Disruption of the M80-Fe ligation stimulates the translocation of cytochrome c to the cytoplasm and nucleus in nonapoptotic cells.
ABSTRACT Native cytochrome c (cyt c) has a compact tertiary structure with a hexacoordinated heme iron and functions in electron transport in mitochondria and apoptosis in the cytoplasm. However, the possibility that protein modifications confer additional functions to cyt c has not been explored. Disruption of methionine 80 (M80)-Fe ligation of cyt c under nitrative stress has been reported. To model this alteration and determine if it confers new properties to cyt c, a cyt c mutant (M80A) was constitutively expressed in cells. M80A-cyt c has increased peroxidase activity and is spontaneously released from mitochondria, translocating to the cytoplasm and nucleus in the absence of apoptosis. Moreover, M80A models endogenously nitrated cyt c because nitration of WT-cyt c is associated with its translocation to the cytoplasm and nucleus. Further, M80A cyt c may up-regulate protective responses to nitrative stress. Our findings raise the possibility that endogenous protein modifications that disrupt the M80-Fe ligation (such as tyrosine nitration) stimulate nuclear translocation and confer new functions to cyt c in nonapoptotic cells.
- [show abstract] [hide abstract]
ABSTRACT: Two unresolved aspects of the role of mitochondria-derived cytochrome c in apoptosis are whether there is a separate pool of cytochrome c within mitochondria that participates in the activation of apoptosis and whether a chemically modified cytochrome c drives apoptosis. These questions were investigated using osteoclasts, because they are rich in mitochondria and because osteoclast apoptosis is critical in bone metabolism regulation. H(2)O(2) production was increased during culture, preceding cytochrome c release; both processes occurred anterior to apoptosis. With the addition of a mitochondrial uncoupler, H(2)O(2) production and apoptosis were blocked, indicating the prominent role of mitochondria-derived H(2)O(2). Trapping H(2)O(2)-derived hydroxyl radical decreased apoptosis. Cytosolic cytochrome c was originated from a single mitochondrial compartment, supporting a common pool involved in respiration and apoptosis, and it was chemically identical to the native form, with no indication of oxidative or nitrative modifications. Protein levels of Bcl-2 and Bc-xL were decreased before apoptosis, whereas expression of wild-type Bcl-2 repressed apoptosis, confirming that cytochrome c release is critical in initiating apoptosis. Cytosolic cytochrome c participated in activating caspase-3 and -9, both required for apoptosis. Collectively, our data indicate that the mitochondria-dependent apoptotic pathway is one of the major routes operating in osteoclasts.AJP Cell Physiology 02/2005; 288(1):C156-68. · 3.71 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Endogenous tyrosine nitration and inactivation of manganese superoxide dismutase (MnSOD) has previously been reported to occur during end-stage human renal allograft rejection. In order to determine whether nitration and inactivation of this critical mitochondrial protein might play a contributory role in the onset of transplant rejection, we employed a rodent model of Chronic Allograft Nephropathy (or CAN). Using this model we followed kidney function from 2-52 weeks post-transplant and correlated graft function with levels of nitration in the renal allograft. Tyrosine nitration of both glomerular and tubular structures occurred at 2 weeks post-transplant. At later times (16 weeks) post-transplant, tyrosine nitration appeared to be confined to tubular structures; however glomerular nitration returned at 52 weeks post-transplant. Interestingly, nitration and inactivation of MnSOD occurs prior to the onset of renal dysfunction in this rat model of chronic allograft nephropathy (2 weeks versus 16 weeks post-transplant). Furthermore, we have identified an additional mitochondrial protein, cytochrome c, as being endogenously nitrated during chronic rejection. The kinetics of cytochrome c nitration lagged behind MnSOD nitration and inactivation (4 weeks compared to 2 weeks); suggesting that loss of MnSOD activity likely contributes to elevation of the nitrating species and further nitration of other targets.Free Radical Biology and Medicine 01/2002; 31(12):1603-8. · 5.27 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Reactive halogen species (RHS; X(2) and HOX, where X represents Cl, Br, or I) are metabolites mediated by neutrophil activation and its accompanying respiratory burst. We have investigated the interaction between RHS and mitochondrial cytochrome c (cyt c) by using electrospray mass spectrometry and electron spin resonance (ESR). When the purified cyt c was reacted with an excess amount of hypochlorous acid (HOCl) at pH 7.4, the peroxidase activity of cyt c was increased by 4.5-, 6.9-, and 8.6-fold at molar ratios (HOCl/cyt c) of 2, 4, and 8, respectively. In comparison with native cyt c, the mass spectra obtained from the HOCl-treated cyt c revealed that oxygen is covalently incorporated into the protein as indicated by molecular ions of m/z = 12,360 (cyt c), 12,376 (cyt c + O), and 12,392 (cyt c + 2O). Using tandem mass spectrometry, a peptide (obtained from the tryptic digests of HOCl-treated cyt c) corresponding to the amino acid sequence MIFAGIK, which contains the methionine that binds to the heme, was identified to be involved in the oxygen incorporation. The location of the oxygen incorporation was unequivocally determined to be the methionine residue, suggesting that the oxidation of heme ligand (Met-80) by HOCl results in the enhancement of peroxidase activity of cyt c. ESR spectroscopy of HOCl-oxidized cyt c, when reacted with H(2)O(2) in the presence of the nitroso spin trap 2-methyl-2-nitrosopropane (MNP), yielded more immobilized MNP/tyrosyl adduct than native cyt c. In the presence of H(2)O(2), the peroxidase activity of HOCl-oxidized cyt c exhibited an increasing ability to oxidize tyrosine to tyrosyl radical as measured directly by fast flow ESR. Titration of both native cyt c and HOCl-oxidized cyt c with various amounts of H(2)O(2) indicated that the latter has a decreased apparent K(m) for H(2)O(2), implicating that protein oxidation of cyt c increases its accessibility to H(2)O(2). HOCl-oxidized cyt c also displayed an impaired ability to support oxygen consumption by the purified mitochondrial cytochrome c oxidase, suggesting that protein oxidation of cyt c may break the electron transport chain and inhibit energy transduction in mitochondria.Journal of Biological Chemistry 09/2002; 277(33):29781-91. · 4.65 Impact Factor
Disruption of the M80-Fe ligation stimulates the
translocation of cytochrome c to the cytoplasm
and nucleus in nonapoptotic cells
Luiz C. Godoya, Cristina Mun ˜oz-Pinedob, Laura Castroc, Simone Cardacia,d, Christopher M. Schonhoffa,e, Michael Kinga,
Vero ´nica To ´rtorac, Mo ´nica Marínc,f, Qian Miaoa, Jian Fei Jiangg, Alexandr Kapralovg, Ronald Jemmersonh,
Gary G. Silkstonei, Jinal N. Patelj, James E. Evansj, Michael T. Wilsoni, Douglas R. Greenk, Valerian E. Kagang,
Rafael Radic, and Joan B. Mannicka,1
aDepartments of Medicine and Cellular Biology, University of Massachusetts Medical School, Worcester, MA 01605;bIDIBELL - Institut d’Investigacio ´
Biome `dica de Bellvitge, Barcelona, Spain;cDepartamento de Bioquímica, Facultad de Medicina, Universidad de la Repu ´blica, Montevideo, Uruguay;
dDepartment of Biology, University of Rome Tor Vergata, 00173 Rome, Italy;eDepartment of Biomedical Sciences, Tufts University Cummings School of
Veterinary Medicine, North Grafton, MA 01536;fSeccio ´n Bioquímica-Biología Molecular, Facultad de Ciencias, Universidad de la Repu ´blica, Montevideo,
Uruguay;gDepartment of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA 15260;hDepartment of Microbiology and Center
for Immunology, University of Minnesota, Minneapolis, MN 55455;iDepartment of Biological Sciences, University of Essex, Colchester CO4 3SQ, United
Kingdom;jDepartment of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605; andkDepartment
of Immunology, St. Jude Children’s Research Hospital, Memphis, TN 38105
Edited by Guido Kroemer, Institut National de la Sante ´ et de la Recherche Me ´dicale, U848, Institut Gustave Roussy, France, and accepted by the Editorial
Board December 29, 2008 (received for review September 18, 2008)
Native cytochrome c (cyt c) has a compact tertiary structure with a
hexacoordinated heme iron and functions in electron transport in
mitochondria and apoptosis in the cytoplasm. However, the pos-
c has not been explored. Disruption of methionine 80 (M80)-Fe
ligation of cyt c under nitrative stress has been reported. To model
this alteration and determine if it confers new properties to cyt c,
a cyt c mutant (M80A) was constitutively expressed in cells.
M80A-cyt c has increased peroxidase activity and is spontaneously
released from mitochondria, translocating to the cytoplasm and
nucleus in the absence of apoptosis. Moreover, M80A models
endogenously nitrated cyt c because nitration of WT-cyt c is
associated with its translocation to the cytoplasm and nucleus.
Further, M80A cyt c may up-regulate protective responses to
nitrative stress. Our findings raise the possibility that endogenous
protein modifications that disrupt the M80-Fe ligation (such as
tyrosine nitration) stimulate nuclear translocation and confer new
functions to cyt c in nonapoptotic cells.
mitochondria ? nitration ? oxidation ? oxidative stress ? peroxynitrite
are occupied, thus preventing its interactions with small ligands
ligation of a subpopulation of cyt c may be endogenously
disrupted in cells. Specifically, tyrosine nitration, M80 oxidation,
and interactions with cardiolipin have been reported to cause
loosening of the tertiary structure of cyt c and disruption of the
M80-Fe ligation. Endogenous nitration of cyt c has been de-
tected in cultured osteoclasts (2) and in kidneys after ischemia/
reperfusion injury and during chronic allograft nephropathy (3,
4). In vitro nitration of cyt c has been shown to disrupt the
M80-Fe ligation, increase the peroxidase activity, and inhibit the
electron transport function of cyt c (5). In addition, oxidation of
M80 by a variety of oxidants, including singlet oxygen and HOCl,
disrupts the M80-Fe ligation (5–8). Finally, approximately 15%
of cyt c tightly binds to cardiolipin in the inner mitochondrial
membrane via both electrostatic and hydrophobic interactions,
resulting in a loosening of the tertiary structure of cyt c and
disruption of the M80-Fe ligation (9–11). Disruption of the
M80-Fe ligation may increase the peroxidase activity of cyt c by
increasing the access of H2O2to the heme iron (1). Indeed, the
subpopulation of mitochondrial cyt c that is tightly bound to
cardiolipin has increased peroxidase activity and may catalyze
hen cytochrome c (cyt c) acts as an electron shuttle in
mitochondria, all 6 coordination positions of its heme iron
cardiolipin peroxidation that is required for cyt c release from
mitochondria during apoptosis (1). This subpopulation of cyt c
may have other unique biological functions due to its altered
conformation that have yet to be identified.
To investigate the possibility that the function of cyt c is
altered by endogenous posttranslational modifications and/or
cardiolipin interactions that disrupt the M80-Fe ligation, we
generated a cyt c mutant with a constitutively disrupted M80-Fe
ligation due to a mutation of M80 to alanine (M80A). We then
investigated the effects of the M80A mutation on the subcellular
localization and function of cyt c.
To determine if disruption of the M80-Fe ligation alters the
intracellular distribution of cyt c, the subcellular localization of
GFP-tagged M80A or WT cyt c expressed in HeLa cells was
analyzed by confocal microscopy. The M80A mutation in iso-
yeast cyt c does not cause major alterations to the tertiary
structure of cyt c despite disrupting the heme ligation (12).
Although WT cyt c was localized primarily in mitochondria,
M80A cyt c had a nonmitochondrial nuclear and cytoplasmic
distribution (Fig. 1A). Similar results were obtained when GFP-
tagged M80A or WT cyt c was expressed in MCF-7 cells (data
not shown). Subcellular fractionation studies confirmed that
M80A cyt c had a predominantly nonmitochondrial localization
(Fig. 1B). The distribution of M80A cyt c was not dependent on
the GFP tag because tetracysteine-tagged M80A cyt c also had
a diffuse cytoplasmic and nuclear distribution by confocal anal-
ysis (data not shown). These findings suggest that the M80A cyt
c mutant is either not imported into mitochondria or is imported
and then rapidly released into the cytoplasm and/or nucleus.
Cyt c is synthesized as an apoenzyme in the cytoplasm,
translocates to mitochondria, and only then acquires a heme
group to form a holoenzyme. Therefore, to determine if M80A
Author contributions: J.E.E., D.R.G., V.E.K., R.R., and J.B.M. designed research; L.C.G.,
C.M.-P., L.C., S.C., C.M.S., M.K., V.T., M.M., Q.M., J.F.J., A.K., and J.N.P. performed research;
L.C.G., R.J., G.G.S., J.E.E., M.T.W., D.R.G., V.E.K., R.R., and J.B.M. analyzed data; and L.C.G.
and J.B.M. wrote the paper.
The authors declare no conflict of interest.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2009 by The National Academy of Sciences of the USA
February 24, 2009 ?
vol. 106 ?
no. 8 ?
is imported into mitochondria before being released into the
cytoplasm, we investigated whether M80A was immunoprecipi-
tated by an antibody specific for heme-bound holocyt c. M80A
cyt c was immunoprecipitated by an antibody specific for holocyt
c, but not by an antibody that recognizes apocyt c (Fig. 1C). In
addition, the molecular weight of immunoprecipitated GFP-
tagged M80A cyt c as determined by liquid chromatography/
mass spectrometry was nearly identical to that of GFP-tagged
WT cyt c, with the observed 60 Da difference attributable to the
mutation from methionine to alanine in M80A. The approxi-
mately 700 Da increase in molecular weight of WT and M80A
cyt c as compared to the predicted molecular weight of the
apocyt c is consistent with the addition of a heme group to both
proteins (supporting information (SI) Fig. S1). Moreover, it has
previously been shown in yeast that mutation of the heme-
coordinating methionine does not inhibit import of cyt c into
mitochondria (13). These findings suggest that M80A cyt c is
imported into mitochondria, acquires a heme group, and then is
spontaneously released. Although mitochondrial release and
nuclear translocation of cyt c has been described during apo-
ptosis (14), our data suggest that this event also occurs in
During apoptosis, cyt c release from mitochondria is depen-
dent on either the Bcl-2 family members Bax and Bak and/or on
the mitochondrial permeability transition pore (MPTP) (15). To
determine if the spontaneous release of M80A cyt c from
mitochondria in the absence of apoptosis was Bax- or Bak-
dependent, GFP-tagged M80A or WT cyt c was expressed in
mouse embryonic fibroblasts (MEFs) obtained from Bax and
Bak double knock-out (DKO) mice (16). M80A cyt c has a
cytoplasmic and nuclear distribution in the Bax/Bak DKO
MEFs, indicating that M80A cyt c release from mitochondria is
Bax/Bak-independent (Fig. S2). Moreover, inhibitors of the
MPTP (cyclosporine A and bongkrekic acid) did not change the
nonmitochondrial distribution of M80A (data not shown), sug-
gesting that its release is also not dependent on the MPTP. A
serine protease-dependent mechanism of mitochondrial cyt c
release has also been reported (17). However, the serine pro-
tease inhibitor 4-(2-aminoethyl)benzenesulfonylfluoride also
did not significantly alter the nuclear and cytoplasmic distribu-
tion of M80A (data not shown). Thus cyt c with a disrupted
M80-Fe ligation may be released from mitochondria in nonapo-
ptotic cells by a mechanism that is independent of Bax/Bak, the
MPTP, and/or serine proteases.
Recent data suggest that an increased peroxidase activity of
cyt c during apoptosis leads to cardiolipin oxidation that is
required for the release of cyt c and other pro-apoptotic factors
from mitochondria (1). Therefore, M80A cyt c may be sponta-
cyt c stably expressed in HeLa cells was determined by confocal microscopy. Mitochondria were labeled with MitoTracker Red (Mito). Colocalization of green
GFP-cyt c staining and red mitochondrial staining is seen as yellow in the merged images. (B) Subcellular localization of WT and M80A cyt c as determined by
subcellular fractionation. Levels of cyt c-GFP (Cyt c-GFP) in equal concentrations of whole cell (Total), cytoplasmic (Cyto), or mitochondrial (Mito) lysates of
untransfected HeLa cells (Control) or HeLa cells expressing WT or M80A cyt c-GFP were assessed on cyt c WBs. As loading controls and to assess the purity of
are representative of 3 separate experiments. c. M80A cyt c is a holoprotein. Lysates of HeLa cells expressing M80A cyt c were immunoprecipitated by antibodies
recognizing apocyt c (Apo), holocyt c (Holo), or by equal concentrations of control antibodies (Ig). A cyt c WB of the immunoprecipitates is shown. MW indicates
molecular weight standards. HC and LC indicate Ig heavy and light chains, respectively.
M80A cyt c is spontaneously released from mitochondria and translocates to the nucleus. (A) The subcellular localization of GFP-tagged M80A and WT
www.pnas.org?cgi?doi?10.1073?pnas.0809279106Godoy et al.
neously released from mitochondria because it has increased
peroxidase activity (as a result of disruption of the Fe-M80
ligation) and oxidizes cardiolipin. In support of this hypothesis,
recombinant M80A cyt c has increased peroxidase activity as
compared to equal concentrations of WT cyt c (Fig. 2A). In
addition, the nitric oxide donor 2-(N,N-Diethylamino)diazeno-
late-2-oxide sodium (DEANO), an inhibitor of the peroxidase
activity of cyt c (18), inhibited the release of M80A cyt c from
mitochondria in a subpopulation of cells (Fig. 2B). Moreover,
knock-down of M80A expression with RNAi protected cells
from tert-Butyl hydroperoxide (tBuOOH)-induced death, as
would be expected if M80A functioned as a peroxidase intra-
cellularly (Fig. S3). These data raise the possibility that the
increased peroxidase activity of M80A cyt c may mediate its
spontaneous release from mitochondria. However, levels of
cardiolipin hydroperoxide were not increased in cells expressing
M80A cyt c as compared to cells expressing WT cyt c (data not
shown). Thus peroxidation of a substrate other than cardiolipin
may be involved in the release of M80A cyt c from mitochondria,
or cardiolipin peroxidation may be rapidly reversed in cells
Normally, release of cyt c into the cytoplasm stimulates
apoptosome formation, caspase activation, and apoptotic cell
death. However, cells expressing M80A cyt c did not undergo
apoptosis, suggesting that M80A cyt c may not activate caspases
as efficiently as WT cyt c. In fact, recombinant M80A cyt c did
not efficiently activate caspase-3 in cytoplasmic lysates and was
not able to competitively inhibit the activation of caspase-3 by
recombinant WT cyt c (Fig. 3A). In addition, SMAC/DIABLO,
a pro-apoptotic protein that is released from mitochondria along
with cyt c during apoptosis, was not released into the cytoplasm
in cells expressing M80A (Fig. 3B). Of interest, endogenous cyt
c was also not released from mitochondria along with M80A cyt
c (Fig. 3B), indicating that M80A cyt c is selectively released.
Consistent with this finding, cells expressing M80A cyt c are
energy-sufficient as expected if endogenous cyt c is retained in
mitochondria. These results suggest that cytoplasmic release of
M80A does not trigger apoptosis both because other pro-apo-
ptotic proteins are not released from mitochondria along with
M80A and because M80A does not efficiently activate the
apoptosome, perhaps due to reduced affinity for Apaf-1. Of
note, similar to what we observed with M80A cyt c (Fig. 3A),
nitrocytochrome c also does not efficiently activate the apopto-
some as we (19) and others (20) have recently reported.
We next determined if M80A cyt c is modeling the physiologic
function of endogenously nitrated cyt c because tyrosine nitra-
dependent. (A) Recombinant M80A has higher peroxidase activity than re-
combinant WT cyt c. The peroxidase activity of recombinant M80A or WT cyt
c was determined by luminol oxidation as described previously (32). (B)
DEANO, an inhibitor of the peroxidase activity of cyt c (18), inhibits M80A cyt
c release from mitochondria. HeLa cells expressing GFP-tagged M80A cyt c
were treated in the presence or absence of DEANO (2 mM) for 8 h. Subcellular
localization of M80A was then determined by confocal microscopy as de-
scribed in Fig. 1A. The results are representative of 4 separate experiments.
The release of M80A from mitochondria may be peroxidase activity-
M80A. Equal concentrations of recombinant M80A or WT cyt c were added to
cytosolic extracts of Jurkat cells in the presence of dATP and ATP. Caspase-3
in combination, in several molar ratios. (B) M80A cyt c expression does not
M80A or WT cyt c or untransfected HeLa cells (Control) were separated into
SMAC/DIABLO and endogenous cyt c (Endog Cyt c) in each fraction were
determined by WB analysis.
Godoy et al.
February 24, 2009 ?
vol. 106 ?
no. 8 ?
tion disrupts the M80-Fe ligation of cyt c (5, 6, 21), and
endogenous nitration of cyt c has been detected in vivo under
conditions of nitrative stress (3, 4). To determine if peroxynitrite
(ONOO?an endogenously produced nitrating agent, induces
nitration of WT cyt c, cells expressing WT cyt c-GFP were
exposed to a low nontoxic dose of ONOO?(25 ?M). Tyrosine
nitration is the only detectable modification of cyt c induced by
low concentrations of ONOO?(5, 6). Cyt c was then immuno-
precipitated from cells and tyrosine nitration was assessed on
anti-nitrotyrosine Western blots. No evidence of cyt c nitration
was seen in cyt c immunoprecipitates of whole cell lysates (data
not shown). However, tyrosine nitration of cyt c that increased
after ONOO?treatment was detected in cyt c immunoprecipi-
tates of nuclear-enriched cellular fractions (Fig. 4A). These
findings raise the possibility that nitrated cyt c translocates to the
nucleus. In support of this hypothesis, ONOO?-induced nitra-
tion of cyt c was associated with the translocation of a subpopu-
To rule out the possibility that the nitration and nuclear
translocation of cyt c is an artifact of GFP-cyt c overexpression,
we analyzed whether mild nitrative stress is associated with a
disruption of the M80-Fe ligation and the nuclear translocation
of endogenous cyt c. To address this issue, nontransfected HeLa
cells were treated with 25 ?M ONOO?, followed by immuno-
precipitation of nuclear nitrated proteins with an anti-
nitrotyrosine antibody, and Western blot analysis with an anti-
cyt c antibody. As shown in Fig. 4C, cyt c is detected in higher
quantity in the nuclear fraction of cells treated with ONOO?.
Detection of cyt c with the anti-nitrotyrosine antibody was
abrogated by pretreatment of the membrane with dithionite,
confirming the nitrated nature of the subpopulation of cyt c
imported into the nucleus. In addition, confocal microscopic
studies of untransfected HeLa cells were performed using an
antibody (1D3) that does not recognize native cyt c but specif-
ically recognizes a cyt c epitope that is exposed when the M80-Fe
detected (Fig. 4C). However, after cells were treated with low
stress. (A) WT cyt c is tyrosine nitrated by nontoxic levels of ONOO?. HeLa cells expressing GFP-labeled WT cyt c were left untreated (Contr) or were treated for
30 sec with ONOO?(25 ?M). The nuclear-enriched fraction of the cells was then immunoprecipitated with an anti-GFP antiserum (GFP) or with equal
concentrations of normal rabbit serum (NRS). Levels of cyt c and nitrotyrosine in the immunoprecipitates were then determined on anti-cyt c (Cyt c WB) or
anti-nitrotyrosine (NT WB) WBs. (B) Nontoxic levels of ONOO?induce the translocation of a subpopulation of WT cyt c from mitochondria to the cytoplasm and
nucleus. HeLa cells expressing GFP-labeled WT cyt c were left untreated or were treated with ONOO?(25 ?M). After 4 h, the subcellular localization of cyt c was
determined by confocal microscopy. The graph shows the mean percentage of cells with nuclear cyt c-GFP staining plus SD in 4 separate experiments. * indicates
Nontransfected HeLa cells were treated with 25 ?M ONOO?for 30 sec, incubated overnight in regular medium and the nuclear fraction was then submitted to
immunoprecipitation with an anti-nitrotyrosine (anti-NT) antibody or a control, irrelevant antibody (IgG). Shown is a cyt c Western blot analysis of the
immunoprecipitates from control (Contr) and peroxynitrite-treated cells (ONOO?). (D) Cells processed as described in (C) were analyzed by confocal microscopy
with the 1D3 antibody, which recognizes an epitope present when the Fe-M80 ligation is disrupted in cyt c (22). Anti-mouse FITC-conjugated antibody was used
to detect 1D3 labeling. Nuclei are stained with DAPI. (E) Cells expressing M80A have higher rates of survival and/or proliferation in response to ONOO?. Equal
using the WST-1 assay. The data indicate the absorption ratio of ONOO?treated/control untreated cells (mean ? SEM) in 8 separate experiments. ˆ indicates P ?
0.05 and * indicates P ? 0.01, two-tailed t test.
www.pnas.org?cgi?doi?10.1073?pnas.0809279106Godoy et al.
doses of ONOO?(25 ?M), 1D3 staining was detected in the
nucleus (Fig. 4D). These findings suggest that mild nitrative
stress disrupts the M80-Fe ligation and leads to the nuclear
translocation of the nitrated subpopulation of endogenous cyt c.
Low, nontoxic levels of ONOO?not only have been shown to
nitrate cyt c (7, 12) but also have been implicated in precondi-
tioning responses that confer increased resistance to oxidative
and nitrative stress (23, 24). To determine if M80A cyt c alters
the response of cells to nitrative stress, cells expressing WT or
M80A cyt c were exposed to a range of concentrations of
ONOO?. M80A-expressing cells had increased rates of survival
when exposed to moderate to highly toxic levels of ONOO?as
compared to cells expressing WT cyt c (Fig. 4E). Thus M80A
may regulate the resistance of cells to nitrative stress. Our
findings raise the possibility that a subpopulation of cyt c is
nitrated when cells are exposed to low levels of nitrative stress,
leading to disruption of the M80-Fe ligation. As a result, this
subpopulation of cyt c translocates from mitochondria to the
cytoplasm and nucleus and regulates the response of cells to
further nitrative and/or oxidative stress.
Our data suggest that the subcellular localization of cyt c is
regulated not only by Bcl-2 family members, the MPTP, and/or
serine proteases, but also by the M80-Fe ligation of cyt c.
Specifically, endogenous disruption of the M80-Fe ligation by
modifications such as tyrosine nitration, M80 oxidation, and/or
of cyt c and induce its translocation from mitochondria to the
cytoplasm and nucleus in nonapoptotic cells. Previous data
suggest that the increased peroxidase activity of cyt c during
apoptosis stimulates its release from mitochondria via cardio-
lipin peroxidation (1). Our findings indicate that mitochondrial
release of cyt c with a disrupted M80-Fe ligation in nonapoptotic
cells may be peroxidase-dependent, although the peroxidation
target remains to be determined. Of interest, NO and related
species can both stimulate (via ONOO?-mediated tyrosine
nitration) and inhibit (via heme nitrosylation) (18, 25) the
peroxidase activity of cyt c. Thus the intracellular redox chem-
istry of NO may precisely regulate the peroxidase activity and
therefore the subcellular localization of cyt c.
It is possible that the M80-Fe ligation also regulates the
subcellular localization and function of cyt c during apoptosis.
The 1D3 antibody, directed against an epitope exposed when the
M80-Fe ligation is disrupted (22), recognizes a subpopulation of
cyt c released from mitochondria during apoptosis. In addition,
detection of heme nitrosylated cyt c during apoptosis also
suggests that the M80-Fe ligation is disrupted in a subpopulation
of cyt c (25). It remains to be determined if the translocation of
cyt c to the nucleus and endoplasmic reticulum during apoptosis
is regulated by protein modifications and/or alterations in the
structure of cyt c (14, 26).
Several groups have shown that low levels of ONOO?are
associated with the development of protective responses to
further oxidative/nitrative stress in models as diverse as isch-
emia-reperfusion-induced heart tissue injury (27, 28), endo-
thelial pathophysiology (29), and transplant-related kidney
dysfunction (24). In addition, it has been demonstrated that
mitochondria produce and are exposed to ONOO?and further
that mitochondrial NOS stimulation causes cyt c release from
mitochondria and that this release can be prevented by
ONOO?scavengers (30). Therefore, it is possible that the
ONOO?-induced disruption of M80-Fe ligation in cyt c, with
subsequent release from mitochondria and nuclear transloca-
tion of cyt c, might be involved in the preconditioning response
to further redox stress. Nevertheless, how the modified form of
cyt c contributes to this protective effect remains to be
Our findings raise the possibility that cyt c has a spectrum of
functions in cells that are dependent on the level of nitrative
and/or oxidative stress. In resting cells, cyt c is predominantly
hexacoordinated and functions in mitochondrial electron trans-
port. In response to low levels of oxidative and/or nitrative stress,
a subpopulation of cyt c may become nitrated and/or oxidized,
translocate to the nucleus and cytoplasm, and function in a
preconditioning response to further nitrative and/or oxidative
stress. When cells are exposed to toxic levels of stress, pro-apo-
ptotic bcl-2 family members may be activated, leading to in-
creased cyt c release from mitochondria and apoptotic cell
In conclusion, cyt c may serve as a redox sensor that
modulates the response of cells to varying levels of stress.
Structural studies of M80A and nitrated cyt c may further
elucidate conformational changes that underlie the functional
alterations in the protein. Future studies may reveal additional
roles for distinct conformations of cyt c in cell physiology and
Materials and Methods
Cell Culture. Cells were grown at 37 °C and 5% CO2in complete medium
[DMEM (Mediatech, Inc.) supplemented with 10% FBS (FBS, Nalge), 0.2 mM
L-glutamine, 10 U/ml penicillin, and 10 ?g/ml streptomycin (Sigma)].
of cell lines with pBABE mouse cyt c-GFP virus was performed as described
previously (31). Experimental details are provided in SI Methods.
Cyt c-GFP Knock-Down by siRNA. Cells were grown in 96-well plates (WST-1
assay) or 35 mm dishes (confocal microscopy). For transfections, GFP-targeted
siRNA (Dharmacon, 50 nM final concentration) was preincubated with Lipo-
fectAMINE (Gibco-Invitrogen) and subsequently added to the cell cultures in
fresh DMEM containing 2% FBS for 12 h. The culture medium was then
replaced with complete medium and protein knock-down was verified after
72–96 h by cyt c Western blot (WB) or confocal analysis.
Peroxidase Activity of WT and M80A cyt c. The peroxidase activity of WT or
M80A cyt c was assayed by luminol oxidation in presence of H2O2as described
Subcellular Fractionation. HeLa cells expressing M80A or WT cyt c were sepa-
rated into mitochondrial and cytoplasmic fractions as described previously
described (34) using antibodies to cyt c (BD Biosciences), SMAC/DIABLO (Pro-
Sciencific, Inc.) and GAPDH (Santa Cruz Biotechnology).
Immunofluorescence Detection of Endogenous cyt c. All steps were carried out
exposure to 25 ?M ONOO?, nontransfected HeLa cells grown on glass cover
slips were fixed with 4% paraformaldeheyde in PBS for 10 min and incubated
for an additional 10 min in 0.4% Triton X-100. Blockage was carried out with
PBS containing 10% FBS, followed by overnight incubation at 4 °C with the
rabbit anti-mouse (Jackson Immunoresearch) was added as the secondary
antibody for 30 min in the dark. PBS washes were carried out between all
medium containing DAPI (Invitrogen). Cells were then analyzed by confocal
microscopy as described in SI Methods.
Cell Viability Assay. Cellular viability after the treatment with tBuOOH or
peroxynitrite was measured using the WST-1 reagent, according to the
manufacturer’s instructions (Roche). The WST-1 assay measures the cleav-
age of tetrazolium salts to formazan by mitochondrial dehydrogenases in
metabolically active cells. Experimental details are provided in the SI
Statistics. Statistical significance was determined using a two-tailed t test for
Godoy et al.
February 24, 2009 ?
vol. 106 ?
no. 8 ?
SI Methods. Details concerning production of recombinant WT and M80A
mutant cyt c, immunoprecipitations, detection of nitrated GFP-tagged cyt c,
preparation of cell-free extracts for caspase 3 assays and confocal microscopy
are provided in SI Methods.
ACKNOWLEDGMENTS. This work was supported by grants from National
Institutes of Health (R01GM065824-05 to J.B.M.), Howard Hughes Medical
Institute and International Center for Genetic Engineering (to R.R.), and
Consejo Superior de Investigaciones Cientı ´ficas (to L.C.).
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www.pnas.org?cgi?doi?10.1073?pnas.0809279106Godoy et al.