Cc.
Data deposition: The atomic coordinates have been deposited in the Protein Data Bank,
www.pdb.org (PDB ID code 2AIU).
‡Z.L. and H.L. contributed equally to this work.
?To whom correspondence may be addressed. E-mail: firstname.lastname@example.org, frankliu@
tsinghua.edu.cn, or email@example.com.
© 2006 by The National Academy of Sciences of the USA
June 13, 2006 ?
vol. 103 ?
no. 24 ?
residues are within the permitted region. The skeleton of T-Cc is
similar to that of horse heart Cc (hh-Cc; Protein Data Bank ID
1HRC) with the greatest rms deviations (rmsd) at the residue
regions 1–5, 21–23, 37–46, 55–58, 85–87, and 96–104. These
differences largely occur at the regions with the differences of the
primary structures of the two proteins. All of the residues in T-Cc
differing from those in S-Cc are located at the distal side (back side
as shown in Fig. 1) of T-Cc, whereas there are few changes at the
proximal side. The prosthetic group, the heme, bonded covalently
to Cys-14 and Cys-17, is buried in the hydrophobic pocket sur-
rounded by the upper part (residues 1–58) and bottom part
by two loop regions from the upper part (residues 14–18, 28–30)
and bottom part (residues 78–83). The inner part of the protopor-
phyrin ring of the heme is involved in a network of hydrogen bonds
with the rest of the protein (Table 1). In T-Cc, the hydrogen bond
interactions around the posterior propionate of the heme show
significant variations compared to those of hh-Cc (Table 1).
in the protein, of which five (Fig. 1) are buried in the internal part
of T-Cc under the van der Waals surface of the protein. These five
of these five water molecules were confirmed by the 2Fo ? Fc
electron density map (Supporting Text and Fig. 8). The other 219
water molecules are all at the surface of the protein, and form a
hydrogen-bond network stabilizing the surface area of T-Cc.
Kinetic Assays. Inthepresenceof170?MH2O2,ferroT-Ccreduced
H2O23-fold faster than S-Cc and hh-Cc (Figs. 2A and 9). From 0
to 30 ?M Cc, the reaction appeared as a first-order reaction where
Vo ? K ? [Cc], where the reaction constants K (?M?2?s?1) for
T-Cc, S-Cc, and hh-Cc are shown in Table 2, with the K value of
T-Cc being about three times that of S-Cc and hh-Cc.
As indicated by the decrease of the Soret band absorption at 408
nm (Fig. 2B), the heme of ferriT-Cc showed much more resistance
to being degraded by excess H2O2 (3 mM) than ferriS-Cc and
ferrihh-Cc. With the same initial concentration, the half lives of
T-Cc, S-Cc, and hh-Cc were 604 s, 410 s, and 300 s, respectively.
Confirming the decrease at 408 nm, the complete spectrum of the
Soret band of T-Cc showed the expected changes with time (Fig.
under the same conditions, the heme of the three ferroCc’s used is
not degraded by H2O2as long as they remain in the ferrous state
(Fig. 2D). The curves of heme degradation and of oxidation of the
behaviors. The first phase, a flat line at 408 nm, indicating no heme
degradation, and a dropping curve at 550 nm, representing con-
tinuous oxidation of the protein to the ferric state, demonstrated
that Cc can protect its heme from H2O2degradation when the
protein is not fully in the ferric form. In the second phase, the fully
ferric Cc (as seen from the flat line at 550 nm) started to be
destroyed by H2O2, generating a slope at 408 nm.
Fig. 2E and Table 2 show that the reduction rate of T-Cc by
ascorbate was ?1?3 and 1?2 that for S-Cc and hh-Cc, respectively.
beef mitochondrial particle preparation were compared by mea-
buffer, but T-Cc was more efficiently oxidized than S-Cc in a more
S-Cc indicated by the lower response to pH changes (Fig. 2F).
Apoptotic Activity of Mouse T-Cc in Xenopus Egg Extracts. The
threshold concentration for T-Cc to fully activate caspase-3, one of
the downstream components from Cc in the intrinsic apoptosis
pathway (23), was 0.1 ?M and 3- to 5-fold lower than that for S-Cc
(0.3–0.5 ?M) (Fig. 3A), indicating that T-Cc has a much higher
apoptotic activity than S-Cc. To further confirm this conclusion,
two other independent hallmarks of apoptosis were used, namely
chromatin condensation (Fig. 3B), which is caused by the loss of
chromatin decondensation activity of nucleoplasmin, and DNA
Table 2. Summary of kinetic assays
Reducing activity, K, sec?1? 10?3
Degradation constant, KD, sec?1? 10?4
Half life of bleaching, T1/2, sec
Ascorbate reduction, KR, sec?1? 10?4
from the curve in Fig. 2B. The linear regions of Fig. 2C (1-20 ?M) yield the
apparent first-order reduction constant, KR.
viewed from the heme opening of the molecule. Backbones are drawn with
are shown in the ball and stick model and colored orange. Five water mole-
cules located in the internal part of the protein are in cyan.
Schematic diagram of T-Cc illustrated from the proximal side, as
Table 1. Heme propionate hydrogen bond interactions
Bank ID 2AIU)
Bank ID 1hrc)
Heme O1A-Try-48 OH*
Heme O1A-Water A OH*
Heme O1A-Water B OH*
Heme O2A-Trp-59 NE1*
Heme O2A-Asn-52 ND2*
Heme O2A-Gly-41 N*
Heme O1D-Thr-49 N
Heme O2D-Thr-49 OG1
Heme O2D-Lys-79 N
Heme O2D-Thr-78 OG1
Value in parentheses is averaged mean for rice, horse, iso-1, and iso-2 Cc.
Water 139 in hh-Cc, respectively.
*The interactions around Arg-38.
†Listed for comparison.
www.pnas.org?cgi?doi?10.1073?pnas.0603327103Liu et al.
fragmentation (Fig. 3C), which is the result of digesting chromatin
by caspase-activated DNase?DFF40 (DNA fragmentation factor,
40 kDa). The differences of activities between T-Cc and S-Cc in
these two experiments were similar to those in the caspase-3
Differences of Internal Water Patterns Between Testicular and So-
matic Cytochromes c. From the comparison of the crystal structures
highly conserved in the vicinity of the heme. Surprisingly we found
that W139 is missing in T-Cc (Fig. 4A), whereas two internal water
molecules are added. One of them (W512) has a high level of
vibration as indicated by the relatively large value of the calculated
B factor. W313 forms three hydrogen bonds to Arg-38. W512,
located just next to W313, also forms a hydrogen bond to Arg-38.
The other new internal water molecule, W308, has a low level of
water molecule which is absent in T-Cc as compared to other
cytochromes c. As shown in Fig. 4A, W308 forms hydrogen bonds
comparing to the standard curve for pNA. (B) Chromatin condensation induced by adding 1 ?M T-Cc and erythrocyte nuclei into Xenopus egg extracts. Samples
were fixed and stained by DAPI at the time indicated for fluorescence observation. (C) DNA fragmentation assays were carried out by adding various
concentrations of T-Cc, S-Cc, and 105chicken erythrocyte nuclei into Xenopus egg extracts, followed by electrophoresis (see Materials and Methods).
Apoptotic activity of T-Cc. (A) Effect of T-Cc and S-Cc on the rate of DEVD-pNA cleavage. Initial rates of DEVD-pNA cleavage were determined by
at 408 nm. Spectra were scanned every 3 min. (D) Time course of ferroT-Cc oxidation (solid line) and inactivation (dashed line) in the presence of 3 mM H2O2.
(E) Reduction titration of T-Cc, S-Cc, and hh-Cc with ascorbate. (F) Activities of purified CcO with T-Cc and S-Cc. Measurements were performed under both low
pH (50 mM phosphate?Tris, pH 6.5) and high pH (25 mM acetate?Tris, pH 7.8) conditions.
Kinetic studies of T-Cc. (A) The initial rates of H2O2reduction by various concentrations of ferroT-Cc, ferroS-Cc, and ferrohh-Cc. (B) Degradation of the
Liu et al.
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to three residues, including two residues invariant in all cyto-
chromes c (Arg-38, Asn-31) and Trp-33, which is substituted by
His-33 in all currently known mammalian S-Cc.
Evolutionary Analysis. The amino acid sequences of 21 Cc genes
were retrieved from the NCBI database. From the evolutionary
a phylogenetic tree (Fig. 5), it turns out that the testicular and
somatic cytochromes c are clustered into two classes (Fig. 5). These
tissue-specific Cc genes are thought to evolve by duplication from
years for every residue difference along two divergent lines of Cc
(26), the 14-aa residue differences between S-Cc and T-Cc implies
that they separated from each other ?150 million years ago.
A fundamental question is why a testis-specific Cc has evolved in
namely, electron transfer, apoptosis and antioxidation (10, 23, 27),
changes in T-Cc as compared to S-Cc.
Has T-Cc particularly evolved to fulfill the high energy require-
activities of T-Cc and S-Cc with CcO in beef mitochondrial particle
sperm is the same to that in somatic cells, the similar rates of
reaction of T-Cc and S-Cc with CcO at pH 6.5 suggest that there is
no significant difference of electron transfer activities between
T-Cc and S-Cc. Likewise, the much higher energy need in motile
sperm is satisfied by the increased concentration of mitochondria
within the midpiece of sperm (28). The energy needs in sperm can
be met even without T-Cc, because mice deficient of T-Cc are still
fertile (20). Moreover, in mammalian cells, the cellular ATP
concentration is sufficiently high (?2 mM) to keep cultured cell
alive for several days upon ATP synthase inhibition (29). Taken
together, these observations argue strongly against the possibility
of sperm. On the other hand, because Cc interacts with CcO by its
proximal side (30), where the residues are the same in T-Cc and
S-Cc (Fig. 1), the very different rates of oxidation of these two
proximal side structure must be involved. However, what these
factors are and how they affect the reaction at different pH values
need further investigation.
The apoptosis of male germ cells is proposed to be a mechanism
regulating the quantity and quality of sperm produced in mamma-
lian testis (20). Although, to date, there is still no direct evidence
that Cc is the principal factor responsible for signaling apoptosis in
constructed as described in Materials and Methods. The clades for testicular
and somatic Cc’s are marked pink and green, respectively.
Evolutionary analysis. Phylogenetic tree for Cc’s from various species,
www.pnas.org?cgi?doi?10.1073?pnas.0603327103Liu et al.
male germ cells, it has recently been demonstrated that the mito-
chondria-dependent pathway of apoptosis plays a key role in the
death processes of male germ cells (31). Moreover, infertile males
show significant elevated levels of Cc, Caspase-9 (the direct down-
Caspase-3 in their semen samples (32). These two observations
strongly underscore the importance of the Cc mediated apoptotic
pathway in the apoptosis of male germ cells. The apoptotic activity
system. This dramatically stronger apoptotic activity of T-Cc might
be important for the apoptosis of male germ cells. A rational
indication is that T-Cc could make male germ cells, as compared to
somatic cells, undergo apoptosis more easily in reacting to ROS
challenges, and therefore play a much more stringent ‘‘quality
control’’ role, as compared to S-Cc. T-Cc has 14 residues differing
from those in S-Cc, almost all of which are located at the distal
surface of the protein (Fig. 1) and might account for the enhanced
apoptotic activity of T-Cc.
Imbalance between ROS production and antioxidant capac-
ity in sperm would lead to increased production of oxidants,
causing mitochondrial and genomic damages and finally re-
sulting in cell death (2–4). Cc works as a ‘‘bodyguard’’
protecting cells from damages by H2O2(10, 15, 16), the major
form of ROS in sperm. The H2O2reduction activity of T-Cc
is 3-fold that of S-Cc. Therefore, in addition to the possible
‘‘quality control’’ role after ROS damages of mitochondria and
nuclear DNA, T-Cc can also prevent these damages by much
more efficiently destroying H2O2. Down-regulation of the
H2O2production in sperm can thus ensure the integrity of the
genome transmitted by sperm.
Interestingly, what confers to T-Cc the much stronger H2O2
reduction activity than S-Cc? The crystal structure of T-Cc reveals
that the environment around Arg-38 of T-Cc is significantly altered
from that of hh-Cc (Fig. 4 A and B). Arg-38 affects the redox
behaviors of Cc by charge-charge interactions with the heme
posterior propionate and?or by hydrogen bonding to the heme
posterior propionate mediated by surrounding water molecules,
namely W125 and W139 (22, 27, 33, 34). The Arg38Ala mutant of
yeast iso-1 Cc shows a significant increase of its reduction potential
(?40 mV) and a more stable oxidized form compared to the
wild-type yeast iso-1 Cc (34). Our experiments also demonstrate
and hh-Cc (Table 2). The ascorbate-binding site is suggested to be
at the crevice defined by Arg-38 (35, 36). Therefore, in the absence
of significant changes elsewhere, the above observations jointly
suggest that the changes of T-Cc from S-Cc around Arg-38,
extraordinarily high activity of T-Cc in H2O2 reduction, and its
resistance to ascorbate reduction.
Whether the testicular form of the Cc gene is present only in
rodent mammals has not been examined directly. However, the
evolutional divergence of T-Cc from S-Cc, occurred ?150 million
years ago as calculated above (Evolutionary Analysis) before the
genome (25). Recently, the coding DNA sequence of a T-Cc like
gene was also isolated from cow cDNA (GenBank accession no.
AAI02715.1). These observations strongly imply that, no matter
whether functional or not, the T-Cc gene is not only present in
rodent mammals but is very prevalent in mammals in general.
Cc serves as an electron carrier in bacteria (27). As evolution
progressed, new roles appeared to be added to this antique protein
in different organisms. There are two main possibilities by which
this can be obtained, either by conserving the essential domains
while mutating others, such as in the case of the apoptotic function
of mammalian somatic Cc, or by gene duplication followed by
divergent mutations to obtain novel functions, such as the Cyt-cd
and Cyt-cp cytochromes c of Drosophila (24, 25). In Drosophila, the
Cyt-cd form is required for the final stage of spermatic terminal
differentiation through an apoptosis-like mechanism (39), whereas
the Cyt-cp isoform is the usual form of Cc used by all tissues of the
gene with independent evolution for ?100 million years, protects
hand and might be adapted to eliminate damaged male germ cells
that T-Cc has evolved to ensure the fidelity and efficiency of the
DNA transmission by sperm.
Materials and Methods
Construction and Expression of Plasmids. The cDNA of T-Cc was
synthesized with eight single-strand primers, each ?80 bp in
length, in a single PCR. The Escherichia coli pBTR expression
system for yeast iso-1 Cc (40) was converted to express a variety
of cytochromes c. The cDNA of T-Cc, S-Cc, and yeast iso-2 Cc
was cloned into pBTR through two BamHI restriction sites (see
Supporting Text). Because of its poor yield in the E. coli system,
S-Cc cDNA was further cloned into YEp13 plasmid with a CYC1
promoter, which was well expressed in the GM-3C-2 (?Ctmp)
yeast strain whose Cc methyltransferase was knocked out by an
efficient site-specific homologous recombination method to
avoid the trimethylation of Lys-72 (41). The directions and
sequences of all constructs were confirmed by DNA sequencing.
cytochromes c, except S-Cc, were overexpressed under the
conditions of 37°C, lack of oxygen, pH 8.0, and in the DH5? E.
coli strain. hh-Cc was purchased from Sigma. For S-Cc, all of the
procedures of expression and pre-HPLC purification were per-
formed according to an established protocol (42).
Purification of Cc. The procedures of purification were based on the
method described by Patel et al. (40). The purity and sequence of
T-Cc protein were also confirmed by peptide mass fingerprint (43).
The concentrations of the various cytochromes c were determined
at 550 nm from the difference in the absorption of the ferrous and
ferric forms of the protein employing the Hitachi 2800 spectropho-
tometer used throughout this study. The ??mM550(reduced ?
oxidized) of yeast Cc is 18.5, and that of the mammalian Cc is 19.6.
The concentrations of the mammalian ferriCc’s were further con-
firmed at 410 nm (??mM410? 106.1).
Reactions of Cc with H2O2, Ascorbate, and Beef Heart Cc Oxidase. The
kinetics of oxidation of ferroCc by H2O2was studied spectropho-
1–30 ?M ferroCc. The degradation of ferriCc heme was estimated
from the dissipation of the Soret band absorption at 408 nm in 50
mM phosphate buffer (pH 6.1) containing 3 mM H2O2. The
degradation constant (Kd) was calculated from a first-order equa-
tion A ? Ao?e?Kdtobtained from the residual absorption versus
For the assay of reduction by ascorbate, different concentrations
of ferriCc ranging from 0.1 to 50 ?M were added to 50 mM
phosphate Na?, 25 mM Na2SO4 (pH 7.0) containing 0.2 mM
ascorbate in a cuvette, which was then sealed to block the access of
air. The initial rate of reduction was measured at 550 nm, and the
different rates obtained were plotted as a function of the Cc
The electron transfer activity of Cc to CcO was measured
spectrophotometrically. The membrane-bound form of the en-
zyme, namely a Keilin–Hartree particle preparation, was prepared
according to the procedure of Ferguson-Miller et al. (44). Initial
rates of oxidation of reduced Cc at two pH values were followed at
550 nm in 25 mM acetate?Tris, pH 7.8 (the standard ionic condi-
tions for polarographic assays), and in 50 mM phosphate?Tris, pH
6.5 (a more acidic buffer reported to be optimal for the spectral
assays). The concentration of beef CcO was calculated from the
Liu et al.
June 13, 2006 ?
vol. 103 ?
no. 24 ?
difference between the oxidized and dithionite-reduced spectra Download full-text
using a ??mM of 24 at 605 nm. Reproducibility of all of the
biochemical assays was confirmed by taking each measurement at
least three times and showed a fluctuation ?8%. Samples with all
components except Cc served as negative controls.
Cell-Free Caspase Activity Assay. The apoptotic activity of Cc in
interacting with apoptotic protease-activating factor 1 (apaf-1) and
substrate, N-acetyl-Asp-Glu-Val-Asp-pNA (DEVD-pNA) from
Sigma, measured spectrophotometrically in a time course at 405
nm. A 70-?l reaction mixture containing 20 ?l of cell extract from
Xenopus egg was incubated with varied concentrations of cyto-
chromes c (from 0.01 to 0.5 ?M) in 25 mM Hepes (pH 7.5), 50 mM
NaCl, 10 mM KCl, 1.5 mM MgCl2, 10% glycerol, and 1 mM DTT
for 30 min, DEVD-pNA was added to a concentration of 100 ?M,
and the increase of absorption at 405 nm was measured every 10 s
for a total of 4,000 s. Using the standard spectral absorption curve
of p-nitroanilide (Sigma), the initial rate of substrate cleavage was
calculated from the slope of the linear portion of the curve of
absorption change versus time.
Apoptosis Induction in Chicken Erythrocyte Nuclei and Assembled
Nuclei. A Xenopus egg extract obtained by ultraspeed centrifuga-
tion, termed extract S-150, and chicken erythrocyte nuclei were
prepared as described by Lu et al. (45). A total of 1 ? 105chicken
erythrocyte nuclei were mixed with 30 ?l of S-150 and a series of
Cc concentrations from 0.01 to 1 ?M, and were incubated at 23°C
for the times shown in Fig. 3B. After DAPI staining, the changes
in the appearance of the chromatin of nuclei were monitored by
using an Olympus IX71 fluorescence microscope and images were
captured by a cooled charge-coupled device camera. For the DNA
fragmentation assay, the reaction mixture was diluted with 10
volumes of 100 mM Tris?Cl (pH 8.0), 5 mM EDTA, 0.2 M NaCl,
0.4% SDS, and 0.2 mg?ml proteinase K and incubated at 37°C for
3 h. The breakage of the DNA, as an indication of apoptosis, was
analyzed by electrophoresis on a 1.5% agarose gel.
Crystallization and X-Ray Data Collection. Crystals of T-Cc were
grown by the hanging drop method with reservoir solutions con-
taining 27.5–22% polyethylene glycol 1000 (wt?vol), 30 mM po-
tassium phosphate (pH 7.1) and drops prepared by mixing 1 ?l of
setup at 16°C, crystals appeared several hours later and kept
growing for 2–3 days. These conditions are similar to those used by
Sanishvili et al. (22) for the crystallization of hh-Cc. The x-ray
diffraction data for T-Cc crystals were collected at 100 K (i.e.,
?173°C) by using a Rigaku R-Axis IV?? image plate with a
Rigaku FRE rotating CuK? anode home x-ray generator at 40 kV
and 50 mA (wavelength ? 1.5418 Å) and diffracted to at least
1.55-Å resolution. All of the intensity data were processed and
scaled by using the HKL2000 program (46). Data collection statistics
are summarized in Table 3.
Model Building and Refinement. ThestructureofT-Ccwassolvedby
the molecular replacement method with the coordinates of
ferrihh-Cc (Protein Data Bank ID 1CRC) (22). Cross-rotation
program CNS (47), and a clear solution for a monomer was found.
The residues that differ between hh-Cc and T-Cc were then
replaced and geometrically adjusted by using the program O (48)
under the guidance of 2Fo? Fcand Fo? Fcdifference maps,
followed by cycles of iterative rebuilding and refinement with the
program CNS, until no better resolution of the protein could be
Phylogenetic Analysis. Aphylogenetictreewasconstructedwiththe
amino acid sequences of various cytochromes c, obtained by
searching the National Center for Biotechnology Information
database (49) using ‘‘cytochrome c’’ as the key word and then
applying the neighbor-joining method with ‘‘the number of differ-
ences’’ model by the MEGA3 program (50).
We thank Prof. A. Grant Mauk (University of British Columbia,
Vancouver) for the generous gift of PBTR plasmid, Dr. Zhigang Lu
(Peking University) for support and valuable comments, and Dr. Ruslan
Sanishvili (Argonne National Laboratory, Argonne, IL) for discussions
on the crystal structures. This project was funded by Key Projects of the
Chinese Ministry of Education Nos. 03180 and 104232, the TransCen-
tury Training Program Foundation for the Talents of the Ministry of
Education, the Tsinghua-Yue-Yuen Medical Sciences Fund, and Na-
tional Natural Science Foundation of China (NSFC) Grants 30225016
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