Low complex I content explains the low hydrogenperoxide production rate of heart mitochondria fromthe long-lived pigeon, Columba livia

Adrian J. Lambert,1 Julie A. Buckingham,1 Helen M.Boysen1 and Martin D. Brand1,2

1Medical Research Council Mitochondrial Biology Unit, Hills Road,

Cambridge, CB2 0XY, UK2Buck Institute for Age Research, 8001 Redwood Blvd, Novato,

CA 94945, USA

Summary

Across a range of vertebrate species, it is known that

there is a negative association between maximum life-

span and mitochondrial hydrogen peroxide production.

In this report, we investigate the underlying biochemical

basis of the low hydrogen peroxide production rate of

heart mitochondria from a long-lived species (pigeon)

compared with a short-lived species with similar body

mass (rat). The difference in hydrogen peroxide efflux

rate was not explained by differences in either superox-

ide dismutase activity or hydrogen peroxide removal

capacity. During succinate oxidation, the difference in

hydrogen peroxide production rate between the species

was localized to the DpH-sensitive superoxide producing

site within complex I. Mitochondrial DpH was significantly

lower in pigeon mitochondria compared with rat, but this

difference in DpH was not great enough to explain the

lower hydrogen peroxide production rate. As judged by

mitochondrial flavin mononucleotide content and blue

native polyacrylamide gel electrophoresis, pigeon mito-

chondria contained less complex I than rat mitochondria.

Recalculation revealed that the rates of hydrogen perox-

ide production per molecule of complex I were the same

in rat and pigeon. We conclude that mitochondria from

the long-lived pigeon display low rates of hydrogen per-

oxide production because they have low levels of com-

plex I.

Key words: comparative biology of aging; complex I; free

radical theory of aging; mitochondria; reactive oxygen

species; superoxide.

Introduction

The term reactive oxygen species (ROS) is usually used to indi-

cate any oxygen-containing molecule capable of initiating some

kind of deleterious reaction. Types of ROS include superoxide

(O2·)), hydrogen peroxide (H2O2), hydroxyl radical (HO·), alkoxyl

radical (RO·) and hydroperoxyl radical (HO2·). ROS are frequently

associated with negative consequences because they play a role

in the aetiology of many diseases; however, they also have

essential functions in cellular signalling pathways and immunity

(Li et al., 1995; Raha & Robinson, 2000; Droge, 2002). The accu-

mulation of unrepaired molecular damage that ROS can cause is

thought to underlie the decline in physiological function

observed in senescence and is the basis of the free radical

hypothesis of aging (Harman, 1956; Beckman & Ames, 1998;

Golden et al., 2002; Brand et al., 2004; Balaban et al., 2005;

Muller et al., 2007).

Pioneering work showed that in isolated mitochondria and

sub-mitochondrial particles, the primary ROS produced is super-

oxide, and complexes I and III of the electron transport chain are

the sites of production (Loschen et al., 1971, 1974; Boveris &

Chance, 1973; Boveris & Cadenas, 1975; Cadenas et al., 1977;

Turrens & Boveris, 1980). This work has since been verified by

several independent laboratories (Turrens, 2003; Brand et al.,

2004; Murphy, 2009) and has led to a consensus view of super-

oxide production, summarized in Fig. 1. A variety of other

enzymes are capable of producing ROS, including succinate

dehydrogenase, a-glycerophosphate dehydrogenase, the elec-

tron transfer flavoprotein quinone oxidoreductase and a-keto-

glutarate dehydrogenase (St-Pierre et al., 2002; Brand et al.,

2004; Starkov et al., 2004; Andreyev et al., 2005). Most of the

superoxide produced inside the mitochondrial matrix is con-

verted into hydrogen peroxide by manganese-containing super-

oxide dismutase. Superoxide produced to the cytosolic side of

mitochondria is also converted to hydrogen peroxide, by cop-

per–zinc-containing superoxide dismutase. Hydrogen peroxide

is metabolized in the mitochondria and cytosol by several

enzymes, including catalase and glutathione peroxidase.

Several independent studies have reported low rates of ROS

production by mitochondria, sub-mitochondrial particles and

cells of long-lived species (Sohal et al., 1990, 1993; Ku & Sohal,

1993; Ku et al., 1993; Herrero & Barja, 1997, 1998, 2000; Barja

& Herrero, 1998; Brunet-Rossinni, 2004; Csiszar et al., 2007;

Lambert et al., 2007; Ungvari et al., 2008). Taken together,

these studies suggest a correlation (and hence possible causality)

between mitochondrial superoxide production and maximum

lifespan. The low rates of ROS production were found in a

Correspondence

Martin Brand, Buck Institute for Age Research, 8001 Redwood Blvd,

Novato, CA 94945, USA. Tel.: +1 (415) 493-3676; fax: +1 (415) 209-2232;

e-mail: mbrand@buckinstitute.org

Accepted for publication 8 November 2009

78 ª 2010 The AuthorsJournal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2010

Aging Cell (2010) 9, pp78–91 Doi: 10.1111/j.1474-9726.2009.00538.xAg

ing

Cell

variety of tissues, at both complexes I and III. The underlying

mechanism responsible for these low ROS production rates by

mitochondria from long-lived species has not been identified.

The purpose of this study was to investigate in detail the possible

causes of low ROS production by mitochondria from long-lived

species. Based on availability, a sevenfold difference in maxi-

mum recorded lifespan, similar body masses and basal metabolic

rates, we chose the laboratory rat and the domesticated pigeon

as short-lived and long-lived model organisms, respectively.

Maximum published lifespans are 5 and 35 years for rat and

pigeon respectively (http://genomics.senescence.info/species).

Results

Pigeon heart mitochondria have lower rates of

hydrogen peroxide efflux than rat heart

mitochondria

It was reported previously that when respiring on succinate,

heart mitochondria isolated from pigeons exhibit significantly

lower rates of hydrogen peroxide efflux than heart mitochondria

isolated from rats (Ku & Sohal, 1993; Herrero & Barja, 1997).

We confirmed these observations (Lambert et al., 2007) and

have sought to explain them mechanistically in this study.

Figure 2A shows the results from a typical hydrogen peroxide

efflux experiment. The rate of fluorescence increase upon addi-

tion of succinate was greater in mitochondria from rats. No dif-

ferences in background rates or standard curves were found

(not shown). Therefore, the differences in the slopes of fluores-

cence versus time translate directly to differences in hydrogen

peroxide efflux rates by the isolated heart mitochondria. The

rates were essentially linear over several minutes; longer incuba-

tions in excess of 5 min resulted in a slow decline in the slopes of

fluorescence versus time in both species, but importantly, the

traces were never seen to overlap.

In most work with isolated mitochondria, bovine serum albu-

min (BSA) is included in the incubation medium to bind free fatty

acids, which are known to uncouple mitochondria (Dedukhova

et al., 1991). Uncoupling results in a lower proton-motive force

which lowers succinate-driven hydrogen peroxide efflux (Kor-

shunov et al., 1997; Lambert & Brand, 2004b). It is possible that

pigeon mitochondrial preparations contain a greater concentra-

tion of fatty acids than rat mitochondrial preparations, are thus

more uncoupled, and so display lower rates of hydrogen perox-

ide efflux (Tretter et al., 2007). To test this possibility, a BSA

titration was performed (Fig. 2B). Increasing the BSA concentra-

tion from zero to the standard concentration of 0.3% increased

the rates of hydrogen peroxide efflux in mitochondria from both

species, but between 0.3 and 0.6% the increases were relatively

minor. This indicates that the normal BSA concentration of

0.3% is not limiting for hydrogen peroxide efflux in the assay.

Importantly, the two data sets did not overlap over the range of

BSA concentrations tested, thus a difference in free fatty acid

concentration and associated difference in uncoupling does not

explain the difference in hydrogen peroxide efflux rates.

Figure 2C shows that the rates of hydrogen peroxide efflux

were not significantly affected by the concentration of mito-

chondria in the assay. From this, we can conclude that the

A

C

B

D

Fig. 1 Modes of superoxide production by the mitochondrial electron transport chain. (A) Pyruvate and malate generate NADH, which induces forward electron

transport and results in relatively low rates of superoxide production from complexes I and III. A membrane potential is generated and oxygen consumption occurs

at complex IV. (B) Inhibited forward electron transport by rotenone results in increased rates of superoxide production from the flavin site of complex I. Proton

pumping is inhibited under these conditions (thus the membrane potential collapses) and the oxygen consumption rate approaches zero. (C) Succinate oxidation

results in a high superoxide production rate from the quinone binding site of complex I. Addition of rotenone diminishes this high rate (D), indicating that the high

rate in (C) originates at complex I during reverse electron transport. Low rates of superoxide production originate at complex III (and perhaps complex II) during

succinate oxidation. The low rate at complex III in (D) is increased markedly by addition of the complex III inhibitor antimycin A, during which the oxygen

consumption rate approaches zero and the membrane potential collapses. Complex I produces superoxide to the matrix side of the inner mitochondrial

membrane, superoxide production by complex III is directed to both the matrix and the intermembrane space (Han et al., 2001; St-Pierre et al., 2002; Muller et al.,

2004).

Mechanism of low ROS production rates, A. J. Lambert et al.

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79

concentration of the amplex red ⁄ horseradish peroxidase (HRP)

detection system was not limiting. Crucially, the rat and pigeon

data sets did not overlap over the range of mitochondrial protein

concentrations tested.

The findings presented here, taken with the independent

results of two other laboratories, indicate that low hydrogen

peroxide efflux rates from pigeon mitochondria compared with

rats is not the result of a system artefact but a genuine biological

phenomenon of the isolated mitochondria.

Low rates of hydrogen peroxide efflux by pigeon

mitochondria are not explained by differences in

anti-oxidant systems

It is established that the primary ROS produced by the mitochon-

drial electron transport chain is superoxide and not hydrogen

peroxide (Loschen et al., 1974; Turrens, 1997; Murphy, 2009).

When measuring superoxide production indirectly as hydrogen

peroxide efflux, it is assumed that matrix superoxide dismutase

(SOD) activity is not limiting, so that all of the superoxide is con-

verted to hydrogen peroxide. This assumption is reasonable as

SOD is present at relatively high (lM) concentrations in the

matrix and the rate constant for the dismutation is very high (kcat

�109

M)1 s)1) (Murphy, 2009). However, the assumption may

not hold when comparing two species, therefore we measured

SOD activity in the mitochondrial preparations to test the possi-

bility that the apparent low hydrogen peroxide efflux rates from

pigeon mitochondria are related to relatively low SOD activity.

This was not the case; as shown in Fig. 3A, SOD activity in bro-

ken pigeon mitochondria was higher than in rat mitochondria.

The calculated SOD activities were 16 ± 3 and 29 ± 3 mU

SOD mg mitochondrial protein)1 for rat and pigeon mitochon-

dria respectively (mean ± S.E.M., P = 0.022 by t-test, n = 6 sep-

arate mitochondrial preparations). This result is in agreement

with published data showing relatively high SOD activity in

pigeon tissues compared with rats, and in long-lived species in

general (Cutler, 1985; Ku & Sohal, 1993; Brown & Stuart,

2007). We conclude that the difference in hydrogen peroxide

efflux rate between rats and pigeons cannot potentially be

explained by differences in matrix SOD activity.

The amplex red ⁄ HRP system detects hydrogen peroxide that

has escaped the mitochondrial matrix. HRP is too large to enter

mitochondria and there is no known system to import it, thus it

functions in the bulk phase only. Amplex red, being a small mol-

ecule and solubilized in DMSO, may possibly enter the mito-

chondria. However, in the absence of HRP, amplex red alone

does not give a fluorescent signal with mitochondria respiring

on succinate (not shown). So if amplex red can enter the mito-

chondrial matrix, it does not react with any endogenous perox-

idases to generate the fluorescent product resorufin. Therefore,

all the fluorescent signal obtained with mitochondria respiring

on succinate derives from escaped hydrogen peroxide reacting

with the amplex red ⁄ HRP detection system.

The mitochondrial matrix contains anti-oxidant systems for

removing hydrogen peroxide, including glutathione peroxidase,

catalase and thioredoxin-dependent peroxide reductases (perox-

iredoxins) (Radi et al., 1991; Antunes et al., 2002; Andreyev

et al., 2005; Salvi et al., 2007). Differences in the activity of

these systems between rats and pigeons might explain the dif-

ferences in hydrogen peroxide efflux rates. It could be that the

rates of superoxide production are the same, but more hydro-

gen peroxide is removed by pigeon mitochondria before it

escapes and is consumed by the amplex red ⁄ HRP detection

system. This possibility was tested by monitoring the removal

rates of hydrogen peroxide by mitochondria in the absence of

A

B

C

Fig. 2 Hydrogen peroxide assays for rat and pigeon mitochondria. Hydrogen

peroxide efflux rate was determined fluorometrically by measurement of

oxidation of amplex red to fluorescent resorufin coupled to the enzymatic

reduction of H2O2 by horseradish peroxidase (HRP). (A) Typical traces of

fluorescence versus time for rat and pigeon heart mitochondria incubated at

0.35 mg mitochondrial protein mL)1 with 0.3% (w ⁄ v) BSA. Succinate (5 mM)

was added at 20 s. (B) Effect of BSA concentration on hydrogen peroxide

efflux rates. Closed symbols: rat; open symbols: pigeon. The mitochondrial

protein concentration was 0.35 mg mL)1. Points are mean ± ranges of n = 2

separate mitochondrial preparations. (C) Effect of mitochondrial protein

concentration on rates of hydrogen peroxide efflux. Closed symbols: rat; open

symbols: pigeon. The BSA concentration was 0.3% (w ⁄ v). Points are

mean ± S.E.M. of n = 3 separate mitochondrial preparations. Two-factor

ANOVA revealed no significant effect of [mitochondrial protein] on rate, and a

significant effect (F1,8 = 35.9, P = 0.004) of species on rate. In all

experiments, the buffer contained 120 mM potassium chloride, 5 mM HEPES,

3 mM EGTA (pH 7.2 at 37 �C), 50 lM amplex red and 6 U mL)1 HRP.

Mechanism of low ROS production rates, A. J. Lambert et al.

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80

succinate. Figure 3B shows that mitochondria could indeed

remove hydrogen peroxide, but the rat and pigeon traces were

virtually indistinguishable. These results indicate that the differ-

ences in hydrogen peroxide efflux rates between rats and

pigeon mitochondria are unlikely to be caused by differences in

hydrogen peroxide removal capacity in the matrix.

The alkylating agent 1-chloro-2,4-dinitrobenzene (CDNB) has

been shown to increase mitochondrial hydrogen peroxide efflux

rates (Zoccarato et al., 1988; Han et al., 2003). These effects

are probably related to its ability to interfere with mitochondrial

anti-oxidant systems. For example, CDNB is known to deplete

mitochondrial glutathione and inhibit thioredoxin reductase

(Arner et al., 1995; Han et al., 2003). To see if the differences in

hydrogen peroxide efflux rate between rat and pigeon mito-

chondria could be removed by CDNB, we performed a CDNB

titration (Fig. 3C). In agreement with previous reports, CDNB

caused an increase in the rate of hydrogen peroxide efflux by

isolated mitochondria respiring on succinate. This increase was

observed in both rat and pigeon mitochondria, but it is clear that

the relationships between [CDNB] and hydrogen peroxide efflux

rate do not overlap, and do not converge at high CDNB concen-

trations. Therefore, the differences in rate of hydrogen peroxide

efflux rate between rat and pigeon mitochondria are not

explained by differences in antioxidant capacity that can be

suppressed by CDNB or by other CDNB-modulated effects.

The above experiments show that the low rate of hydrogen

peroxide efflux from pigeon mitochondria is not explained by

differences in anti-oxidant systems. The most likely explanation

for this low rate is a low rate of superoxide production by the

electron transport chain.

The difference in hydrogen peroxide efflux rate

between rat and pigeon mitochondria originates at a

DpH sensitive site within complex I of the

mitochondrial electron transport chain

During succinate oxidation, electrons enter complex II and are

transported down the electron transport chain, leading to pro-

ton pumping by complexes III and IV. The resulting proton-

motive force and reduced coenzyme Q pool drives electrons

thermodynamically uphill into complex I, reducing NAD+ to

NADH. This process of reverse electron transport is inhibited by

the complex I inhibitor rotenone. Hydrogen peroxide efflux rates

from isolated mitochondria respiring on succinate are relatively

high and these high rates are diminished markedly by rotenone.

This phenomenon has been observed by several laboratories

using mitochondria from a variety of tissues (Hansford et al.,

1997; Votyakova & Reynolds, 2001; Kushnareva et al., 2002;

Liu et al., 2002; Lambert & Brand, 2004b; Ohnishi et al., 2005;

Zoccarato et al., 2007). The standard interpretation of this

observation is that the majority of superoxide is produced at

complex I during reverse electron transport from succinate to

NAD+. Figure 4A shows that the difference in hydrogen perox-

ide efflux rate between rat and pigeon mitochondria seen in the

presence of succinate was removed by the addition of rotenone.

A

B

C

Fig. 3 (A) Superoxide dismutase (SOD) activity in lysed rat and pigeon heart

mitochondria. Superoxide was generated using a xanthine ⁄ xanthine oxidase

system and detected by monitoring the change in absorbance because of the

reduction of acetylated cytochrome c. The ability of mitochondria to inhibit

this reaction reports the activity of SOD in the sample. Using a 96-well plate,

each 200 lL assay (performed in duplicate wells) contained either 0.4 mg

mitochondrial protein or a known activity of commercial copper–zinc bovine

SOD. Stars: 0, 0.02 or 2 units of bovine SOD per well as indicated, points are

means (n = 2 separate wells, error bars omitted for clarity). Additional assays

containing 0.002 and 0.2 units of bovine SOD were performed (omitted for

clarity). Closed symbols: rat; open symbols: pigeon. Each rat and pigeon data

point is the mean ± S.E.M. of n = 5 separate mitochondrial preparations,

with each assay performed in duplicate. (B) Hydrogen peroxide removal

capacity of rat and pigeon mitochondria. Known amounts of hydrogen

peroxide were added to mitochondria (0.35 mg mitochondrial protein mL)1)

and its disappearance over time was followed by addition of the amplex

red ⁄ HRP detection system and measurement of fluorescence. At t = 0 min,

the detection system was added prior to the hydrogen peroxide. The amount

of H2O2 in nmol per well at each time point was calculated from a standard

curve. Using a 96-well plate, each 200 lL assay was performed in

quadruplicate. Closed symbols: rat; open symbols: pigeon; circles: 0.2 nmol

H2O2 added; squares: 0.04 nmol H2O2 added. Each rat and pigeon data point

is the mean ± S.E.M. of n = 6 separate mitochondrial preparations. In control

experiments without mitochondria, there was no degradation of H2O2 (not

shown). (C) Effect of CDNB on rates of H2O2 efflux. Closed symbols: rat; open

symbols: pigeon. Points are mean ± S.E.M. of n = 4 separate mitochondrial

preparations. Two-factor ANOVA revealed a significant effect (F3,24 = 7.05

P < 0.002) of [CDNB] on rate, and a significant effect (F1,24 = 23.9,

P < 0.0001) of species on rate. Incubation conditions were 0.35 mg

mitochondrial protein mL)1 in KHEB buffer (37 �C) with 5 mM succinate,

50 lM amplex red and 6 U.mL)1 HRP.

Mechanism of low ROS production rates, A. J. Lambert et al.

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This confirms our earlier observation and those of Herrero and

Barja (Herrero & Barja, 1997; Lambert et al., 2007). We con-

clude that the difference in hydrogen peroxide efflux rate

between rat and pigeon mitochondria originates as a difference

in superoxide production rate at complex I during reverse elec-

tron transport from succinate to NAD+. We assume that there

was enough rotenone present to inhibit complex I completely in

both species, as a rotenone concentration of 2 lM is in tenfold

excess of the 0.2 lM required to fully inhibit complex I under

similar conditions to those used in the assay (Lambert & Brand,

2004a).

It has been demonstrated in brain, liver and skeletal muscle

mitochondria that hydrogen peroxide efflux from mitochondria

oxidizing succinate is diminished in the presence of 100 nM of

A B

C D

E F

Fig. 4 (A) Effect of rotenone on rates of hydrogen peroxide efflux from rat and pigeon heart mitochondria. Filled bars: rat; open bars: pigeon. Incubation

conditions were 0.35 mg mitochondrial protein mL)1 in KHEB buffer with amplex red and HRP. The succinate and rotenone concentrations were 5 mM and 2 lM

respectively. Bars are means ± S.E.M. of n = 9 separate mitochondrial preparations. Two-factor ANOVA revealed a significant effect of rotenone (F1,32 = 134,

P < 0.0001) and a significant difference by Bonferroni post t-test (*P < 0.001). (B) Main panel: conditions identical to those in panel A, except 100 nM nigericin

was present. Filled bars: rat; open bars: pigeon. Bars are mean ± S.E.M. of n = 6 separate mitochondrial preparations. Two-factor ANOVA revealed a significant

effect of rotenone (F1,20 = 34.1, P < 0.0001) and no significant difference between species. Significant difference versus absence of nigericin by post hoc two-

factor ANOVA (F1,26 = 59.1, **P < 0.0001). Inset: effect of nigericin concentration on rate of hydrogen peroxide efflux. Closed symbols: rat; open symbols: pigeon.

Each point is the mean of n = 5 separate mitochondrial preparations, error bars omitted for clarity. (C) Measurement of mitochondrial membrane potential and

DpH in mitochondria respiring on succinate. The plot shows a typical trace of TPMP+ concentration versus time for rat heart mitochondria (0.35 mg mitochondrial

protein mL)1) incubated in KHEB buffer. TPMP+ (4 · 125 nM), succinate (5 mM), nigericin (100 nM) and FCCP (2 lM) were added as indicated. (D) Same as panel C,

except with pigeon mitochondria. (E) Plot of rate of hydrogen peroxide efflux from rat and pigeon mitochondria versus DpH. Closed symbols: rat; open symbols:

pigeon. Incubation conditions were 0.35 mg mitochondrial protein mL)1 in KHEB buffer with amplex red, HRP and 5 mM succinate. DpH was varied by nigericin

titration to concentrations of 0, 1.25, 2.5, 5 and 100 nM. Each point is the mean ± S.E.M. of n = 5 separate mitochondrial preparations. Hydrogen peroxide efflux

rates significantly different (*P = 0.039), DpH not significantly different (t-test). (F) Effect of mitochondrial matrix pH (pHin) on rates of hydrogen peroxide efflux.

Closed symbols: rat, open symbols, pigeon. Incubation conditions were 0.35 mg mitochondrial protein mL)1 in KHEB buffer with amplex red, HRP and 5 mM

succinate. The pH of the mitochondrial matrix was varied by using KHEB buffer with pH values (pHout) of 6.6, 7.2 and 7.8. The values of pHin were calculated using

the formula pHin = pHout + DpH (in pH units). Each point is the mean ± S.E.M. of n = 3 separate mitochondrial preparations.

Mechanism of low ROS production rates, A. J. Lambert et al.

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82

the proton ⁄ potassium exchanger nigericin (Liu, 1997; Lambert

& Brand, 2004b; Zoccarato et al., 2007; Selivanov et al., 2008).

We concluded that within complex I, there is a site of superoxide

production that is dependent on the pH gradient across the

mitochondrial inner membrane (DpH), because 100 nM nigericin

collapses DpH to zero (Lambert & Brand, 2004b). We show here

that nigericin also has a significant inhibitory effect on hydrogen

peroxide efflux in heart mitochondria from both the rat and the

pigeon (compare Fig. 4A, B). The differences in hydrogen perox-

ide efflux rates between rats and pigeons were abolished by

nigericin (Fig. 4B), suggesting that the differences originate at a

DpH-sensitive superoxide producing site within complex I. The

inset in panel B of Fig. 4 shows a plot of hydrogen peroxide

efflux rate versus nigericin concentration for both rat and

pigeon. We conclude that the nigericin concentration was in

excess at 100 nM and the nonlinearity of the titrations indicate

that it is unlikely that nigericin is simply acting as a direct anti-

oxidant.

Because superoxide production rate depends on DpH, the dif-

ferences between rat and pigeon may be due to differences in

DpH itself. We determined DpH using a triphenylmethylphos-

phonium cation (TPMP+) electrode; typical experiments are dis-

played in Fig. 4C, D. TPMP+ is a lipophilic cation that

accumulates in the mitochondrial matrix according to the mem-

brane potential, Dw. When nigericin is added, DpH collapses to

zero and is converted to the electrical component of proton-

motive force, Dw. When DpH is zero, Dw is equal to the total pro-

ton-motive force, Dp. DpH is simply calculated as DpH = Dp

(presence of nigericin) ) Dw (absence of nigericin), as previously

described (Lambert & Brand, 2004b). Comparing Fig. 4C, D, it

can be seen that the deflection in the trace upon addition of

nigericin was smaller in pigeon mitochondria, suggesting that

DpH was lower. Table 1 shows that DpH in pigeon mitochondria

was indeed significantly lower than DpH in rat mitochondria, as

determined by TPMP+ distribution. To confirm this result, further

experiments were conducted using radiolabelled acetate, which

distributes across the mitochondrial inner membrane according

to DpH. Table 1 shows that using this method, DpH was again

significantly lower in pigeon mitochondria. We conclude that

DpH is significantly lower in pigeon mitochondria than rat mito-

chondria respiring on succinate.

If the difference between rat and pigeon hydrogen peroxide

efflux rate was because of a difference in DpH, then equalizing

DpH should equalize the rates. This hypothesis was tested by

nigericin titration; Fig. 4E. Similar to the results found using rat

skeletal muscle mitochondria (Lambert & Brand, 2004b), the

plot of hydrogen peroxide efflux rate versus DpH displayed satu-

ration-type behaviour. Above approximately 20 mV for rat mito-

chondria and 10 mV for pigeon mitochondria, changes in DpH

had diminishing effects on the rates of hydrogen peroxide

efflux. Addition of 1.25 nM nigericin to rat mitochondria

resulted in a DpH of 14 ± 3 mV, which was not significantly dif-

ferent from the DpH of 18 ± 3 mV in pigeon mitochondria at

zero nigericin concentration. However, the corresponding rates

of hydrogen peroxide efflux were significantly different. There-

fore, equalizing the values of DpH in rat and pigeon mitochon-

dria did not equalize the rates of hydrogen peroxide efflux, in

fact the rates only began to equalize at very low values of DpH.

We conclude that the differences in hydrogen peroxide efflux

rate are not explained by the lower value of DpH, even though

they originate at the DpH sensitive site within complex I and DpH

is lower in pigeon mitochondria.

Nigericin affects the pH of the mitochondrial matrix (pHin), as

well as DpH. This is because protons are pumped out from the

matrix to give a pH gradient that is acid outside, so collapse of

DpH acidifies the matrix. We previously showed that hydrogen

peroxide efflux rate was not dependent on pHin in rat skeletal

muscle mitochondria respiring on succinate and concluded that

nigericin lowers hydrogen peroxide efflux rate by lowering DpH

(Lambert & Brand, 2004b). Figure 4F shows that pHin does not

affect hydrogen peroxide efflux rate in heart mitochondria from

either the rat or the pigeon, and that the two data sets do not

overlap. This indicates that the effects of nigericin on hydrogen

peroxide efflux rates are mediated by DpH and not by pHin, in

mitochondria from both species.

In addition to DpH, superoxide production also depends on

Dw. No significant differences in this parameter were found

(Table 1), which excludes differences in Dw as an explanation for

the differences in superoxide production rate.

Low rates of hydrogen peroxide efflux by pigeon

mitochondria are not explained by differences in

redox potentials of the electron carriers within

complex I

The rate of superoxide production depends on the concentra-

tions of oxygen and X where ‘X’ is the reductant of oxygen (Tur-

rens, 1997). In complex III, ‘X’ is believed to be a semiquinone at

centre o in the enzyme (Turrens et al., 1985; Cape et al., 2007).

In isolated complex I, ‘X’ is the reduced flavin moiety, FMNH2

(Kussmaul & Hirst, 2006). In intact, proton-pumping mitochon-

dria, it is not clear which site is responsible for superoxide pro-

duction by complex I, it may be the flavin mononucleotide

Table 1 Bioenergetic parameters of rat and pigeon heart mitochondria

Rat Pigeon

Dw (mV)† 144 ± 7 152 ± 6

DpH (mV)† 45 ± 4 18 ± 3*

Dp (mV)† 189 ± 8 170 ± 5

DpH (mV)� 44 ± 4 28 ± 2*

Mitochondrial matrix volume

(lL mg mitochondrial protein)1)�

1.48 ± 0.22 1.44 ± 0.24

Mitochondrial membrane potential (Dw), pH gradient across the inner

mitochondrial membrane (DpH), proton-motive force (Dp) and

mitochondrial matrix volume in rat and pigeon mitochondria.

*Significant difference, rat versus pigeon (P < 0.003).

†Determined using a TPMP+ electrode, values are mean ± standard errors

for n = 5 separate mitochondrial preparations.

�Determined using radioisotopes, values are mean ± standard errors for

n = 8 separate mitochondrial preparations.

Mechanism of low ROS production rates, A. J. Lambert et al.

ª 2010 The AuthorsJournal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2010

83

(FMN) site, one of the iron–sulphur centres, the quinone (Q)

binding site or a combination of these sites. The structure of

hydrophilic arm of complex I from Thermus thermophilus (Saza-

nov & Hinchliffe, 2006) shows that most of the internal electron

carriers (the iron-sulphur centres) are shielded from solvent, and

are thus unlikely to donate electrons to oxygen and produce

superoxide. The most likely superoxide-producing carriers are

therefore at the solvent-exposed substrate binding sites, which

are the FMN at the NADH binding site, and the Q-binding site

(Hirst et al., 2008). Our current model proposes that the Q-bind-

ing site dominates during reverse electron transport (Lambert &

Brand, 2004a; Lambert et al., 2008a,b). We investigated the

possibility that relatively low rates of hydrogen peroxide efflux

rates in pigeon mitochondria are related to a relatively low con-

centration of ‘X’ within pigeon complex I, assuming ‘X’ to be in

the Q-binding site. The degree of reduction of the Q-binding site

is determined by the QH2 ⁄ Q ratio, the NADH ⁄ NAD+ ratio and

the proton-motive force.

The NADH ⁄ NAD+ ratio was determined by fluorescence. We

defined the NADH ⁄ NAD+ pool as fully reduced (100% reduc-

tion) in the presence of pyruvate, malate and rotenone, and fully

oxidized (0% reduction) in the presence of succinate and car-

bonylcyanide p-trifluoromethoxy phenylhydrazone (FCCP)

(Fig. 5). In the presence of succinate alone (where the differ-

ences in hydrogen peroxide efflux rate are observed), the

NADH ⁄ NAD+ pool in rat mitochondria was 92.6 ± 1.7%

reduced and not significantly different from that in pigeon mito-

chondria (89.4 ± 1.7% reduced), n = 5 separate mitochondrial

preparations. To calculate the redox potential of the NADH ⁄ -NAD+ pools (ENADH=NADþ ), we applied the Nernst equation:

ENADH=NADþ ¼ Em �RT

nFln½NADH�½NADþ�

� �

where Em is the standard midpoint potential of the NADH ⁄ -NAD+ couple ()320 mV), R ⁄ F is the Nernst equation constant,

T is the temperature, n is the number of electrons transferred

(two in this case) and the ratio [NADH] ⁄ [NAD+] is calculated

from the percentage of reduced NADH, stated above. The

calculated NADH ⁄ NAD+ redox potentials were )357 ± 5 and

)350 ± 3 mV for rat and pigeon mitochondria respectively,

and not significantly different (n = 5 separate mitochondrial

preparations). At the particular excitation and emission wave-

lengths employed, the measured fluorescence will report both

NADH and NADPH. We assume that any contribution by

NADPH to the observed changes in fluorescence do not alter

our conclusions significantly. Previous work indicated that this

is not an unreasonable assumption (Katz et al., 1987).

The redox potentials of the QH2 ⁄ Q pools were calculated from

the equation EQH2=Q ¼ ENADH=NADþ þ nDp where n is the number

of protons pumped per electron transferred (which is two for

complex I) and Dp is proton-motive force (determined previ-

ously, shown in Table 1). The calculated QH2 ⁄ Q redox potentials

were +21 ± 5 and )4 ± 3 mV for rat and pigeon mitochondria

respectively (P = 0.004, n = 5 separate mitochondrial prepara-

tions). The more negative redox potential of the Q pool in

pigeon mitochondria suggests that the Q binding site within

complex I may be more reduced than the Q binding site of com-

plex I in rat mitochondria. A more reduced Q binding site would

be consistent with a high superoxide production rate from that

site, but the superoxide production rate is low in complex I of

pigeon mitochondria. Therefore, the differences in the redox

state of the Q pool are unlikely to explain the differences in

superoxide production rates.

We conclude that a difference in the redox poise of the most

likely complex I superoxide production site (the Q binding site), is

not likely to explain the difference in superoxide production rate

between rat and pigeon mitochondria.

Pigeon mitochondria contain less complex I, which

explains their low rate of hydrogen peroxide efflux

Previous data suggested that pigeon mitochondria may contain

less complex I per mg mitochondrial protein than rat mitochon-

dria (St-Pierre et al., 2002). This would in effect mean that

pigeon mitochondria contain less of the superoxide producing

site, and this may in part or fully explain the low rates of hydro-

gen peroxide efflux seen in pigeon mitochondria. We first deter-

mined the amount of complex I in isolated rat and pigeon

mitochondria using the same method as St-Pierre et al. (2002).

This method is based on the amount of acid-extractable

FMN, because complex I is the only mitochondrial enzyme to

contain this moiety. In agreement with St-Pierre et al. (2002), a

A

B

Fig. 5 Determination of NADH redox potential in mitochondria respiring on

succinate. The plots show typical traces of NAD(P)H fluorescence versus time

for rat (A) and pigeon (B) heart mitochondria (0.35 mg mitochondrial

protein mL)1) incubated in KHEB buffer. Solid line: 2 lM rotenone added at

t = 0, pyruvate and malate (2.5 mM each) added at t = 20 s. Dashed line:

succinate (5 mM) added at t = 20 s, FCCP (2 lM) added at t = 80 s. Control

experiments indicated that excitation and emission spectra for NAD(P)H were

not different between the various conditions and not different between

species (not shown).

Mechanism of low ROS production rates, A. J. Lambert et al.

ª 2010 The AuthorsJournal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2010

84

significantly lower amount of FMN mg mitochondrial protein)1

was found in pigeon mitochondria (Fig. 6A). To confirm this

result, mitochondria were analysed by blue native polyacryl-

amide gel electrophoresis (PAGE) and the results are displayed in

Fig. 6A, B. We conclude from this data that pigeon mitochon-

dria contain significantly less complex I than rat mitochondria.

We hypothesized that if pigeon mitochondria contain less com-

plex I than rat mitochondria, then the maximal rates of electron

transfer from NADH to oxygen should be relatively low in pigeon

mitochondria. This was indeed the case, as shown in Fig. 6C.

We also hypothesized that the rates of reverse electron trans-

port from succinate to NAD+ should be relatively low in pigeon

mitochondria and this was indeed the case also (Fig. 6D). The

data in Fig. 6C, D provide indirect evidence for low complex I

content in pigeon mitochondria, as other components in the sys-

tem (complexes III and IV, the pyruvate ⁄ malate carrier and so

on) may exert a degree of control over oxygen consumption

and ⁄ or reverse electron transport. However, taken together, the

data presented in Fig. 6A–D provide compelling evidence that

pigeon mitochondria contain fewer complex I molecules per mg

mitochondrial protein than mitochondria from the rat.

Knowledge of the rates of hydrogen peroxide efflux per mg

mitochondrial protein and complex I content per mg mito-

chondrial protein allows the rates to expressed in terms of

complex I. Figure 7A shows the data in Fig. 4A normalized to

the complex I content of the mitochondria. The differences in

hydrogen peroxide efflux rate became nonsignificant after this

normalization. Similarly, the differences between rat and

pigeon displayed in Figs 2C, 3C and 4E became nonsignificant

(not shown). Because pigeon mitochondria contain less com-

plex I than rat mitochondria, the rates of hydrogen peroxide

efflux per mg mitochondrial protein during forward electron

transport should be lower in pigeon mitochondria. This was

the case (Fig. 7B) and the rates per nmol complex I were not

different (Fig. 7C).

We conclude that pigeons contain fewer molecules of com-

plex I in their heart mitochondria than rats. Pigeon mitochondria

therefore contain fewer DpH-sensitive superoxide producing

sites and thus display low rates of hydrogen peroxide efflux.

Discussion

The rates of ROS production by isolated mitochondria vary

greatly, depending on a variety of factors. The main factors are

the combination of substrates, ionophores and inhibitors pre-

sented to the mitochondria; this combination determines which

complexes receive electrons, the values of DpH and Dw, the

redox state of the electron carriers within each complex, and in

the case of complex I, the direction of electron flow. Substrates

that induce forward electron flow into complex I, such as pyru-

vate plus malate, result in low rates of ROS production (Fig. 1A).

This low rate can be increased several fold by complex I inhibi-

tors such as rotenone or piericidin (Fig. 1B). Very high rates are

seen when mitochondria are presented with the complex II sub-

strate, succinate (Fig. 1C). These rates are diminished by rote-

A

B 1 2 3 4 5 6 7 8

C

D

Fig. 6 (A) Complex I content in rat and pigeon mitochondria. Acid-

extractable flavin mononucleotide (FMN) and blue-native (BN) PAGE were

performed as described in Materials and methods. Filled bars: rat; open bars:

pigeon. Each bar is the mean ± S.E.M. of n = 27 and 15 separate

mitochondrial preparations for FMN extraction and BN PAGE respectively.

Significant difference (*P < 0.04) by t-test. (B) Representative gel from a BN

PAGE experiment. Lanes 1–3: 2, 5 and 10 lg respectively, of purified bovine

complex I; lane 4: empty; lanes 5 and 6: 20 lg of pigeon mitochondrial

protein; lanes 7 and 8: 20 lg of rat mitochondrial protein. I, V and IV:

complexes I, V and IV respectively. (C) Rates of uncoupled respiration in rat

and pigeon mitochondria. Inset: typical trace of % air-saturated oxygen in the

chamber versus time. Incubation conditions were 0.5 mg mitochondrial

protein mL)1 in KHEB buffer. Pyruvate and malate (2.5 mM) each were added

at 300 s, FCCP (2 lM) was added at 800 s. Main panel: rates of oxygen

consumption in the presence of pyruvate, malate and FCCP. Each bar is the

mean ± S.E.M. of n = 3 separate mitochondrial preparations. Significant

difference (*P = 0.013) by t-test. (D) Rates of reverse electron transport, as

determined by NADH reduction by succinate in rat and pigeon mitochondria.

Rates were derived from experiments of the type depicted in Fig. 5. Each bar

is the mean ± S.E.M. of n = 5 separate mitochondrial preparations.

Significant difference (*P < 0.0001) by t-test.

Mechanism of low ROS production rates, A. J. Lambert et al.

ª 2010 The AuthorsJournal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2010

85

none, indicating that most of the superoxide originates at com-

plex I during succinate oxidation (Fig. 1D).

Several studies have reported that mitochondria and cells

from long-lived species display low rates of ROS production

when compared with short-lived species (Sohal et al., 1990,

1993; Ku & Sohal, 1993; Ku et al., 1993; Herrero & Barja, 1997,

1998, 2000; Barja & Herrero, 1998; Brunet-Rossinni, 2004;

Csiszar et al., 2007; Lambert et al., 2007; Ungvari et al., 2008).

Some of these results were confounded by body mass effects

and potentially confounded by phylogeny. However, after

correction for body mass effects by analysis of residuals and cor-

rection for phylogeny by phylogenetic independent contrasts,

we found a significant association between succinate-induced

ROS production rate and maximum lifespan (Lambert et al.,

2007). We also showed that the differences between species

were located at complex I during succinate-supported reverse

electron transport. Therefore, we focused on this mode of ROS

production in this study.

We first carried out controls to establish that the differences in

ROS production between rat and pigeon heart mitochondria

were genuine and not because of some artefact of the system.

We then tested what we considered to be the most likely expla-

nations of the differences in rates of ROS production. Initially,

these potential explanations were: differences in anti-oxidant

capacity; differences in components of the proton-motive force,

Dw and DpH, and differences in the redox state of the superox-

ide producing sites within complex I. Differences in some of

these parameters were indeed discovered, but they did not

explain the differences in ROS production rate. The low rates of

ROS production by pigeon heart mitochondria were best

explained by a low amount of superoxide producing sites.

Preliminary data suggested that this explanation also applied to

the low ROS production rates of heart mitochondria from other

long-lived species reported by Lambert et al. (2007). This leads

to the possibility that the rate of superoxide production per

molecule of complex I is not different across a diverse range of

taxa. This suggests that the rates of superoxide production by

complex I are conserved and that evolution changes mitochon-

drial ROS production rates by changing complex I content.

Therefore, selection for low mitochondrial complex I content

may have positive consequences for longevity.

The increased availability of genomic data allows the explora-

tion of trait variation and lifespan using many more species than

is usually possible with functional studies. Indeed, several corre-

lations between mitochondrial genomic properties and lifespan

have been found, although some of the results are contradic-

tory. In a survey of 248 animals, it was discovered that mitoc-

hondrially encoded cysteine predicts animal lifespan

(Moosmann & Behl, 2008). Long lifespan also correlates posi-

tively with the rate of amino acid substitution per site within dif-

ferent mtDNA-encoded peptides and the rate of evolution of

cytochrome b (Rottenberg, 2006, 2007). Re-analysis of this

work resulted in the opposite conclusion: that lifespan correlates

negatively with evolutionary rate of mitochondrial proteins (Gal-

tier et al., 2009). Maximum lifespan correlates positively with

mitochondrial cytosine and negatively with adenine or thymine

content (Lehmann et al., 2006, 2008). Among mammals, pri-

mates are particularly long-lived and accelerated evolution of

the electron transport chain has been reported for this order

(Grossman et al., 2004). There is strong evidence therefore, for

a role of mitochondria in the determination of lifespan across

diverse taxa, although many questions remain. How these geno-

mic properties of long-lived species manifest at the functional

level of mitochondria to result in increased longevity is not clear

and more work in this important area is required.

A

B

C

Fig. 7 (A) Rates of hydrogen peroxide efflux expressed per nmol of complex

I. The rates with succinate from Fig. 4A were normalized to the amount of

complex I as measured in the same samples by FMN extraction. Bars are

means ± S.E.M. of n = 9 separate mitochondrial preparations. (B) Rates of

hydrogen peroxide efflux per mg mitochondrial protein during forward

electron transport. Pyruvate and malate were present at 2.5 mM each and

rotenone was present at 2 lM. (C) Data in panel B normalized to the average

complex I content as determined by FMN extraction. For panels B and C, each

bar represents the mean ± S.E.M. of n = 4 separate mitochondrial

preparations.

Mechanism of low ROS production rates, A. J. Lambert et al.

ª 2010 The AuthorsJournal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2010

86

We observed from the blue-native polyacrylamide gel electro-

phoresis (BN PAGE) results that pigeon mitochondria contain

about the same amounts of complexes III and IV, and signifi-

cantly higher amounts of ATP synthase than rat mitochondria.

Although rats and pigeons have similar basal metabolic rates,

the maximal energetic demands of pigeons may be higher than

rats because they fly. As complex I exerts little flux control over

cellular oxygen consumption or ATP synthesis [less than 15% in

hepatocytes (Brown et al., 1990)], the main energetic conse-

quence of lowered complex I content in pigeons may not be

diminished respiration or ATP synthetic rate, but decreased lev-

els of the product of complex I, reduced ubiquinone, at a given

ATP demand. The low content of complex I in pigeons and other

long-lived species may therefore be a mechanism for decreasing

ROS production not only from complex I itself, but also from

complex III, in which ROS production decreases strongly when Q

is very oxidized.

It is not known if complex I undergoes reverse electron

transport in cells in the same way that it does in isolated mito-

chondria. As cells do not use succinate exclusively, it is unlikely

that rates of ROS production in vivo are the same as those

observed in vitro. Succinate induces reverse electron transport

by reducing the Q pool and maintaining a high proton-motive

force. In cells, mitochondria mostly use not only pyruvate, but

also glycerol-3-phosphate and fatty acids, which also reduce

the Q pool and generate a high proton-motive force, thereby

mimicking the succinate condition except that forward elec-

tron transport would be in effect. With the Q pool relatively

reduced, forward electron transport in complex I may stall,

resulting in high rates of superoxide production and we sug-

gest that succinate-supported superoxide production during

reverse electron transport may mimic this condition. If the

superoxide-producing site within complex I that is active dur-

ing succinate oxidation is also active in cells, then rotenone

should decrease the rates of ROS production in intact cells.

Some reports indicate that this does indeed happen (Li &

Trush, 1998; Schuchmann & Heinemann, 2000; Vrablic et al.,

2001; Parthasarathi et al., 2002), but other studies report an

increase in ROS production in cells treated with rotenone (Bar-

rientos & Moraes, 1999; Nakamura et al., 2001; Siraki et al.,

2002; Li et al., 2003). In general, therefore, it is not clear what

sites of superoxide production are active in cells and more

work is required in this area.

Experimental procedures

Materials

Amplex red (10-acetyl-3,7-dihydroxyphenoxazine) was pur-

chased from Invitrogen (Paisley, UK). Premade polyacrylamide

gels (Cat. no. 161-1104) were from Bio-Rad Laboratories

(Hemel Hempstead, UK). [14C]Sucrose and [3H]water were from

Amersham (Little Chalfont, UK), [3H]acetate was from Perkin

Elmer (Beaconsfield, UK). Purified bovine complex I was a gift

from Judy Hirst (MRC Mitochondrial Biology Unit, Cambridge,

UK). All other chemicals were from Sigma (Poole, UK) or BDH

(Hull, UK).

Animals and isolation of heart mitochondria

Female Wistar rats (Rattus norvegicus) were from Charles River

UK and maintained under barrier conditions for 1–4 weeks prior

to use. Approximate age and body mass at time of use were

10 weeks and 200 g respectively. Mixed-sex pigeons (Columba

livia) were obtained from a local supplier and examined by a vet-

erinarian. All birds were deemed to be healthy young adults as

judged by appearance, body mass (approximately 300 g) and

behaviour.

Animals were killed by cervical dislocation, and the hearts

immediately excised and placed in ice-cold ‘STE’ isolation buf-

fer (250 mM sucrose, 5 mM Tris and 2 mM EGTA, pH 7.4 at

4 �C). Standardized isolation methodologies were used as pre-

viously described (Tyler & Gonze, 1967), with modifications.

Briefly, heart tissue was chopped with scissors and minced

with a scalpel blade prior to incubation with protease in isola-

tion buffer. The tissue was homogenized and the mitochondria

were isolated by differential centrifugation. Rat mitochondria

were prepared from the pooled hearts from four individuals;

pigeon mitochondria were prepared from the heart of a single

individual. On any given day, rat and pigeon mitochondria

were prepared in parallel, to allow experiments to be per-

formed in parallel. Mitochondrial protein content was deter-

mined by the biuret assay using BSA as standard. The

preparations resulted in approximately 0.5 mL of mitochondria

(in STE buffer) at a concentration of 20–30 mg mitochondrial

protein mL)1.

Measurement of hydrogen peroxide efflux

Hydrogen peroxide efflux rate was determined fluorometrically

by measurement of oxidation of amplex red to fluorescent res-

orufin coupled to the enzymatic reduction of H2O2 by HRP.

Unless otherwise stated, mitochondria were incubated at

0.35 mg mitochondrial protein mL)1 in standard ‘KHEB’ buffer

containing 120 mm potassium chloride, 3 mM HEPES, 1 mM

EGTA and 0.3% BSA (w ⁄ v) (pH 7.2 and 37 �C). All incubations

also contained 50 lM amplex red, 4 U mL)1 HRP and 30 U mL)1

SOD. The reaction was initiated by addition of succinate (5 mM)

and the increase in fluorescence was followed at excita-

tion ⁄ emission wavelengths of 563 and 587 nm respectively.

Appropriate correction for background signals and standard

curves generated using known amounts of H2O2 were used to

calculate the rate of H2O2 production in nmol min)1 mg mito-

chondrial protein)1.

Superoxide dismutase activity

Superoxide dismutase activity was determined using a modified

(Flohe & Otting, 1984) version of the assay described by McCord

& Fridovich (1969). The basic buffer comprised 50 lM xanthine

Mechanism of low ROS production rates, A. J. Lambert et al.

ª 2010 The AuthorsJournal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2010

87

and 0.25 mg mL)1 acetylated cytochrome c in 120 mM potas-

sium chloride, 3 mM HEPES and 1 mM EGTA (pH 7.2 and 37 �C).

Mitochondria were lysed in STE buffer containing 0.5% (w ⁄ v)

sodium deoxycholate to give a mitochondrial protein concentra-

tion of 2 mg mL)1. Standard bovine CuZnSOD was also pre-

pared in STE ⁄ deoxycholate buffer to give a range of known SOD

activities per mL. Samples and standards were added to the

basic reaction buffer and superoxide generation was initiated by

addition of xanthine oxidase to a concentration of 0.01 U mL)1.

The increase in absorbance (caused by superoxide reducing the

acetylated cytochrome c) was followed at 550 nm, the inhibi-

tion of this increase reflects activity of SOD in the sample or stan-

dard.

Hydrogen peroxide catabolism

Mitochondria (0.35 mg mitochondrial protein mL)1 in KHEB)

were incubated with known amounts of H2O2, the amount of

H2O2 remaining after a given time was determined by the addi-

tion of the amplex red ⁄ HRP detection system and measurement

of fluorescence.

Membrane potential, pH gradient and proton-motive

force

Mitochondrial membrane potential, Dw, was determined using

an electrode sensitive to the TPMP+ (Brand, 1995). The electrode

was calibrated by sequential 125 nM additions of TPMP+ up to

500 nM. The reaction was initiated by addition of succinate

(5 mM) and Dw was measured upon reaching the steady state.

The chemical component of proton-motive force, DpH, was

then measured as the change in Dw after DpH was converted to

Dw by addition of 100 nM nigericin. After each run, the uncou-

pler FCCP was added to a concentration of 2 lM to release the

TPMP+ and allow for correction of any small drift in the TPMP+

electrode. Potentials were calculated as described (Brand,

1995), on the basis that proton-motive force (Dp) = Dw + DpH

(all in mV, giving positive signs to electrical potentials that were

positive outside and pH gradients that were acid outside).

TPMP+ binding correction factors used were 0.4 for rat and 0.54

for pigeon (Brookes et al., 1998).

DpH was also determined using [3H]acetate as described

(Brand, 1995). Briefly, to determine the mitochondrial matrix

volume, water and pellet spaces were measured using [3H]water

and [14C]sucrose. The accumulation ratio of a weak acid (ace-

tate) was then measured using [3H]acetate and [14C]sucrose.

The Nernst equation was applied to the accumulation ratio to

calculate DpH. Incubation conditions were 0.35 mg mitochon-

drial protein mL)1 in standard KHEB buffer at 37 �C.

Oxygen consumption

Oxygen consumption was measured using a Clark-type elec-

trode. Coupled (‘state II’) respiration was initiated by addition of

pyruvate and malate (2.5 mM each), then uncoupled respiration

was initiated by addition of 2 lM FCCP. Incubation conditions

were 0.35 mg mitochondrial protein mL)1 in standard KHEB

buffer at 37 �C.

NADH redox state and rate of reverse electron

transport

NAD(P)H fluorescence in intact mitochondria was determined at

excitation and emission wavelengths of 365 and 450 nm respec-

tively. Incubation conditions were 0.35 mg mitochondrial pro-

tein mL)1 in standard KHEB buffer at 37 �C. The NADH pool

was defined as fully reduced in the presence of pyruvate, malate

plus rotenone; and fully oxidized in the presence of succinate

plus FCCP. The rate of formation of NADH during succinate oxi-

dation was taken to represent the rate of reverse electron trans-

port.

Determination of mitochondrial complex I content

Flavin mononucleotide content of mitochondria was determined

as described (Bessey et al., 1949; Burch, 1957) with modifica-

tions. Frozen–thawed mitochondrial samples (0.2–0.5 mg mito-

chondrial protein) in 50 lL of 0.01 M HCl were mixed with

450 lL of 11% (w ⁄ v) trichloroacetic acid (TCA) and incubated

on ice for 15 min. A series of FMN standards (0–5 lM) were pre-

pared in 0.01 M HCl and processed in parallel with the samples.

Following centrifugation (11 000 g) for 5 min, 2 · 40 lL aliqu-

ots of the supernatant were transferred to 2 separate 96-well

plates. The first plate was sealed with parafilm and incubated in

the dark at 37 �C for 18 h. Two hundred microlitres of 0.2 M

potassium phosphate (pH 6.8) was added to each well of the

second plate and the fluorescence was measured at excitation

and emission wavelengths 450 and 525 nm respectively. This

fluorescence was used to calculate the FMN concentration ‘Ri’.

After incubation, the first plate was processed identically to the

second plate and the FMN concentration ‘Rt’ was calculated.

The concentration of flavin adenine dinucleotide, FAD, is given

by [FAD] = (Ri ) Rt) ⁄ 0.35. We used a denominator of 0.35 as

opposed to the published value of 0.85 (Burch, 1957) to account

for the final pH of our TCA ⁄ phosphate buffer mixture (Bessey

et al., 1949). The FMN concentration is given by

[FMN] = Rt ) [FAD]. Complex I content per mg mitochondrial

protein was calculated on the basis of 1 FMN moiety per mole-

cule of complex I.

Blue-native polyacrylamide gel electrophoresis was performed

as described (Schagger & von Jagow, 1991; Nijtmans et al.,

2002) with minor modifications. Frozen–thawed mitochondrial

samples (0.5 mg mitochondrial protein) were suspended in

50 lL extraction buffer (0.75 M aminocaproic acid and 75 mM

Bis–Tris, pH 7 at 4 �C) with 12.5 lL of 10% (w ⁄ v) laurylmalto-

side and incubated on ice for 15 min. After centrifugation

(100 000 g) for 15 min, 5 lL of the supernatant was taken for

protein determination. Then 50 lL of the remaining supernatant

was removed and mixed with 3.75 lL Serva Blue G solution [5%

(w ⁄ v) Serva Blue G in 500 mM aminocaproic acid]. Electrophore-

Mechanism of low ROS production rates, A. J. Lambert et al.

ª 2010 The AuthorsJournal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2010

88

sis was performed using commercial 4–15% linear gradient

polyacrylamide gels, with 20 lg of extracted mitochondrial pro-

tein loaded per lane. Purified bovine complex I was processed in

parallel with the samples. The cathode buffer comprised 50 mM

Tricine, 15 mM Bis–Tris and 0.02% (w ⁄ v) Serva Blue G, pH 7 at

4 �C, the anode buffer was 50 mM Bis–Tris, pH 7 at 4 �C. Initial

electrophoresis conditions were 100 V for 60 min, after which

the gel was run overnight at 40 V using cathode buffer without

Serva Blue G (all at 4 �C). Gels were scanned and analysed using

Image J software.

Statistics

Values are given as mean ± standard error (or ± range, if

n = 2). Unless stated otherwise, n is the number of separate

mitochondrial preparations. The significance of differences

between means was assessed by ANOVA or Student’s t-test.

P-values of < 0.05 were taken to be significant.

Acknowledgments

This work was supported by funding from a Research into Aging

fellowship to AJL, the Medical Research Council and the Well-

come Trust (066750 ⁄ B ⁄ 01 ⁄ Z) to MDB. We thank Judy Hirst for

providing isolated complex I and Jason Treberg for useful

discussion.

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