Comparative Biochemistry and Physiology Part B 129 Ž2001. 129᎐137
Sulfide oxidation coupled to ATP synthesis in chicken
liver mitochondria
Rothsovann Yong, Dennis G. Searcy U
Biology Department, Uni¨ ersity of Massachusetts, Amherst, MA 01003, USA
Received 12 September 2000; received in revised form 26 January 2001; accepted 29 January 2001
Abstract
Chicken liver mitochondria consumed O 2 at an accelerated rate when supplied with low concentrations of hydrogen
sulfide. Maximum respiration occurred in 10 ␮M sulfide, and continued more slowly up to concentrations as high as 60
␮M. Sulfide oxidation was coupled to adenosine triphosphate ŽATP. synthesis, as shown by firefly luciferase luminescence and by measurement of the mitochondrial membrane electrochemical gradient. Synthesis of ATP required low,
steady-state concentrations of sulfide Ž- 5 ␮M., which were maintained by use of a syringe pump. The ratio of
consumed O 2 to sulfide changed at low sulfide and O 2 concentrations, indicating alternative metabolic reactions and
products. In low concentrations of sulfide, presumably most similar to physiological, the O 2rsulfide ratio was 0.75. This
is the first report of sulfide oxidation linked to ATP synthesis in any organism not specifically adapted to a sulfide-rich
environment. We suggest that this may be a widespread mitochondrial trait, and that it is consistent with the hypothesis
that mitochondria originated from sulfide-oxidizing symbionts. 䊚 2001 Elsevier Science Inc. All rights reserved.
Keywords: Mitochondria Žchicken.; Sulfide oxidation; Adenosine triphosphate synthesis; Mitochondria Ževolutionary origin.
1. Introduction
Although H 2 S is an inhibitor of aerobic respiration ŽNational Research Council Subcommittee
On Hydrogen Sulfide, 1979; Nicholls and Kim,
1981; Grieshaber and Volkel,
1998., it is nonethe¨
less detoxified by oxidative mitochondrial
metabolism ŽBaxter et al., 1958.. High concentrations of sulfide1 inhibit respiration, whereas low
U
Corresponding author. Tel.: q413-545-2679; fax: q413545-3243.
E-mail address: [email protected] ŽD.G. Searcy..
1
Aqueous sulfide is typically a mixture of H 2 Sq HSy; p K a
s 7.0 ŽNational Research Council Subcommittee On Hydrogen Sulfide, 1979.. At pH 7.4 it is 29% H 2 S and 71% HSy. In
chemical reactions we show it as HSy.
concentrations stimulate O 2 consumption. The
concentration that causes inhibition varies widely
between species, being highest in those organisms
adapted to sulfidic environments. Typically, the
onset of inhibition occurs in the range 10᎐50 ␮M
sulfide ŽBagarinao and Vetter, 1990; Grieshaber
and Volkel,
1998..
¨
Sulfide oxidation coupled to adenosine triphosphate ŽATP. synthesis has been reported in several organisms adapted to sulfide-rich environments. These include three distantly related
groups of animals: annelid worms, mollusks and
vertebrates Žreviewed in Grieshaber and Volkel,
¨
1998.. Such widespread distribution might be explained by evolutionary convergence, or could be
a shared primitive trait. The latter would be consistent with the ‘sulfide hypothesis’ for mitochon-
1096-4959r01r$ - see front matter 䊚 2001 Elsevier Science Inc. All rights reserved.
PII: S 1 0 9 6 - 4 9 5 9 Ž 0 1 . 0 0 3 0 9 - 8
130
R. Yong, D.G. Searcy r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 129᎐137
drial origin, which postulates that mitochondria
evolved from sulfide-oxidizing symbiotic bacteria
ŽSearcy, 1992; Searcy et al., 1999.. If that is true,
then ATP-linked sulfide oxidation should be a
widespread trait among eukaryotes. Nonetheless,
sulfide oxidation coupled to ATP synthesis has
not been reported in any organism not specifically
adapted to a sulfide-rich environment ŽPowell and
Somero, 1986; Bagarinao and Vetter, 1990..
Even moderate concentrations of sulfide may
uncouple ATP synthesis. Thus, in previous experiments ATP synthesis may have been undetected
because sulfide concentrations were too high. To
test that, a syringe pump can be used to add
sulfide at rates below that of maximum consumption, thus maintaining a low, steady-state concentration.
For detection of ATP production firefly luciferase can be used ŽLemasters and Hackenbrock, 1978.. Since luminescence Žor ATP production. might occur for a variety of reasons, it is
important to confirm that sulfide oxidation correlated with mitochondrial membrane electrical
polarization. For that purpose we used the dye
safranin, which accumulates inside mitochondria
that have a strong inside-negative electrical potential resulting in easily measured changes in
˚
optical absorbance ŽAkerman
and Wikstrom,
¨
1976; Schweizer and Richter, 1996.. None of these
tests are quantitative Žsee Section 4., but they can
be used to test whether ATP synthesis occurs and
whether or not it is coupled to sulfide oxidation
by a chemiosmotic mechanism.
Among previous studies there has been disagreement concerning the immediate products of
sulfide oxidation. Most often it has been reported
that sulfide is oxidized to thiosulfate, S 2 O 32y
ŽBaxter et al., 1958; Koj et al., 1967; Bartholomew
et al., 1980; Grieshaber and Volkel,
1998.. How¨
ever, in the mudflat worm Arenicola marina
ŽPhylum Annelida. sulfide-dependent O 2 consumption did not always correlate with S 2 O 32y
production ŽTable 2 in Volkel
and Grieshaber,
¨
1996.. In another study that employed radioactive
sulfide and several different species of marine
invertebrates Žnot isolated mitochondria., sulfide
was oxidized to SO42y, SO 32y, S 2 O 32y, elemental
sulfur, plus approximately 1r3 unidentifiable
compounds ŽPowell et al., 1980.. Mitochondria
from the sulfide-adapted clam Solemya reidi oxidized H 2 S to S 2 O 32y with near 100% yield, but
when the same analytical techniques were applied
to rat liver mitochondria none of the products of
sulfide oxidation could be identified ŽO’Brien and
Ž1960. suggested that in rat
Vetter, 1990.. Sorbo
¨
liver the initial product of sulfide oxidation is
., and that S 2 O 32y forms seconpolysulfide ŽS 2y
n
darily. Thus, it appears likely that the products of
sulfide oxidation differ between species, or
between different environmental conditions.
We used chickens for these experiments because they are not adapted to sulfidic environments. Also, they were available on local farms,
where procurement of tissues for research could
be combined with use of the animals for food.
2. Materials and methods
2.1. Isolation of mitochondria
Roosters Ž Gallus gallus. were obtained from
the Tilson Poultry Research Center, University of
Massachusetts, and from local farms. Within 1
min after the animals were slaughtered their livers were removed, placed in cold 0.15 M NaCl,
0.015 M sodium citrate ŽpH 8., and cut into
pieces Ž; 1 g..
Mitochondria were isolated by a procedure
modified from Rasmussen et al. Ž1997.. Ten grams
of liver were placed into 4 vols. 100 mM KCl, 5
mM MgSO4 , 1 mM sodium ethylenediaminetetraacetate ŽEDTA., 0.2% wrv bovine serum albumin Žfatty acid poor., 1 mM ATP Žadded fresh
as dry powder. and 50 mM trisŽhydroxymethyl.aminomethane chloride ŽTris᎐HCl buffer,
pH 7.4.. The tissue was minced with scissors and
then homogenized with three to five strokes of a
motor-driven glass-Teflon homogenizer. The resulting suspension was centrifuged at 200 = g
Ž1600 rev. miny1 , 5 min., and the supernatant
centrifuged again at 2800 = g Ž6000 rev. miny1 , 10
min.. The pellet was resuspended in 40 ml of the
above medium without ATP and serum albumin,
and centrifuged at 4200 = g Ž7300 rev. miny1 , 10
min.. The final pellet was resuspended in 4 vols.
225 mM d-mannitol, 75 mM sucrose, and contained approximately 20 mg protein mly1 .
2.2. O2 consumption
Oxygen concentrations were measured using a
polarographic O 2 probe ŽModel 53,YSI Inc., Yellow Springs, Ohio.. The Pt cathode was polarized
R. Yong, D.G. Searcy r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 129᎐137
y0.8 v relative to an Ag anode, and covered by
an O 2-selective Teflon membrane. Temperature
was thermostatted to 30.0⬚C. The electrode chamber contained 3 ml of 225 mM d-mannitol, 75
mM sucrose, 10 mM potassium phosphate, 10
mM sodium diethylenetriaminepentaacetate
ŽDTPA., 11 mM MgSO4 and 20 mM Tris᎐HCl
ŽpH 7.35.. DTPA was included to inhibit the
catalytic activity of Fe and Mn ions ŽBuettner et
al., 1983., which otherwise can account for significant sulfide oxidation.
The O 2 probe was calibrated by including 1 mg
catalase in a 3-ml deoxygenated chamber buffer
Žsee above., and then adding measured amounts
of H 2 O 2 . The H 2 O 2 concentration was measured
spectrophotometrically Ž ␧ 240nm s 39.4 My1 cmy1 ;
Nelson and Kiesow, 1972.. By this measure,
chamber buffer equilibrated with air at 30⬚C contained 218 ␮M O 2 . We detected no non-linearity
in the response of the O 2 probe at either high or
low concentrations of O 2 , and no interference by
sulfide. Non-enzymic O 2 consumption in the
absence of mitochondria was subtracted from the
data. E.g., in 3 ml buffer containing 10 ␮M sulfide the background O 2 consumption was 0.12
nmol miny1 , compared to 68 nmol O 2 miny1
consumed by mitochondria provided with succinate.
In the early experiments ŽFigs. 1 and 2., sulfide
and other substrates were added by microliter
syringe and by a pipettor. The stock solution of
sulfide was freshly prepared 2 mM Na 2 S in 1 mM
131
Fig. 2. Rates of initial O 2 consumption by mitochondria in
various concentrations of sulfide. Each datum was measured
on a separate sample of mitochondria Žapprox. 1 mg protein
mly1 . in approximately 200 ␮M O 2 . Symbols: Ž䢇. mitochondria with 10 mM sodium succinate and 500 ␮M ADP, plus
varying concentrations of sulfide; Ž`. no addition of succinate
nor ADP, and therefore this is respiration upon sulfide; 1 mM
KCN, plus succinate and ADP. See Section 2 for additional
details. Error bars show S.E., n s 6.
sodium DTPA. Sulfide delivery was verified by
adding it directly into 5 ml of 5 mM ZnCl 2 ᎐10
mM NaOH, followed by chemical assay for sulfide
ŽSearcy and Lee, 1998..
In later experiments sulfide was gradually added
using an adjustable syringe pump equipped with a
1 ml glass syringe filled with 2 mM Na 2 S᎐1 mM
DTPA. Thick-walled polyethylene tubing Ždesigned for high pressure work. was used between the
syringe and the sample chamber so as to minimize
O 2 and H 2 S diffusion through the tubing wall.
Sulfide delivery was tested by delivery into
ZnCl 2 ᎐NaOH solution as described above, and
over the range 1 ␮l to 100 ␮l miny1 did not
deviate from that expected.
2.3. ATP production
Fig. 1. Representative recordings from a polarographic O 2
probe. Mitochondria were combined with succinate, ADP, or
sulfide, each added quickly by a pipettor. Final concentrations
are shown in the figure. Mitochondrial protein concentrations
were: ŽA. 1.0 mg; ŽB. 0.63 mg; and ŽC. 5.0 mg mly1 . Each
experiment started at wO 2 x of approximately 200 ␮M. T s 30⬚C.
Firefly luciferin-luciferase was used ŽLemasters
and Hackenbrock, 1978.. In a 5-ml polyethylene
test tube, freshly isolated mitochondria Žs 1 mg
protein. were suspended in 1 ml of 225 mM
d-mannitol, 75 mM sucrose, 10 mM K 2 HPO4 , 10
mM DTPA, 15 mM MgSO4 and 20 mM Tris᎐HCl
ŽpH 7.35.. After adding adenosine monophosphate to 100 ␮M and adenosine diphosphate to
15 ␮M, the suspension was incubated 2 min at
30⬚C. Finally, we added 150 nmol luciferin and 20
132
R. Yong, D.G. Searcy r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 129᎐137
␮g luciferase ŽSigma Chemical Company.. The
suspension was placed into a luminometer
equipped with a photomultiplier tube and thermostatted to 30⬚C. Sodium sulfide Ž1 mM in 1
mM Na 5 DTPA. was added using a syringe pump,
as described above. Mixing was provided by gentle aeration.
2.4. Mitochondrial membrane potential
˚
The safranin method of Akerman
and Wikstrom
¨
Ž1976. and Schweizer and Richter Ž1996. was used
to measure mitochondrial membrane potential.
Mitochondria Ž4 mg protein. were suspended in 2
ml 10 ␮M safranin, 225 mM d-mannitol, 75 mM
sucrose, 1 mM potassium phosphate, 1 mM potassium ethyleneglycoltetraacetate ŽEGTA., and 10
mM potassium N-Ž2-hydroxyethyl.piperazine-N⬘Ž2-ethanesulfonate . ŽHEPES buffer, pH 7.35..
Stirring and aeration were accomplished by
bubbling air up the back side of the cuvette, and
kept out of the light beam by tipping the entire
spectrophotometer forward approximately 30⬚.
The temperature was 25⬚C. Instead of using two
wavelengths as originally described, a single wavelength was measured at 532 nm. Changes in optical density caused by light scattering were measured in identical mitochondrial suspensions with
no safranin, and subtracted from the data.
3. Results
3.1. Sulfide oxidation
Fig. 1 shows representative recordings of mitochondrial O 2 consumption. Chicken liver mitochondria supplied with succinate and ADP consumed 36 " 7 ŽS.D., n s 6. ␮mol O 2 miny1 Žg
protein.y1 . The respiratory control ratios Žstate
3rstate 4. of the mitochondria were in the range
2᎐3, which is similar to previously published values for liver mitochondria respiring on succinate.
ŽSee Section 4..
Instead of succinate, when provided with 10
␮M sulfide the chicken mitochondria consumed
7.2" 2.5 ŽS.D., n s 6. ␮mol O 2 miny1 gy1, which
compares with 14.3 ␮mol miny1 gy1 for the sulfide-adapted fish Fundulus par¨ ipinnis and 5.5 for
the non-adapted fish Citharichthys stigmaeus
ŽBagarinao and Vetter, 1990.. When compared
with gill mitochondria from sulfide-adapted bi-
valve Geukensia demissa ŽParrino et al., 2000.,
chicken mitochondria respired 57% faster on succinate plus ADP, but respired 73% slower on
sulfide. Thus, chicken liver mitochondria use sulfide relatively slowly, as might be expected for an
organism from a non-sulfidic environment.
When sulfide was added in small batches, O 2
consumption first accelerated, and then slowed
again after the sulfide had been consumed ŽFig. 1,
Trace C.. Similar stepwise responses to sulfide
addition have been reported previously for mitochondria from several sulfide-adapted animals
ŽPowell and Somero, 1986; Bagarinao and Vetter,
1990; Volkel
and Grieshaber, 1994, 1997; Parrino
¨
et al., 2000.. Adding ADP to chicken mitochondria respiring on sulfide resulted in a slight decrease in the rate of O 2 consumption, which was
unexpected, and discussed later below.
Fig. 2 shows the rates of O 2 consumption over
a range of sulfide concentrations, both with and
without succinate. When supplied with only sulfide, O 2 consumption was maximal at 10 ␮M,
which compares with approximately 5 ␮M for G.
demissa mitochondria ŽParrino et al., 2000., and
20 ␮M for the sulfide-adapted fish F. par¨ ipinnis
ŽBagarinao and Vetter, 1990.. Chicken mitochondria respiring on succinate tended to be inhibited
by even 1 ␮M sulfide, although inhibition was not
statistically significant until ) 5 ␮M ŽFig. 2.. Oxygen consumption was completely inhibited by
KCN regardless of whether the substrate was
succinate or sulfide, similar to mitochondria from
the fish F. par¨ ipinnis. That differs from the invertebrates G. demissa and A. marina, which have
branched electron transport chains and at least
one terminal oxidase that is cyanide-resistant
ŽVolkel
and Grieshaber, 1996; Parrino et al.,
¨
2000..
3.2. Stoichiometry of O2 and sulfide
The data in Fig. 3 show that the amount of O 2
consumed per mole of sulfide added was affected
by both the O 2 concentration and the rate of
sulfide addition ŽFig. 3.. When O 2 concentrations
were near atmospheric and the sulfide was added
rapidly so that its concentration was near 15 ␮M,
the O 2rsulfide ratio was close to 1.0. That replicates previous studies on other organisms, which
almost all were done in similar conditions. When
sulfide was added more slowly, so that its concentration in the mitochondrial suspension remained
R. Yong, D.G. Searcy r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 129᎐137
133
3.4. Membrane potential during sulfide oxidation
Fig. 3. Ratio of O 2 consumption to sulfide consumption in
relation to O 2 concentration and rate of Na 2 S addition. Other
conditions as in Fig. 2, using 1 mg mitochondrial protein in 3
ml and no succinate nor ADP. Each addition totaled 45 nmol
Na 2 S, delivered by syringe pump. Then incubation was continued until O 2 consumption returned to its basal rate. Background O 2 consumption has been subtracted from the calculations. Concentrations of O 2 are median values between those
at the beginning and end of each respiratory pulse.
low, the O 2rsulfide ratio was approximately 0.75
and was nearly independent of O 2 concentration.
In low O 2 concentration, the O 2rsulfide ratio was
even lower. The observations suggest that the
products of sulfide oxidation may change, depending on circumstances.
3.3. ATP production
Production of ATP during sulfide oxidation was
detected by using firefly luciferase. When the
sulfide was added slowly using a syringe pump,
the mitochondria produced ATP ŽFig. 4.. When it
was added more rapidly, no ATP was detected.
When the experiment involved sequential additions of sulfide, luminescence decreased with each
addition ŽFig. 4, trace A.. That is a property of
the assay and not indicative of decreased ATP
production ŽLemasters and Hackenbrock, 1978..
As a control, sulfide added to buffer without
mitochondria resulted in no luminescence. When
mitochondria were present, sulfide-dependent
luminescence was abolished by either cyanide or
by the uncoupler carbonyl cyanide p-Žtrifluoromethoxy.phenylhydrazone ŽFCCP.. These observations are consistent with a respiratory chemiosmotic mechanism of ATP production.
Mitochondrial membrane potential was measured using the dye safranin. In Fig. 5, a downward deflection in absorbance indicates stronger
membrane electrical polarization, inside negative
˚
ŽAkerman
and Wikstrom,
¨ 1976.. In Fig. 5 the
numbers indicate the rate of Na 2 S addition in
nmol miny1 . Trace A shows that membrane energization occurred when Na 2 S was added at a rate
of 10 nmol miny1 , and that the energized state
could be sustained for relatively long periods. In
contrast, when added at a rate of 21 nmol miny1 ,
the mitochondria initially became energized but
then lost it. Presumably, accumulation of sulfide
resulted in uncoupling. At still higher rates of
Na 2 S addition, the mitochondria became energized and then de-energized even more quickly
Žnot shown..
Trace B is a control showing that also ATP can
cause membrane energization, as expected when
the ATP synthase is run in the direction of hydrolysis. The brief polarization caused by each addition of ATP indicates that there was substantial
ATPase activity present, or that some mitochondria were uncoupled. As a control, the uncoupler
FCCP eliminated the membrane potentials induced by either sulfide or ATP.
Trace C shows again a rapid rate of sulfide
addition, but in this case the pump was shut off
before uncoupling occurred. In this experiment
the deflection caused by sulfide oxidation was
greater than that caused by adding ATP.
Fig. 4. Mitochondrial ATP production measured by firefly
luciferase luminescence. The samples contained 1 mg mitochondrial protein, 15 ␮M ADP, 100 ␮M AMP, 20 ␮g luciferase and 150 ␮M luciferin in a final volume of 1 ml.
Numbers indicate rates of Na 2 S addition in nmol miny1 ,
using a syringe pump. ŽA. Complete system; ŽB,C., with inhibitors added as indicated. Line B is displaced for display,
Ts 30⬚C.
134
R. Yong, D.G. Searcy r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 129᎐137
Fig. 5. Mitochondrial membrane potential measured by safranin absorbance. A downward deflection of absorbance indicates increased
membrane electrical potential, inside negative. ŽA᎐D. Separate runs, each starting at time zero. The numbers above each trace indicate
the rates of Na 2 S addition in nmol miny1 , using a syringe pump filled with 2 mM Na 2 S. Each sample Ž2 ml. contained 4.0 mg
mitochondrial protein and 10 ␮M safranin. T s 25⬚C.
Trace D ŽFig. 5. shows that at certain rates of
sulfide addition there were oscillations in membrane potential, possibly related to the mitochondrial volume oscillations that have been described
previously and reviewed by Bernardi et al. Ž1982..
4. Discussion
4.1. Rates of sulfide and O2 consumption
Mitochondrial preparations are evaluated by
their specific respiratory rates and Respiratory
Control Ratio. When supplied with succinate plus
ADP, chicken mitochondria consumed 36 ␮mol
O 2 miny1 Žg protein.y1 , which compares with
approximately 25 ␮mol miny1 gy1 for liver mitochondria from various vertebrate animals ŽJohnson and Lardy, 1967; Bagarinao and Vetter, 1990..
In contrast, rates for muscle mitochondria are
typically higher ŽRasmussen et al., 1997..
The respiratory control ratio is the rate of O 2
consumption immediately after ADP addition
ŽState 3. divided by the rate after respiration has
again slowed ŽState 4., and is a measure of both
inner membrane permeability and ATPase activity. Published values are in the range 1᎐40 ŽTyler
and Gonze, 1967; Toth et al., 1986.. Our mitochondrial preparations had respiratory control ra-
tios in the range 2᎐3. In general, lower values are
observed with mitochondria from liver compared
to those from muscle, and when using succinate
rather than substrates such as malate and pyruvate. Nonetheless, liver mitochondria were adequate for the research described here, and much
more easily isolated than are muscle mitochondria.
4.1.1. Relati¨ e rates cf. succinate
Provided with only 10 ␮M sulfide, rooster liver
mitochondria used O 2 at a rate 20% of that on
measured on succinate plus ADP. That is significantly slower than reported for mitochondria from
sulfide-adapted bivalves and fish, which respire on
sulfide at rates up to 170% of that on succinate
ŽPowell and Somero, 1986; Bagarinao and Vetter,
1990; Parrino et al., 2000.. It is evident that
chickens are not sulfide-adapted organisms.
4.1.2. Rates of O2 consumption
When using 10 ␮M sulfide as the electron
donor, the rate of O 2 consumption by chicken
mitochondria was 7.2" 2.5 S.D. Ž n s 6. ␮mol O 2
miny1 Žg protein.y1 . That was uncoupled respiration, as explained later below.
In concentrations up to 50 ␮M sulfide ŽFig. 2.,
chicken liver mitochondria continued to consume
O 2 , which was true also of rat liver mitochondria
R. Yong, D.G. Searcy r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 129᎐137
ŽBartholomew et al., 1980.. That is substantially
higher than the concentration that inhibits purified cytochrome oxidase, which is - 1 ␮M sulfide
ŽNicholls and Kim, 1981; Vetter et al., 1991.. The
explanation is not known.
4.1.3. Rates and stoichiometry of sulfide
consumption
In addition to the rate O 2 consumption discussed above, the rate of sulfide consumption can
be estimated from Fig. 1, trace C, by knowing how
much sulfide was added and how much time was
required to oxidize it. Thus, chicken liver mitochondria consumed sulfide at rates between 7 and
10 ␮mol sulfide miny1 Žg protein.y1 . That is
within the range reported for most animal mitochondria Ž4᎐18 ␮ mol sulfide min y 1 gy 1 ;
Grieshaber and Volkel,
1998., although substan¨
tially slower than for the mudflat worm A. marina
Ž30 ␮mol sulfide miny 1 gy 1 ; Volkel
and
¨
Grieshaber, 1997..
4.2. ATP production
ATP was detected using firefly luciferase, which
was used because of its sensitivity and specificity.
There are two difficulties with the luciferase technique when used in the presence active mitochondria. First, there is significant background ATP
production by the enzyme adenyl kinase, which
catalyzes the reaction: 2 ADPl AMPq ATP.
Since at least some ADP must be added as a
substrate for mitochondrial ATP synthesis, this is
unavoidable. However, the problem can be ameliorated by adding excess AMP, shifting the equilibrium to the left ŽLemasters and Hackenbrock,
1978.. Nonetheless, whenever ADP was added
there was substantial background luminescence,
even in the absence of respiratory substrate.
A second problem is that the luciferase reagent
is progressively desensitized by reaction with ATP
ŽLemasters and Hackenbrock, 1978.. Typically,
each addition of ATP produces 50% less luminescence than the previous addition. When using
luciferase reagent to measure ATP concentrations in deproteinized tissue extracts this is not a
problem, since each aliquot of reagent is used
only once. However, when the reagent is combined with mitochondria there is background ATP
production by adenyl kinase, so that it becomes
difficult to know how far reagent desensitization
135
has progressed. Thus, when used with mitochondria, the luciferase assay is not quantitative.
Chemiosmotic ATP production uses an electrochemical Hq gradient across the mitochondrial inner membrane. By using the dye safranin
we could detect the electrical component of this
gradient ŽFig. 5.. The gradient that occurred during sulfide oxidation was greater than that caused
by adding ATP without sulfide, confirming that
sulfide oxidation energizes the mitochondrial
membrane sufficiently to drive ATP synthesis.
If ADP is added to coupled mitochondria supplied with Krebs cycle intermediates, then as ATP
synthesis occurs one expects an increase in O 2
consumption. After the added ADP has been
phosphorylated, the rate of O 2 consumption
should return to its resting rate, and that is commonly observed when oxidizing organic substrates
such as succinate, malate, or pyruvate. Nonetheless, when chicken mitochondria were respiring
on sulfide and then given ADP there was a slight
decrease in the rate of O 2 consumption. In contrast, in most previous studies on sulfide-adapted
animals, when ADP was added to mitochondria
respiring upon sulfide the rate of respiration increased ŽPowell and Somero, 1986; Volkel
and
¨
Grieshaber, 1997; Parrino et al., 2000.. One exception was mitochondria from the fish F. par¨ ipinnis; while respiring on 50 ␮M sulfide, the addition of ADP decreased O 2 consumption, whereas
in 30 ␮M sulfide adding ADP stimulated it
ŽBagarinao and Vetter, 1990.. Thus, stimulation
of sulfide-specific respiration may occur only at
the lowest sulfide concentrations, which may be
lower than can be tested with our current instruments. We are building a more sensitive device
that should be able to get around that limitation.
4.2.1. Uncoupling concentration
The concentration of sulfide that resulted in
uncoupling was lower than can be reliably assayed
chemically, but it can be estimated from the
safranin experiment as follows. When sulfide was
added at a rate of 10 nmol miny1 to 2 ml of
mitochondrial suspension, the mitochondria
maintained an elevated membrane potential for
extended times, suggesting that the mitochondria
were able to consume the sulfide as rapidly as it
was added. However, when sulfide was added at
21 nmol miny1 the membrane potential was lost
after 1 min, suggesting that sulfide accumulated
and exceeded the uncoupling concentration. Dur-
136
R. Yong, D.G. Searcy r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 129᎐137
ing the first minute approximately half the added
sulfide should have been consumed, and therefore net accumulation would have been ; 5 ␮M.
Thus, 5 ␮M sulfide is our estimate of the minimum concentration that causes uncoupling, which
is an upper limit.
A similar pattern, but shifted to higher sulfide
concentration, has been described in the worm
A. marina ŽVolkel
and Grieshaber, 1996, 1997..
¨
The concentration that half-uncoupled ATP synthesis was 25 ␮M sulfide, although the rate of
uncoupled O 2 consumption was not significantly
affected even at 400 ␮M sulfide. The authors
suggested that rapid, uncoupled sulfide oxidation
serves the purpose of sulfide detoxification.
Thus, there appear to be at least two modes of
sulfide oxidation, coupled and uncoupled, with
differing rates between them. For chicken mitochondria, estimated from the safranin data, the
maximum rate of coupled sulfide oxidation was
approximately 2.5 ␮mol sulfide min y 1 Žg
protein.y1 , whereas the rate of uncoupled oxidation was 7᎐10 ␮mol miny1 gy1 .
The mechanism of uncoupling is not known.
One might conjecture that H 2 S might be able to
shuttle Hq across a membrane in a manner similar to dinitrophenol, which would be a simple
physical phenomenon. However, in Fig. 5, line d,
the membrane potential varies during constant
sulfide input, suggesting that coupling may be
switched off and on. Thus, uncoupling may involve an actively regulated metabolic switch, such
as a gated Hq leakage channel.
4.3. O2 r sulfide stoichiometry
In relatively high substrate concentrations Žexceeding 10 ␮M sulfide and 100 ␮M O 2 ., the mole
ratio of O 2 to sulfide consumption was near 1.0
ŽFig. 3.. That replicates previous studies which
typically have used sulfide concentrations between
20 and 100 ␮M ŽPowell and Somero, 1986;
Bagarinao and Vetter, 1990; O’Brien and Vetter,
1990; Volkel
and Grieshaber, 1994, 1996.. Such
¨
concentrations are not unreasonable for animals
adapted to sulfidic environments. An O 2rsulfide
ratio of 1.0 is consistent with the sulfur product
being S 2 O 32y, which has been confirmed chemical
analysis in several different species ŽBagarinao
and Vetter, 1990; O’Brien and Vetter, 1990;
Volkel
and Grieshaber, 1996..
¨
Sulfide oxidation donates electrons into the mi-
tochondrial electron transport chain near Respiratory Complex III. The electrons react ultimately
with O 2 in Respiratory Complex IV, or perhaps
in a cyanide-resistant terminal oxidase ŽPowell
and Somero, 1986; Grieshaber and Volkel,
1998;
¨
Parrino et al., 2000.. In either case, the site of
sulfide reaction is separate and distinct from that
of O 2 reaction ŽGrieshaber and Volkel,
1998..
¨
Thus, it is difficult to imagine how highly oxygenated products such as SO42y and S 2 O 32y can
be immediate products of sulfide oxidation.
The variable O 2rsulfide stoichiometry shown in
Fig. 3 suggests that the products of sulfide oxidation might vary in different substrate concentrations. Potential oxidation products include thiyl
0
.
radical ŽHS ., polysulfides ŽHSy
n , and S , as well
2y
2y
as S 2 O 3 and SO4 . Reduction products of O 2
might include O 2᎐, H 2 O 2 and H 2 O. The ratio 0.75
O 2r1 HSy is shown in Fig. 3, and might be
explained by the following hypothetical reaction.
䢇
3O 2 q 4 HSyq 3 Hqª HSy
4 q 3 H 2 O2
Ž1.
Although there is no additional evidence for
the above reaction, it is attractive because poly. is a substrate for cytoplasmic sulsulfide ŽHSy
4
fide production ŽSearcy and Lee, 1998.. Thus,
small amounts of sulfur can cycle repeatedly
between the mitochondrion and cytoplasm, similar to what might have occurred the hypothetical
symbiosis that gave evolutionary origin to mitochondria ŽSearcy, 1992..
4.4. Conclusion
Mitochondria from chickens are not adapted to
sulfidic environments, but nevertheless can oxidize sulfide with coupled ATP synthesis. Presumably, that has not been observed previously in
non-sulfide-adapted species because the sulfide
concentrations were too high. We suggest that
coupled respiration on sulfide may be a
widespread and primitive trait of mitochondria.
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