Cytochrome bc1 regulates the mitochondrial permeability transition by two distinct

pathways

Jeffrey S. Armstrong, Hongyuan Yang, Wei Duan, and Matthew Whiteman

Department of Biochemistry, National University of Singapore, Singapore, 117597

Running Title: Cytochrome bc1 and the mitochondrial permeability transition

Correspondence and reprint requests:

1Jeffrey S. Armstrong Ph.D.

Department of Biochemistry

National University of Singapore

Kent Road, Singapore

Phone: +65 6874 5996

Fax: +65 6779 1453

E-mail: bchjsa@nus.edu.sg

JBC Papers in Press. Published on September 10, 2004 as Manuscript M408882200

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

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SUMMARY

The mitochondrial permeability transition (MPT) pore is a calcium-sensitive channel in the

mitochondrial inner membrane that plays a crucial role in cell death. Here we show that

cytochrome bc1 regulates the MPT in isolated rat liver mitochondria (RLM) and in CEM and

HL60 cells by two independent pathways. Glutathione (GSH) depletion activated the MPT via

increased production of reactive oxygen species (ROS) generated by cytochrome bc1. The ROS

producing mechanism in cytochrome bc1 involves movement of the “Rieske” Iron Sulfur Protein

(ISP) subunit of the enzyme complex, since inhibition of cytochrome bc1 by pharmacologically

blocking ISP movement completely abolished ROS production, MPT activation and cell death.

The classical inhibitor of the MPT, cyclosporine A (CsA), had no protective effect against MPT

activation. In contrast, the calcium-activated, CsA regulated, MPT in RLM was also blocked

with inhibitors of cytochrome bc1. These results indicate that electron flux through cytochrome

bc1 regulates two distinct pathways to the MPT, one unregulated and involving mitochondrial

ROS and the other regulated and activated by calcium.

Keywords: redox, glutathione, stigmatellin, iron sulfur protein, mitochondrial permeability

transition, cytochrome bc1,

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INTRODUCTION

Mitochondria play a vital role in cell fate by their regulation of energy metabolism and their

involvement in cell death by apoptosis and necrosis (1-3). Mitochondrial function, including ion

transport, biogenesis and ATP formation, requires an intact mitochondrial transmembrane

potential (∆ψm) which depends upon the generation of an electrochemical proton gradient

(∆µΗ+) across the mitochondrial inner membrane. The ∆µΗ+ is generated by three multisubunit

protein complexes localized in the mitochondrial inner membrane including respiratory complex

I (NADH:ubiquinone dehydrogenase), complex III (cytochrome bc1), and complex IV

(cytochrome c oxidase) (4, 5). A crucial event that occurs in mitochondria when a cell dies is

loss of the ∆µΗ+ and subsequent collapse of the ∆ψm (6) which can occur due to opening of high

conductance permeability transition pores (MPT) in the mitochondrial inner membrane which

allow the nonselective diffusion of solutes (<1500 Da) across the membrane with resulting

organelle swelling and membrane rupture (6-9). The MPT is known to be activated by Ca2+, and

reactive oxygen species (ROS) and inhibited by the potent immunosuppressive agent

cyclosporine A (CsA) (7-10). While many studies have considered that the MPT is due to the

formation of a preformed pore complex between the mitochondrial inner and outer membranes

involving the adenine nucleotide translocator (ANT), the voltage dependent anion channel

(VDAC), cyclophilin D (CyD), and a number of accessory proteins (11-15), an alternative view

has been that the MPT is not the result of opening of a pre-formed pore, but the result of

increased membrane permeability due to oxidative damage to pre-existing membrane proteins

include the ANT (8, 9).

The mechanism by which CsA inhibits the MPT has been attributed to its inhibitory effect

on the peptidyl-prolyl isomerase (PPIase) activity of CyD which is believed to be required for

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the formation of an ANT/ CyD protein complex required for MPT activation (14-17). However,

this MPT model has been questioned by a report showing when CyD was overexpressed and

targeted to mitochondria, it protected cells from oxidants indicating that it was inhibiting rather

than activating the MPT (18). Although, in direct contrast to this Li et al., recently found that

overexpression of mitochondrial CyD rendered mitochondria more susceptible to the MPT, and

cells more sensitive to oxidant mediated injury (19). The controversy concerning the role of the

ANT and CyD in the MPT is further compounded, firstly, by the recent finding that the MPT

was found to be inducible in mitochondria taken from the livers of mice with genetically

inactivated ANT isoforms (20), and, secondly, although CsA blocks the MPT in some cases, it is

ineffective at MPT inhibition in others (21, 22). This led He and Lemasters to propose an

alternative model of the MPT that would account for some of these inconsistencies (9, 11). Their

model envisaged both a CsA “regulated” and an “unregulated” form of MPT based on a study

showing two possible conductance modes for the MPT. One mode was activated by Ca2+ and

inhibited by CsA and the other was unregulated (23, 24).

The dualistic model of He and Lemasters, thus, reconciles two apparently divergent ideas on

the MPT where both the ANT and CyD play an important regulatory role. However, other

investigators have considered models of MPT, that although may involve the ANT and CyD,

feature mitochondrial respiratory components other than the ANT as crucial MPT regulators, for

example, work of Fontaine and colleagues clearly showed that the MPT is regulated by electron

flux through the NADH:ubiquinone-dehydrogenase and that various classes of quinone analogs

were important MPT regulatory molecules (25-27). Whereas, we have previously shown that

mitochondrial reactive oxygen species (ROS) activates the MPT in vivo after glutathione (GSH)

depletion and proposed the redox target(s) include the ANT (28). These varied, and somewhat

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controversial reports, on the molecular composition and regulation of the MPT indicate that

definitive knowledge on this phenomenon is lacking and suggest that concerted efforts should be

made to advance our understanding of this crucial mitochondrial event associated with cell death.

In this study we show that cytochrome bc1 is a key regulatory component of the MPT in

rat liver mitochondria (RLM) and in leukemic CEM and HL60 cells. Our results indicate a

fundamental role for ISP subunit movement in ROS-mediated MPT activation in cells while

investigations with RLM indicate that cytochrome bc1 may possess a MPT channel-like function

suggesting that cytochrome bc1 is involved in two distinct pathways to the MPT.

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MATERIALS AND METHODS

Materials: All chemicals were reagent grade and were obtained from Sigma (St Louis, MO).

Tetra-methyl rhodamine methyl ester (TMRM) and dichlorodihydrofluorescein diacetate

(DCFDA) was obtained from Molecular Probes (Eugene Oregon). Bongkrekic acid (BgK) was

obtained from Calbiochem (La Jolla, CA). Stigmatellin was obtained from Fluka Biochemika

and 2-methoxy antimycin A3 (2-MeAA) was obtained from BIOMOL Research Laboratories,

PA, USA.

Cell culture: CEM and HL60 cells (HL60 overexpressing Bcl-2 protein were used because of

their sensitivity and reproducibility to GSH induced redox stress (28, 29) were cultured in RPMI

with 10 % FCS and supplements as previously described (29). Cells were passaged daily to

maintain them in log-phase and kept at a concentration between 2.5- 5 x105/ ml.

Mitochondrial isolation and swelling test:

Rat liver mitochondria (RLM) were isolated by conventional differential centrifugation from the

livers of male adult Sprague Dawley rats fasted overnight. Large-amplitude swelling was

measured by spectrophotometry in a Beckman DU 640 by recording absorbance change at 540

nm. Isolated rat liver mitochondria (~1 mg/ml) were suspended in mitochondrial isolation buffer

consisting of Mannitol (220 mM), sucrose (70 mM), Hepes (2mM), EGTA (0.5 mM), BSA

(0.1%) for all experiments.

Measurement of GSH: CEM cells were treated with DEM (5 mM) for 0, 15, 30, 45 and 60 min.

GSH levels were measured by the monochlorobimane method (30). The amount of acid-

insoluble protein was determined by the Bradford method with γ-globulin as a standard.

Measurement of ROS production: After treatment with DEM (1 mM) for 0, 10, 20, 30, 40 and 50

min, cells were stained with 10 µM DCFDA for 15 min, washed with phosphate-buffered saline

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containing 10 mM glucose and analyzed immediately by flow cytometry using WinMidi

software. DCFDA is a non-fluorescent ester of the dye fluorescein. DCFDA is cleaved by

intracellular esterases and entrapped within the cell as the oxidant-sensitive DCF. ROS oxidize

DCF to the fluorescent product fluorescein (31). The green fluorescence of fluorescein was

measured using the FL-2 setting (log mode) after cell debris were electronically gated out. In

each analysis, 10,000 events were recorded.

Measurement of mitochondrial membrane potential (∆ψm): ∆ψm was measured with the

fluorescent lipophilic cationic dye tetramethyl rhodaminemethyl ester TMRM (250 nM) which

accumulates within mitochondria according to the ∆ψm. After treatment with DEM for 0, 30, 60

90 and 120 min, cells were stained with TMRM for 15 minutes and red fluorescence was

measured by flow cytometry using the FL-3 setting. The protonophore CCCP (10 µM) was used

to dissipate the chemiosmotic proton gradient (∆µH+) and served as a control for loss of ∆ψm. In

each analysis, 10,000 events were recorded.

Electron microscopic examination of cells: HL60 or CEM cells in the logarithmic proliferation

phase, were treated with DEM (5 mM) +/- the respiratory complex inhibitors stigmatellin (5µM),

antimycin A (5µM) or 2-MeAA (5µM) as described in figure legends. Cells were, 1) fixed with

2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4 at room temperature for 1 h, 2) washed

with 0.1 M cacodylate buffer and post-fixed with 1% osmium tetraoxide in 0.1 M cacodylate

buffer and 3) dehydrated with graded series of ethanol, and embedded in LX112. Thin sections

were prepared, and stained with uranyl acetate. Specimens were examined on a JEOL 1000X

electron microscope operating at 80 KV.

Cytochrome bc1 activity measurements: Cytochrome bc1 activity in CEM cells was determined

as described previously (29). Briefly, cytochrome bc1 complex activity from digitonin

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permeabilized and sonicated CEM and HL60 cell fractions were assayed in 50 mM potassium

phosphate pH 7.0, 250 mM sucrose, 0.2 mM EDTA, 1 mM NaN3, 0.1% (w/v) and 0.01%

Tween-20 at 23°C, using 50 µM 2,3-dimethoxy-5-methyl-6n-decyl-1, 4-benzoquinol

(decylubiquinol) (dUb) as substrate and 50µM cytochrome c. Decylubiquinonol was synthesized

in the laboratory from decylubiquinone by reduction with sodium borohydride (NaBH4) (31).

Reduction of cytochrome c was monitored in a spectrophotometer at 550 versus 539 nm in dual

wavelength mode. Data are expressed as percentage of control activity and were determined

from five individual isolations that were assayed in triplicate.

Statistical analysis: Statistical analyses were performed using Student’s t test for unpaired data,

and p values <0.05 were considered significant. Data are presented as mean ± SEM.

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RESULTS

GSH depletion mediates ROS increase in CEM and HL60 cells that is decreased by

inhibition of cytochrome bc1: Figure 1A shows representative ROS production, and figure 1B

shows mean ROS production, respectively in CEM cells after treatment with 5 mM DEM ±

respiratory complex inhibitors. Increased DCF fluorescence, a measure of ROS production (29,

32), was determined by a shift in DCF fluorescence to the right after DEM treatment (Figure 1B,

panel 1). Figures show that DEM treatment causes a time-dependent increase in ROS production.

GSH levels were determined on aliquots of CEM cells treated with 5 mM DEM. Figure 1B (inset

panel) shows that a time dependent loss of GSH occurs in CEM cells after cells were treated with

5 mM DEM. Approximately 90% of GSH is lost after DEM treatment for 30 min at which time

ROS increase occurs (Figure 1B).

The distal Qo site of cytochrome bc1 is the predominant site of ROS production after GSH

depletion: To determine the major mitochondrial site of ROS-production after GSH depletion in

CEM cells, we pharmacologically inhibited respiratory complex I (NADH ubiquinone-

dehydrogenase), respiratory complex II (succinate dehydrogenase; SDH) and respiratory

complex III (cytochrome bc1) since these respiratory sites are well known to be involved in

mitochondrial ROS production (33-36). Cells were co-incubated with DEM + either rotenone (5

µM), thenoyltrifluoroacetone (TTFA) (5 µM), stigmatellin (5 µM), myxothiazol (5 µM),

antimycin A and 2-MeAA (control for antimycin A which does not inhibit respiration). Rotenone

selectively blocks NADH ubiquinone-dehydrogenase (37), TTFA blocks SDH (38), stigmatellin

and myxothiazol block the ubiquinol oxidation (Qo) site of cytochrome bc1 at the distal and

proximal niches respectively (39) and Antimycin A blocks the Qi site of cytochrome bc1 (33,

34). Myxothiazol, which binds to the proximal niche of the Qo ubiquinol oxidation site of

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cytochrome bc1 and antimycin A which binds to the Qi site inhibited ROS formation (Figure 1

A, panels 2 and 4), whereas, stigmatellin, which binds to the distal niche of the Qo site

completely blocked DEM-mediated ROS increase (Figure 1 A, panel 3). The experiment with

stigmatellin was extended up to 90 mins but we did not observe any increase in ROS production

(data not shown). To confirm that antimycin A inhibited ROS production we used the structural

analog 2-MeAA as a control (this compound does not inhibit respiration) (40). 2-MeAA did not

inhibit ROS production in DEM treated CEM cells compared to antimycin A (Figure 1 A, panel

5). Rotenone and TTFA did not significantly alter ROS increase compared to DEM treatment

alone concentrations (Figure 1 A, panels 6 and 7). These results indicate that the Qo distal site of

cytochrome bc1 is a key source of ROS production since its inhibition abolished ROS

production. However, since inhibition of the proximal Qo and the Qi sites of cytochrome bc1

reduced ROS production, indicates that this respiratory site is key ROS producing site

additionally implicates the cytochrome bc1 in ROS production. An identical set of experiments

was performed using HL60 cells with similar results (data not shown).

Cytochrome bc1 activity required for ROS production after GSH depletion: The role of

cytochrome bc1 in ROS production in CEM cells was confirmed by determining cytochrome bc1

activity in isolated membrane fractions from CEM cells incubated with DEM, DEM +

stigmatellin, DEM + myxothiazol, DEM + rotenone, DEM + TTFA, DEM + antimycin A and

DEM + 2-MeAA. Figure 1C shows mean ± SEM percentage activity of cytochrome bc1 in CEM

cells treated with DEM ± respiratory inhibitors. Results show that cytochrome bc1 enzyme

activity was not significantly different in CEM cells treated with DEM ± rotenone, TTFA or 2-

MeAA compared to controls, whereas, the compounds stigmatellin, myxothiazol and antimycin

A inhibited cytochrome bc1 enzyme activity approximately 95%.

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ROS production by cytochrome bc1 mediates cell death by activation of the MPT: ∆ψm was

determined in CEM cells, after treatment with DEM (5 mM) to deplete GSH, by monitoring the

fluorescence of the cationic potentiometric dye TMRM. Figure 2A shows representative TMRM

flow cytometric histograms of the CEM cell population monitored every 30 min for 150 mins ±

inhibitors used in the ROS experiments. Loss of ∆ψm is indicated by a shift left of the cell

population on the x axis of the histogram (log scale). Figure 2B shows the percentage (mean ±

SEM) of CEM cells with intact ∆ψm after treatment with DEM ± inhibitors. Figure 2A shows

that the cytochrome bc1 inhibitors stigmatellin (5 µM) (panel 3), and antimycin A (5 µM) (panel

4) and the ANT inhibitor BgK (50 µM) (panel 8) blocked loss of ∆ψm after GSH depletion

(figures 2A and 2B) whereas, rotenone (panel 7), TTFA (panel 6) and the antimycin A analog 2-

MEAA (panel 5) did not prevent loss of ∆ψm. Surprisingly, myxothiazol (Figure 2A, panel 2 and

figure 2B, top), which reduced ROS production, did not prevent DEM-mediated loss of ∆ψm,

paradoxically, this inhibitor increased the rate of loss of ∆ψm compared to DEM treatment alone.

The classical MPT inhibitor CyA did not inhibit loss of ∆ψm induced by GSH depletion, even at

concentrations up to 10 µM (data not shown).

Inhibition of cytochrome bc1 with stigmatellin or antimycin A protects mitochondrial

ultrastructure in response to GSH depletion: To confirm the protective effect of stigmatellin

and antimycin A on mitochondrial integrity, electron microscopy (EM) was performed to

visualize mitochondrial ultrastructure after GSH depletion. Figure 2C shows representative

electron micrographs of mitochondrial ultrastructure in control cells (top panel, left), cells treated

with DEM for 150 min h (top panel, right), cells treated with DEM for 150 min + 5 µM

stigmatellin (bottom panel, left) and cells treated with DEM for 150 min + 5 µM antimycin A

(bottom panel, right). Figure indicates that stigmatellin and antimycin A preserved mitochondrial

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ultrastructure structure compared to DEM treatment alone. Structural and functional studies were

also performed using the HL60 B cell line to determine whether the results observed with CEM

cells were a general or cell specific phenomenon. HL60 cells were treated with DEM (5 mM)

and EM and ∆ψm were determined every 30 min for a total of 150 min. Results show a time-

dependent loss of ∆ψm that corresponds with significant ultrastructural changes in mitochondria.

At 150 min, mitochondrial ultrastructure in HL60 cells was similar to that observed in CEM

cells, including increased electron opacity of mitochondrial inner membrane and cristae. These

structural changes were prevented by co-incubation of cells with DEM + stigmatellin or

antimycin A, but not 2-MeAA (figure 3C).

Cytochrome bc1 inhibition is required for protection against ROS mediated cell death:

Since inhibition of cytochrome bc1 prevented loss of ∆ψm, we determined cell viability after

incubation of CEM cells with 5 mM DEM ± 5 µM of cytochrome bc1 inhibitors (stigmatellin,

antimycin A and myxothiazol) or cyclosporin A (1-10 µM). Cell viability was performed every

30 min for 150 min using trypan blue exclusion. Cells treated with either DEM alone or DEM +

rotenone, TTFA or 2-MeAA lost cell viability over a similar time, whereas BgK prevented loss

of cell viability (Figure 4A). Of the cytochrome bc1 inhibitors, stigmatellin blocked loss of cell

viability induced by DEM and antimycin A reduced the rate at which cells died whereas

myxothiazol did not prevent loss of cell viability (Figure 4B). These results indicate that the

inhibition of ROS formation by stigmatellin or antimycin A protects against redox-dependent

cell death. BgK, also prevented loss of cell viability, however, myothiazol, which reduced ROS

production but did not preserve ∆ψm after GSH depletion, did not prevent loss of cell viability.

CyA, even over a range of concentrations (1-20 µM), did not prevent loss of cell viability after

GSH depletion (data not shown). Cell death was not inhibited by broad spectrum caspase

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inhibitor zVADfmk or the caspase 3 inhibitor DEVD-CHO suggesting the predominant death

pathway was by necrosis (data not shown).

Inhibition of cytochrome bc1 blocks the Ca2+ activated MPT in RLM: Since inhibition of

cytochrome bc1 prevented the redox-activated MPT in cells in situ, we determined whether

inhibition of cytochrome bc1 would also prevent the MPT induced by Ca2+ in RLM. Figure 5A

shows a representative example of the effect of different respiratory complex inhibitors on the

Ca2+ activated MPT in RLM. Stigmatellin (1µM, trace a) and rotenone (1 µM, trace b)

completely prevented mitochondrial swelling induced by 100 µM Ca2+ with similar potency as

CsA (1 µM, trace c). TTFA did not prevent mitochondrial swelling (trace d). Figure 5B shows a

representative example of the effects of antimycin A (1 µM and 10 µM, traces a and c

respectively) and myxothiazol (1 µM, trace b) on the Ca2+ activated MPT in RLM. Figure 5B,

trace d shows the effect of 2-MeAA on Ca2+ induced swelling of RLM this trace also represents

the profile of RLM swelling induced by Ca2+ alone. These results indicate that electron flux

through cytochrome bc1, and the NADH:ubiquinine dehydrogenase regulates the Ca2+-activated

MPT in RLM.

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DISCUSSION

In this study we show that mammalian cytochrome bc1 is a key regulator of the MPT in

CEM and HL60 cells and RLM. In cells, mitochondrial ROS generated during ISP movement at

the ubiquinol oxidation (Qo) site of cytochrome bc1 activates the MPT which is insensitive to

CsA and results in necrosis. In contrast, in RLM the MPT is activated by Ca2+ load and is

blocked by inhibitors of the Qo site of cytochrome bc1 but is sensitive to CsA, These results

indicate that cytochrome bc1 plays a key role in regulation of the regulated MPT in RLM and the

unregulated MPT in cells.

DEM was used to deplete cellular GSH and induce redox stress in CEM and HL60 cells as

previously shown (28, 29). The increase in mitochondrial ROS in CEM cells was blocked by

inhibition of cytochrome bc1 but not NADH:ubiquinone dehydrogenase or SDH (Figure 1 B).

Since these respiratory complexes have each been associated with ROS production (34), our

results indicate that under these experimental conditions, cytochrome bc1 is the principal

mitochondrial site of ROS formation as previously found (28, 29). Inhibition of cytochrome bc1

at either the Qo or Qi sites of cytochrome bc1 inhibited ROS production (measured by the

relative DCF fluorescence of cells) however, the distal Qo niche inhibitor stigmatellin,

completely blocked ROS production whereas, myxothiazol a proximal Qo niche inhibitor and

antimycin A which inhibits the Qi site of cytochrome bc1 reduced but did not block ROS

formation (Figure 1B; bottom panel). These results indicate that mitochondrial ROS are

produced principally at the Qo site of cytochrome bc1. The difference in the Qo site binding

mechanism of the inhibitors myxothiazol and stigmatellin may be important in elucidating the

the mechanism involved in ROS formation at the Qo site, since these two inhibitors have

dramatically different effects on the mobility of the extramembrane domain of the iron-sulfur

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protein (ISP) (41-43). Stigmatellin imobilizes the ISP domain on the surface of cytochrome b

inhibiting the Q cycle, whereas myxothiazol allows movement of the ISP but inhibits the enzyme

by competitive inhibition at the ubiquinone oxidation site (44). These observations suggest that

ROS formation by cytochrome bc1 involves the mobility of the ISP as a key feature. Although

the generation of superoxide by cytochrome bc1 is not new, the concept that ISP movement is

mechanistically linked to ROS during the Q cycle is noteworthy since, to our knowledge, this is

the first report to propose the ISP as directly involved in ROS production. In support of this

novel idea, superoxide production has recently been investigated in isolated cytochrome bc1

complexes from Saccharomyces cerevisiae (S. cerevisiae) by Muller et al., 2003 who also

showed that stigmatellin completely blocked superoxide production, whereas myxothiazol only

partially prevented superoxide formation (39). Also, a report by Sun and Trumpower, also using

bovine heart and S. cerevisiae cytochrome bc1 complexes also showed that stigmatellin

eliminated superoxide formation compared to myxothiazol, and antimycin A (45).

We next considered that if the mechanism of ROS formation involved ISP mobility,

inhibition of ISP movement should, not only prevent ROS formation, but also prevent the

toxicity from uncontrolled ROS production. Cell viability experiments clearly showed that ROS

inhibition of ISP mobility with stigmatellin preserved cell viability compared to DEM alone

treated cells (Figure 4A). The Qi site inhibitor antimycin A also preserved cell viability, although

to a lesser extent than stigmatellin, however, surprisingly myxothiazol, which reduced ROS

formation, did not inhibit cell death (Figure 4A). To investigate this we first confirmed the

efficacy of the respiratory complex inhibitors on cytochrome bc1 enzyme activity using

membrane fractions of CEM cells treated with DEM ± inhibitors. Results showed that

cytochrome bc1 activity was almost completely (~ 95%) inhibited in cells treated with DEM ±

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either stigmatellin, myxothiazol or antimycin A (Figure 1C). We next considered that

myxothiazol could be intrinsically toxic at the concentration used in the experiment. To test this,

we treated cells with a combination of DEM and stigmatellin + myxothiazol expecting that if

myxothiazol were intrinsically toxic, inhibition of ROS production by stigmatellin would fail to

rescue cells. We found that cells were rescued from loss of viability induced by DEM+

myxothiazol by co-incubation with stigmatellin indicating that myxothiazol was not intrinsically

cytotoxic under the conditions of the experiment (data not shown). Next we considered that the

specific topological sites of ROS production by mitochondria in the presence of myxothiazol or

antimycin A could account for the observed ROS reduction using both inhibitors and yet

significant differences in cytotoxicity. Since myxothiazol inhibits superoxide release by

cytochrome bc1 into the intermembrane space and cytosol, but does not prevent formation of

superoxide release into the mitochondrial matrix, ROS will continue to be released into the

mitochondrial matrix resulting in increased intramitochondrial oxidation (see scheme 1:

Topology of mitochondrial superoxide production in the presence of antimycin A and

myxothiazol (adopted from Trumpower 1990 and Boveris and Cadenas 2000, ref 46, 47).

Moreover, since myxothiazol is not specific for cytochrome bc1, but also inhibits the

NADH:ubiquinone dehydrogenase and increases superoxide formation in the mitochondrial

matrix side, significantly increased mitochondrial protein oxidation might be expected using this

inhibitor (48- 50).

SCHEME 1

inner membrane

Antimycin A

myxothiazolmatrix

Inter-membrane space

Q.- Q.-Q Q

O2 O2.-

O2 O2.-

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There is some evidence for this proposition, since ∆ψm of CEM cells was lost at a significantly

earlier time after treatment with DEM + myxothiazol compared DEM alone (Figures 3B and

3C). The decreased level of ROS detection in CEM cells treated with DEM + myxothiazol

compared to DEM alone is considered to be due to the fact that the DCF probe predominantly

detects cytosolic ROS formation and not mitochondrial ROS (Figure 1B, panel 2)(51). These

results, therefore, indicate that cytochrome bc1 is crucial in the redox-activated MPT and that

increased ROS production in the mitochondrial matrix is likely to be the major cell compartment

where ROS are produced and are responsible for loss of ∆ψm and the MPT.

If ROS production in the matrix and subsequent oxidative damage to matrix proteins were

involved in the redox-MPT, we would expect that cytochrome bc1 inhibition would preserve

mitochondrial ultrastructure and ∆ψm which we determined in two independent cell lines

including CEM and HL60 cells. Results clearly showed that GSH depletion caused loss of ∆ψm

together with significant mitochondrial ultrastructural changes in mitochondria compared to

mitochondria in control cells (Figure 2A and 3A). These GSH dependent changes included a

characteristic increased electron density of inner mitochondrial membranes and cristae in both

HL60 and CEM cells (Figure 2C and 3A). The ultrastructural changes resulting from GSH

depletion were not observed in mitochondria of cells co-incubated with DEM and stigmatellin or

antimycin A (Figure 2C and 3B and C) but were apparent in mitochondria of cells treated with

the antimycin A analog 2-MeAA (Figure 3C). The ANT has been previously implicated as a

key protein target in the redox-MPT, since the BgK, which inhibits the ANT at the matrix side,

also blocked loss of ∆ψm and cell death (Figure 3B). Our results suggest that ANT is a key

protein involved in the redox-MPT and that the ATP/ADP binding site of the ANT which is

excluded by binding the ligand BgK is a key MPT oxidative target protein (52). The redox-MPT

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is characterized as unregulated since CsA, which was used over a wide range of concentrations

(1-20 µM), did not inhibit ROS production, or prevent loss of ∆ψm and cell death (data not

shown) (23, 24). Our results suggest that cytochrome bc1–dependent ROS production activates

the MPT described by the model proposed by Kowaltowski et al, in 2001 (8) and recapitulates

the unregulated MPT model proposed by He and Lemasters (23, 24).

Since cytochrome bc1 was clearly involved in the unregulated MPT by its ROS producing

activity, we next determined whether this respiratory complex was a regulator of the Ca2+-

dependent MPT. For these studies we isolated RLM, by standard procedures, and performed the

classical mitochondrial swelling test as an indicator of MPT activation in response to either

increased Ca2+ load. Stigmatellin prevented large amplitude swelling of RLM induced by 100

µM Ca2+ with similar (equimolar) efficacy as CsA. Myxothiazol and antimycin A also inhibited

swelling of RLM but to a lesser extent (Figures 5A and 5B). 2-MeAA was used as a control for

antimycin A, did not prevent Ca2+ induced large amplitude swelling in RLM compared to

antimycin A (Figure 3C). Taken together, our results indicate that two distinct pathways to the

MPT exist, as previously suggested (8, 9,11-15, 23, 24) and that cytochrome bc1 may be a key

regulator factor in both pathways.

Although the proteins composing and regulating the MPT are still unknown, previous

reports clearly indicate that the MPT is regulated by electron flux through the

NADH:ubiquininone dehydrogenase in cells as well as isolated mitochondria (25-27). Our study,

using inhibitors of 1) the Qo ubiquinol catalytic site, 2) the Qi site and 3) ISP movement show

that cytochrome bc1 is a key regulatory component of the MPT by its ability to generate ROS

and its potential to activate the MPT in response to increased Ca2+. Metabolic flux control theory

shows that the NADH:ubiquininone dehydrogenase and cytochrome bc1 may be associated as a

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single enzyme with coenzyme Q as a common substrate (53, 54). Schagger (1999) has proposed

that these two respiratory components form a stable core respirasome in humans (54) and similar

respiratory complex associations have been found in plant mitchondria (55). Based on these

notions, our work, and the investigations of others we propose a novel MPT model regulated by

a respiratory supercomplex formed by NADH:ubiquininone dehydrogenase and cytochrome bc1

as well as the ANT (Figure 6). The role of quinones in this model is implicated from the work of

Fontaine and colleagues (26, 27) as well as from our previous work (29).

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ACKNOWLEDGEMENTS.

We would like to acknowledge Yee Liu Chua for her technical assistance with this work and the

Electron Microscopy unit at the National Universtiy of Singapore (NUS). Acknowledgements

also to ARF GRANT # R183000103112 (JSA) (NMRC/0474/2000, NMRC/0481/2000,

NMRC/0635/2002) (MW) and the NUS Office of Life Science (R183000603712) (MW) for their

generous research support.

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FIGURE LEGENDS:

Figure 1.

A, Representative flow cytometric analysis of CEM cells stained with DCFDA and analyzed

using FL2-H channel as described in the methods section. CEM cells were treated with DEM (5

mM) (panel 1) + myxothiazol (5 µM) (panel 2); stigmatellin (5 µM) (panel 3); Antimycin A (5

µM) (panel 4); 2-MeAA (5 µM) (panel 5); TTFA (5 µM) (panel 6) and rotenone (5 µM) (panel

7) for 0, 10, 20, 30, 40, and 50 min, washed in PBS and suspended in PBS containing 10 mM

glucose. Cells were loaded with DCFDA (10 µM) for 15 min and green fluorescence was

measured by flow cytometry using the FL2-H setting. Figure shows representative example of 3

independent experiments. In each analysis, 10,000 events were recorded.

B, Mean DCF fluorescence intensity (arbitrary units) of CEM cells treated with DEM (5 mM) ±

stigmatellin (5 µM); myxothiazol (5 µM); TTFA (5 µM); rotenone (5 µM); antimycin A (5 µM)

or 2 MeAA (5 µM) for 0, 10, 20, 30, 40, and 50 min washed in PBS and suspended in PBS

containing 10 mM glucose. After treatment cells were loaded with DCFDA (10 µM) for 15 min

and green fluorescence was measured by flow cytometry as described above. In each analysis,

10,000 events were recorded. Data are expressed as mean ± S.E.M (n = 3). Inset panel, CEM

cells were treated with 5 mM DEM or RPMI (control) for 0, 15, 30, and 45 min. GSH levels

were measured on aliquots of cells (approximately 4 x 106/ ml) using monochlorobimane. GSH

concentration was plotted as nmol/mg protein. Data are expressed as mean ± SEM (n=3).

C, Relative activity of cytochrome bc1 in CEM cells treated with DEM (5 mM) ± 5 µM

myxothiazol, 5 µM stigmatellin, 5 µM antimycin A, 5 µM 2-MeAA, 5 µM rotenone, or 5 µM

TTFA for 150 min. Cytochrome bc1 activity measurements were made as described in the

methods and materials. Results are expressed as percentage of control cytochrome bc1 activity ±

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SEM and were determined from 3 independent isolations each assayed in triplicate.

Figure 2.

A, Representative TMRM flow cytometric histograms of CEM cells treated with DEM (5 mM)

(panel 1) + myxothiazol (5 µM) (panel 2); stigmatellin (5 µM) (panel 3); antimycin A (5 µM)

(panel 4); 2-MeAA (5 µM) (panel 5); TTFA (5 µM) (panel 6); rotenone (5 µM) (panel 7) and

BgK (50 µM) (panel 8) for 0, 30, 60, 90, 120, and 150 min washed in PBS and suspended in PBS

containing 10 mM glucose. Cells were loaded with TMRM (250 nM) for 15 minutes and red

fluorescence was immediately measured by flow cytometry using the FL-3 setting as described

in the methods section. Figure shows representative example of 3 independent experiments. In

each analysis, 10,000 events were recorded

B (top), Percentage of CEM cells with an intact ∆ψm determined by relative TMRM

fluorescence intensity. Cells were treated with DEM (5 mM) ± myxothiazol (5 µM) (panel 2),

stigmatellin (5 µM) (panel 3); antimycin A (panel 4); and 2-MeAA (5 µM) for 0, 30, 60, 90, 120,

and 150 min washed in PBS and suspended in PBS containing 10 mM glucose. Cells were

loaded with TMRM (250 nM) for 15 minutes and red fluorescence was immediately measured

by flow cytometry using the FL-3 setting as described in the methods section. Data are expressed

as mean ± S.E.M (n = 3). In each analysis, 10,000 events were recorded. (bottom) Percentage of

CEM cells with an intact ∆ψm determined by relative TMRM fluorescence intensity. Cells were

treated with DEM (5 mM) ± TTFA (5 µM); rotenone (5 µM), and BgK (50 µM) for 0, 30, 60,

90, 120, and 150 min washed in PBS and suspended in PBS containing 10 mM glucose. Cells

were loaded with TMRM (250 nM) for 15 minutes and red fluorescence was immediately

measured by flow cytometry using the FL-2 setting as described in the methods section. Data are

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expressed as mean ± S.E.M (n = 3). In each analysis, 10,000 events were recorded

C, Figure shows the effects of DEM treatment on CEM mitochondrial ultrastructure. Electron

microscopy (TEM) was performed as described in the methods and materials section. Figure

shows the effects of untreated cells (top panel, left); DEM treated cells (top panel, right), DEM +

stigmatellin (5 µM) (bottom panel, left), antimycin A (bottom panel, right).

Figure 3.

A, Figure shows the effects of DEM treatment on HL60 mitochondrial ultrastructure. EM was

performed as described in the methods and materials section. Cells were treated with control

vehicle (RPMI media) or DEM (5 mM) and analyzed by EM at 0, 30, 60, 90, 120, and 150 min.

Inset panels show representative ∆ψm of cell population at times corresponding to the EM

analyses.

B, Top panel shows the effects of DEM (5 mM) on HL60 mitochondrial ultrastructure in the

presence of stigmatellin (5 µM) taken 150 min after treatment. Inset panel shows the

representative ∆ψm of HL60 cell population at a time corresponding to the EM analysis. In the

analysis, 10,000 events were recorded. Bottom panel: shows bar graph of ∆ψm of HL60 cells

treated with 5 mM DEM for 0, 30, 60, 90, 120 and 150 ± 5 µM stigmatellin. Figure shows the

percentage cells ± SEM (n= 3) with intact ∆ψm. In each analysis, 10,000 events were recorded

C, Figure shows the effects of antimycin A and 2-MeAA on ultrastructure of mitochondria in

DEM treated HL60 cells. Panels at right show the ∆ψm of the HL60 cell population at a time

corresponding to the EM analysis: a) control , b) DEM + antimycin A (5 µM) c) DEM + 2-

MeAA (5 µM) and d) CCCP (10 µM). Figure shows representative examples of at least 3

independent experiments. In each analysis, 10,000 events were recorded.

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Figure 4.

A, CEM cells were incubated with 5 mM DEM ± either 5 µM rotenone, 5 µM TTFA, or 50 µM

BgK and cell viability was performed every 30 min up to 150 min using trypan blue exclusion

technique. Data are expressed as mean ± S.E.M (n = 3).

B, CEM cells were incubated with 5 mM DEM ± either 5 µM stigmatellin, 5 µM myxothiazol, 5

µM antimycin A, or 5 µM 2-MeAA and cell viability was performed every 30 min up to 150

min using trypan blue exclusion technique. Data are expressed as mean ± S.E.M (n = 3).

Figure 5.

A, Figure shows the effect of inhibition of NADH:ubiquinine dehydrogenase, SDH and the

the Qo distal site of cytochrome bc1 on the Ca2+ activated MPT in RLM. RLM (~1 mg/ml)

were incubated briefly with control vehicle DMSO (Ca2+ alone trace), stigmatellin (1µM) (trace

a), rotenone (1µM) (trace b), CsA (1 µM) (trace c) or TTFA (1µM) (trace d). 100 µM Ca2+ was

added to mitochondrial suspensions and absorbance change was monitored at 540 nm. Large

amplitude mitochondrial swelling is indicated by a decrease in absorbance at 540 nm. The

cytochrome bc1 inhibitors stigmatellin and the NADH:ubiquinone dehydrogenase inhibitor

rotenone blocked mitochondrial swelling induced by 100 µM Ca2+. The SDH inhibitor TTFA

did not prevent mitochondrial swelling in response to 100 µM Ca2+. Figure shows representative

traces from at least 3 independent experiments.

B, Figure shows the effect of inhibition of the Qi and the Qo proximal sites of cytochrome

bc1 on the Ca2+ activated MPT in RLM RLM (~1 mg/ml) were incubated briefly with control

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vehicle DMSO (Ca2+ alone trace), antimycin A (1µM) (trace a), myxothiazol (1µM) (trace b),

antimycin A (10 µM) (trace c) or 2-MeAA (1-10 µM) (trace d). 100 µM Ca2+ was added to

mitochondrial suspensions and absorbance change was monitored at 540 nm. Large amplitude

mitochondrial swelling is indicated by a decrease in absorbance at 540 nm. The cytochrome bc1

inhibitors myxothiazol and antimycin A inhibited mitochondrial swelling induced by 100 µM

Ca2+. The antimycin A analog 2-MeAA did not prevent mitochondrial swelling in response to

100 µM Ca2+. Figure shows representative traces from at least 3 independent experiments.

Figure 6

Figure shows a model of cytochrome bc1 involvement in regulated and unregulated MPT:

Model depicts the unregulated MPT activated by mitochondrial ROS generated by cytochrome

bc1 in the mitochondrial matrix and the regulated MPT activated by electron flux through

cytochrome bc1. In the model we show the idea that a respiratory supercomplex formed by

NADH:ubiquininone dehydrogenase and cytochrome bc1 is involved in regulation of the MPT.

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FIGURE 1

0 10 20 30 40 50 600

25

50

75

100DEMDEM + MYXDEM + STIGDEM + AADEM + 2-MeAADEM + TTFADEM + ROTENONE

Time (mins)

DC

F Fl

uore

scen

ce (a

.u.)

B

0 10 20 30 40 500

5

10

15

time (min)

GSH

nm

ol/m

g pr

otei

n

0

25

50

75

100

controlDEM+ROT (5 µM)DEM+AA (5 µM)DEM+2AA (5 µM)DEM+TTFA (5 µM)DEM+MYX (5 µM)DEM+STIG (5 µM)DEM

cyto

chro

me

bc1

activ

ity(%

con

trol

)

** ** **

C

0

10

20

30

40

50

PANEL 1 2 3 4 5 6 7

proximal Oo distal Oo OiCytochrome bc1

SDH

DCF Fluorescence (a.u.)Log scale

NADH: ubiquinone

dehydrogenasemyxothiazol stigmatellin antimycin A 2-Me AA TTFA rotenone

A

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FIGURE 2

0.5 µM 0.5 µM

DEM

0.5 µM

0.5 µM

0.5 µM

0.5 µM

control

DEM + antimycin A DEM + stigmatellin

DEM

C

0 30 60 90 120 1500

25

50

75

100

DEM alone+ myxothiazol+ stigmatellin+ antimycin A+ 2-Me AA

time (min)

perc

enta

ge c

ells

with

inta

ct∆ψ

m

inhibition of cytochrome bc1

Qo distal

Qi

Qo proximal

B

0 30 60 90 120 1500

25

50

75

100

DEM alone+ TTFA+ rotenone+ BgK

time (min)

perc

enta

ge c

ells

with

inta

ct∆ψ

m

inhibition of NADH:ubiquinone dehydrogenase,SDH and ANT

ANT

SDH and NADH:ubiquinone dehydrogenase

0

30

60

90

120

150

DEM + MYX + STIG + AA + 2-MeAA + TTFA + rotenone + BgK

min

utes

PANEL 1 2 3 4 5 6 7 8

A

TMRM Fluorescence (a.u.)Log scale

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FIGURE 3

C Antimycin A 2-MeAA

control

DEM + antimycin A

TMRM fluorescence (a.u.)Log scale

DEM + 2-MeAA

CCCP

a

b

c

d

x 4750 x 4750

x 47500 x 47500

A B

x 47,500

DEM + 10 µM stigmatellin

90 120 150

0 30 60

x 47500 x 47500

x 47500

x 47500

x 47500 x 47500

0 30 60 90 120 1500

25

50

75

100

**

time (min)

% c

ells

with

inta

ct∆ψ

m

150 + stigmatellin

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FIGURE 4

A

B

0 30 60 90 120 150 180 2100

25

50

75

100

+ stigmatellin+ myxothiazolantimycin A2-MeAA

time (min)

viab

ility

(% c

ontr

ol)

inhibition of cytochrome bc1

0 30 60 90 120 150 180 2100

25

50

75

100

DEM alone+ rotenone+ TTFA+ BgK

time (min)

viab

ility

(% c

ontr

ol)

Inhibition of NADH:ubiquinone dehydrogenaseSDH and ANT

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FIGURE 5

A

B

30 90 150 210 270 330 3900.12

0.22

0.32

0.42

0.52 (a, b, c)

(d)

(Ca2+ alone)

Ca2+

OD

@ 5

40 n

m

30 90 150 210 270 330 3900.12

0.22

0.32

0.42

0.52

Time (seconds)

OD

@ 5

40 n

m

(d)

(a)

(b)

(c)

Ca2+

(Ca2+ alone)

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FIGURE 6

ANT ANT

Qo

Mitochondrial inner membrane

Cyclophilin D

H2O, ions

Unregulated MPT

cytochrome bc1

Qi

Mitochondrial inter membrane space

Matrix

Q.-

Regulated MPT

O2.-

Qo

NADH: ubiquinonedehydrogenase

Qo Q

ROS Ca2+ and ubiquinones

cytochrome bc1

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Jeffrey S. Armstrong, Hongyuan Yang, Wei Duan and Matthew Whitemanpathways

Cytochrome bc1 regulates the mitochondrial permeability transition by two distinct

published online September 10, 2004J. Biol. Chem. 

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