Sulfide consumption by mussel gill mitochondria is not strictly tied to oxygen reduction: measurements using a novel polarographic sulfide sensor
Department of Biology, University of Alabama at Birmingham, 1300 University Boulevard, Birmingham AL 35294-1170, USA
* Author for correspondence (e-mail: dwkraus{at}uab.edu)
Accepted 26 July 2004
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The ribbed mussel Geukensia demissa, an inhabitant of sulfide-rich coastal sediments, consumes sulfide in a chemolithoheterotrophic metabolic strategy. Gill mitochondria use sulfide as respiratory substrate for ATP production, and sulfide consumption is sufficiently rapid and so kinetically complex that only continuous real-time detection captures these events. Under normoxic conditions, oxygen and sulfide consumption are matched. Under hypoxic to anoxic conditions, however, sulfide consumption continues without commensurate oxygen consumption, and these results can be duplicated at higher oxygen conditions by selective blockade of terminal oxidases. These metabolic capabilities depend on prior environmental sulfide exposure, which suggests substantial mitochondrial metabolic plasticity. The recent finding that endogenous sulfide is a critical cell signaling molecule in all organisms suggests that the metabolic pathways that tightly control cellular sulfide levels are widespread. Sensors that accurately report sulfide concentrations under physiologically relevant conditions are valuable tools with which to explore the expanding role of sulfide in biological systems.
Key words: sulfide, mitochondria, oxygen, sensor, ribbed mussel, Geukensia demissa.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Besides being a potent respiratory toxin, hydrogen sulfide is also a source
of reducing equivalents, and many organisms possess mechanisms that enable
them to take advantage of sulfide's reductive poise. Most organisms that use
sulfide to their energetic advantage are chemolithoautotrophic prokaryotes,
with metabolic strategies to capture energy from sulfide oxidation to support
oxidative phosphorylation and carbon dioxide fixation
(Atlas, 1996). A select few
prokaryotes are chemolithoheterotrophs that obtain reduced carbon compounds
heterotrophically and utilize the reducing potential of hydrogen sulfide for
oxidative phosphorylation (Kuenen et al.,
1985
). Mitochondria from some metazoans also possess this ability
(Powell and Somero, 1986
;
reviewed in Grieshaber and Völkel,
1998
). We have shown that the gills of the ribbed mussel
Geukensia demissa use sulfide-based ATP production to support ciliary
beating, thus exhibiting metazoan chemolithoheterotrophy (Doeller et al.,
1999
,
2001
). In this case, electrons
resulting from sulfide oxidation are fed into the mitochondrial electron
transport chain, most likely at cytochrome c, for the production of
ATP, with ATP/oxygen atom and sulfide/oxygen molecule ratios both of one
(Parrino et al., 2000
;
Doeller et al., 2001
).
The process of sulfide-based metazoan chemolithoheterotrophy is influenced
by several factors, one being oxygen. The initial steps of sulfide oxidation
catalyzed by sulfide oxidases use H2O as the oxidant, produce
thiosulfate and may not be dependent on molecular oxygen. However, when
sulfide becomes the respiratory substrate, instead of reduced coenzymes such
as NADH from carbohydrate combustion, electrons may enter the mitochondrial
electron transport chain at cytochrome c causing the P/O ratio to
change from approximately three to one. Under steady state cellular conditions
with no change in ATP demand, oxygen consumption rate must then increase by
threefold in order to maintain the same ATP turnover rate. This has been
documented for intact mussel gills
(Doeller et al., 1999). This
increased need for oxygen, coupled with oxygen-limiting conditions in reduced
sulfide-rich sediments, most likely makes sulfide-driven oxidative
phosphorylation sensitive to oxygen partial pressure. In this paper, we
address the dependency of mussel gill mitochondrial sulfide consumption on
oxygen partial pressure with simultaneous measurements of oxygen and sulfide
consumption.
Environmental sulfide exposure also influences the process of sulfide-based
metazoan chemolithoheterotrophy. Intact gills from Geukensia demissa
maintained in low sulfide conditions exhibited a decreased oxygen consumption
rate in response to sulfide; gills from Mytilus edulis collected from
and maintained in sulfide-free conditions exhibited an even lower oxygen
consumption rate in response to sulfide
(Lee et al., 1996). In this
paper, we also address the dependency of G. demissa gill
mitochondrial sulfide consumption on the history of environmental sulfide
exposure by the whole animals.
Sulfide disappearance from solution in a closed container (i.e.
respirometer chamber) can result from abiotic sulfide oxidation, catalyzed by
a number of chemical agents (Chen and
Morris, 1972). If biological samples are present in the chamber,
enzymatic sulfide oxidation (sulfide consumption) and sulfide binding to
molecules such as transport proteins, will also contribute to a decrease in
solution sulfide concentration.
The rate of sulfide disappearance can be measured indirectly or directly.
Indirect methods include the measurement of oxygen consumption rate, often
combined with the rate of production of sulfide oxidation products such as
thiosulfate (for intact mussel gills, see Doeller et al.,
1999,
2001
). Direct methods to
measure solution sulfide levels include HPLC
(Fahey et al., 1981
) and
colorimetric assays (Svenson,
1980
; Cline, 1969
).
These measurements are not typically in real time, however, since samples are
taken at specified intervals and processed for later determination of sulfide
content. Because sulfide consumption in intact gills and by isolated
mitochondria can be rapid, these are not methods of choice. In this study we
describe the development of a sulfide respirometric method to obtain
continuous real-time measurements of sulfide consumption, accomplished using a
polarographic sulfide sensor fitted into the sensor port of a respirometer.
With sulfide respirometry, the kinetics of changes in sulfide concentration in
physiological solutions such as seawater and mitochondrial respiration buffer
are continuously recorded. Sulfide respirometry indicates that gill
mitochondrial sulfide consumption shows conformity to oxygen partial pressure,
exhibiting multiphase kinetics, and it also occurs in the absence of oxygen.
Additionally, the process of sulfide-supported chemolithoheterotrophy in gills
is dependent on environmental sulfide exposure.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Polarographic sulfide sensor design and calibration
The polarographic sulfide sensor (PSS;
Fig. 1), based on a microsensor
used to measure sulfide levels within bacterial mats
(Jeroschewski et al., 1996),
was designed with dimensions equal to that of the polarographic oxygen sensor
(POS; model 2120 Orbisphere, Geneva, Switzerland) used in the Oroboros
Oxygraph respirometer (Innsbruck, Austria), in order to place the PSS into the
POS port for sulfide respirometry. The housing was machined from polyether
ether ketone (PEEK, Victrex USA Inc., Rockford, MI, USA). Both anode and
cathode were fashioned from platinum wire (1 and 0.5 mm diameter,
respectively; Blankinship Porter, Birmingham, AL, USA) and cemented into the
PEEK housing with epoxy (Scotch-Weld 2216, 3M, St Paul, MN, USA). The
electrolyte, consisting of 0.05 mol l1
K3[Fe(CN)6] in alkaline carbonate buffer pH 10
(Fig. 1, insert), was held in
the sensor tip reservoir with a two-layer membrane made of a
H2S-permeable membrane (MEM 213, 25 µm thick, MemPro, Troy, NY,
USA), cemented (Silicone Adhesive RTV 167, GE, Waterford, NY, USA) to a
H2S-impermeable membrane (25 µm thick saran, mylar or FEP). The
membrane was held onto the PSS tip between an O-ring and an adapter ring. The
term sulfide refers to the total of H2S, HS and
S2. As H2S diffuses into the electrolyte, it
dissociates into H+ and HS, which reduces
ferricyanide to form ferrocyanide and sulfur. The anode current is created as
ferrocyanide donates an electron to the anode, polarized at 0.1 V, and
ferrocyanide is then restored to ferricyanide. Sulfide-dependent changes in
anode current were converted to proportional voltage with a modified POS
meter, and the output voltage was recorded digitally (Virtual Bench, National
Instruments, Austin, TX, USA). Relative H2S permeabilities of
various membranes were determined as PSS signal magnitude and response
time.
|
In general, the PSS was calibrated with stepwise increases in sulfide
concentration while positioned in the respirometer chamber containing 3 ml
stirred (500 r.p.m.) 20 mmol l1 Tris-buffered zero-grade
argon (<0.0003 kPa oxygen)-equilibrated analytical grade purified water
(Solution 2000, Jasper, GA, USA), pH 7.0, 20°C. Sulfide stocks of 10
mmoll1 are made by dissolving fresh crystals of
Na2S in 20mmoll1 Tris-buffered analytical grade
water. First, the buffer solution in a 20 ml Pyrex syringe was vigorously
sparged with argon for at least 10 min to achieve anoxia. Liquid from the
syringe was then used to dissolve the crystals in a conical Pyrex centrifuge
tube filled with a continuous stream of argon, pH was adjusted to 7 with
dilute HCl, and the solution was immediately drawn back into the syringe free
of bubbles and sealed with a rubber serum stopper. Samples of the anoxic
Na2S stock were obtained with a gas tight syringe (Hamilton, Reno,
NV, USA) through the stopper and injected into the respirometer. Dilute
sulfide stocks of 0.1 mmol l1 were made by injecting the
concentrated stock into a syringe containing anoxic buffered water through the
sealing stopper. Stock solutions were calibrated with the standard 2-PDS assay
(Svenson, 1980) Samples of the
sulfide stock were dissolved into an excess of the reagent 2-PDS so that all
sulfide reacted to form stoichiometric amounts of the product 2-thiopyridone.
The optical density at 343 was divided by the extinction coefficient of 8.08
mmol l1 cm1 to determine the concentration
of the product 2-thiopyridone (Jensen et
al., 2000
; path length was 1 cm). The expected concentration of
sulfide was regressed against the measured concentration of 2-thiopyridone,
yielding a slope that was typically within ±2% of ideal.
To compare the PSS with a standard colorimetric method in terms of its
ability to accurately follow the kinetics of changing sulfide concentration,
the sulfide contents of 5 ml air-equilibrated solutions were assayed by both
the PSS and the 2-PDS method (Svenson,
1980). To also determine if the presence of either the PSS or a
POS would differentially alter chamber sulfide levels, sulfide solutions were
placed in three Pyrex chambers, one with the PSS, one with a POS, and one a
blank chamber without sensors. The chambers were unstoppered to allow rapid
sample removal while allowing sulfide concentration to decline smoothly by
volatilization and oxidation processes. After the initial addition of 100
µmol l1 Na2S into the chambers, 20 µl
discrete samples were removed at periodic intervals and immediately injected
into the 2-PDS reagents. Reaction mixtures were incubated and read at 343 nm
to determine sulfide concentration as a function of time.
To determine pH effects on the PSS signal, the pH of the solution to receive the sulfide stock was adjusted with 20 mmol l1 Tris, Hepes or MES to cover pH within the range of 5.5 to 8.5. The anoxic sulfide stock solution was also prepared with 20 mmol l1 of the same buffer at the tested pH. At each pH, the PSS signal was recorded after equilibration with stepwise additions of sulfide stock aliquot samples.
Because of the instability of sulfide in aerated solutions, even in the presence of metal chelators such as DTPA, PSS signal drift was determined in a flow-through system in which spontaneous sulfide oxidation was limited by syringe pump (model 22, Harvard Apparatus, Holliston, MA, USA) delivery of sulfide stock at a constant rate to a 3 ml constant volume chamber, with excess volume removed by aspiration. The sulfide stock solution was 7.5 mmol l1 Na2S in 500 mmol l1 Tris-buffered anoxic analytical grade water, pH 7.0. Delivery rates were adjusted between 0.1 and 0.2 ml h1 to create a steady state sulfide concentration between 20 and 100 µmol l1, determined by 2-PDS. PSS signal drift was measured for up to 10 h under aerated conditions.
Oxygen and sulfide respirometry
Respiration rates of excised gills and isolated mitochondria were
determined in a dual-chamber respirometer as reported previously (see
Doeller et al., 2001, for
gills and Parrino et al.,
2000
, for mitochondria;
Gnaiger, 2001
), except that
one chamber sensor port was fitted with a POS and the other with the PSS.
Experimental procedures
Intact gill sulfide and oxygen consumption as a function of PO2
To obtain nearly identical gill pieces for use in each respiration chamber,
a small section (about 1 cm anterior to posterior length) of the demibranch
was excised from freshly collected or sulfide-maintained mussels and then
halved into two hemibranch sections. Each hemibranch section, approximately 1
cm2 and 10 mg dry mass, was placed on a perforated 316 stainless
steel support in 5 ml stirred seawater in each respiration chamber.
Experimental interventions were added nearly simultaneously (<30 s delay)
to both PSS and POS chambers. Typically, gills were allowed to consume nearly
all the oxygen, then the partial pressure of oxygen
(PO2) in the chamber was elevated by lifting
the stoppers, allowing air to fill a gas space above the liquid. As the
desired PO2 was reached, stoppers were again
lowered to eliminate the gas space, and respiration measurements were resumed.
Some experiments were begun at low PO2 by
initially flushing argon into the gas space above the liquid while the
stoppers were slightly elevated. The experimental sulfide concentration used
with intact gills was 100 µmol l1, which caused maximal
sulfide-stimulated oxygen consumption (Lee
et al., 1996). To determine anoxic sulfide consumption rates,
sulfide was injected into the chamber after PO2
had reached zero. Measurements using heat-killed gills microwaved for 60 s, or
chambers alone after the gills were removed, were used to determine background
or spontaneous sulfide oxidation rates.
Gill mitochondrial sulfide and oxygen consumption as a function of PO2
Mitochondria, isolated as previously described
(Parrino et al., 2000) from
gills of freshly collected, sulfide-maintained or sulfide-free mussels, were
added to 11.5 ml respiration buffer at a concentration of 0.52
mg protein ml1. Protein was determined using the
Folinphenol reagent and bovine serum albumin to construct the standard
curve (Brookes et al., 2003
).
Before experimental interventions to respiration chambers, the respiratory
control ratio (RCR) for each mitochondrial preparation was determined as the
ratio of State 3 respiration, measured with 4 mmol l1 malate
as substrate and 25 µmol l1 ADP, and State 4 respiration,
measured after ADP is consumed. After RCR determination, the chamber was
cleaned and another aliquot sample of mitochondria added to the respiration
buffer. To achieve State 3 respiration with sulfide as sole respiratory
substrate, 25 µmol l1 ADP was injected into the
respiration buffer first, then sulfide was immediately injected at one of
three concentrations, 56, 1013, or 1820 µmol
l1. To determine the effect of
PO2 on sulfide consumption rate, mitochondria
were allowed to consume all the oxygen in the chamber as sulfide was injected
repeatedly. To determine anoxic sulfide consumption rates, sulfide was
injected at PO2=0.
Gill mitochondrial sulfide consumption with inhibited terminal oxidase enzymes
The roles of both classical and alternative terminal oxidase pathways in
sulfide consumption were evaluated using selective inhibitors. Mitochondria
were first stimulated with sulfide to achieve State 3 respiration rates in
non-limiting oxygen conditions. Sulfide consumption rates were then determined
at three sulfide concentrations in the presence of 1 mmol l1
KCN to inhibit cytochrome c oxidase, and then after the addition of 1
mmol l1 SHAM to inhibit the putative alternative
oxidase.
Data presentation and statistical analysis
For both oxygen and sulfide measurements, the respirometer software
(Oroboros DatLab, Innsbruck, Austria) was used for baseline correction,
derivatization of the parent traces, rate calculations, and integration of the
derivative traces to determine the quantity of oxygen and sulfide consumed.
Data are presented as mean ± standard deviation (S.D.;
N=number of repetitions). Two-sample comparisons were made with the
paired or unpaired one-tailed t-test assuming equal variance
(Microsoft Excel). Significance was assigned at the 5% level.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The PSS signal up to approximately 150 µmol l1 sulfide can be fitted by linear regression (Fig. 2). At higher sulfide concentrations, the PSS response is best fitted by a second order polynomial equation. The curvilinear nature of the calibration curve extends to at least 1 mmol l1 sulfide and is highly repeatable. Because anoxic sulfide stocks prepared early in the day gave identical calibration curves 4 h later, it is unlikely that the curvilinear response is the result of spontaneous sulfide oxidation in the stock solution or in the anoxic chamber. Results from tests using different electrolyte concentrations suggest that the nonlinear response at higher sulfide concentrations may result in part from saturation of the sensor electrolyte. Sulfide levels used in gill experiments are below 200 µmol l1 and in mitochondrial experiments are below 25 µmol l1. A typical stepwise calibration experiment (Fig. 2, insert) illustrates a response time of approximately 30 s to reach 90% of the new signal, followed by an unchanging signal indicating slow spontaneous sulfide oxidation under anoxic conditions. Replacement of the solution with fresh sulfide-free aerated buffer returned the signal to baseline in about 30 s.
|
The PSS accurately followed the kinetics of changing sulfide levels, as determined by the standard 2-PDS method (Fig. 3). Although the loss of sulfide due to volatilization was typically threefold more rapid than the oxidation rate in closed chamber measurements, the presence of a PSS or POS did not appear to accelerate sulfide loss. Furthermore, the PSS sulfide consumption rate, calculated from the microamp current using Faraday's constant, is in the pmol s1 range, thus negligible during these experiments, and, from tests of membrane permeability, H2S will not readily cross the POS membrane. We therefore argue that the spontaneous sulfide oxidation rate would be equivalent in all three chambers, independent of sensor type.
|
The sulfide species able to diffuse across the sensor membrane is
H2S. For a given sulfide concentration, H2S formation is
favored as solution pH is lowered, leading to a rise in PSS signal
(Fig. 4). The titration of
sulfide to lower pH follows a single protonation of HS, with pK for the
H2S/HS couple near 6.9 depending on solution
composition (Millero 1986;
Millero and Hershey, 1989
; for
correct equation coefficients, see Kraus et al., 1996). Under the conditions
used for the pH titration of the PSS signal, the observed pK was 6.75
(Fig. 4, insert). However, at
constant solution pH, the PSS exhibits linearity up to 200 µmol
l1 total sulfide.
|
Some general performance characteristics of the PSS are provided in
Table 2. The detected sulfide
species is H2S, and ionized sulfide species do not appear to pass
through the H2S-permeable membrane. At neutral pH, with the meter
amplifier at maximum gain, 0.1 µmol l1 sulfide produces a
signal noticeably above baseline noise. This lower level of detection is
comparable to several of the more sensitive sulfide spectrophotometry methods
(see Lawrence et al., 2000;
Table 1). Repeated sulfide (100
µmol l1) injections into previously cleaned anoxic
chambers produced signals differing by only ±2%. The PSS functions best
at pH favoring the H2S species, but provides reliable signals up to
pH 8.5. Nointerference was observed with other common biologically produced
sulfide oxidation products (Doeller et
al., 2001
), nor was the PSS sensitive to oxygen, NO or
H2O2. However, 1 mmol l1 KCN produced
a small (<10 mV) positive offset of the PSS signal, presumably as HCN
diffused through the membrane and altered electrolyte redox chemistry. This
offset was easily subtracted as a baseline correction.
|
Respiratory studies
Gill respiration
Upon the addition of sulfide, intact gills increased oxygen consumption
rate by three- to fourfold, as previously reported
(Lee et al., 1996; Doeller et
al., 1999
,
2001
). Representative sulfide
and oxygen traces over time for intact gills are shown in
Fig. 5. Sulfide consumption
rate of gill sections under aerated conditions was 0.94±0.16 nmol
sulfide s1 (N=7), approximately 50-fold faster than
0.020± 0.005 nmol sulfide s1 (N=6), the
spontaneous rate of sulfide oxidation under aerated conditions in chambers
without gills or with heat-killed gills. The initial rate of gill sulfide
consumption under anoxic conditions, determined as the average derivative for
the first 3050% of the trace, was 0.044±0.16 nmol sulfide
s1 (N=5), approximately 5% of the normoxic gill
sulfide consumption rate but approximately sevenfold faster than the
spontaneous anoxic sulfide oxidation rate of 0.006±0.002 nmol sulfide
s1 (N=3).
|
The rate of sulfide consumption was a function of both ambient oxygen tension and sulfide concentration. At higher initial oxygen levels, sulfide was consumed more rapidly. If sulfide was repeatedly injected at identical starting air saturation levels, sulfide consumption rates were consistent. Six injections of 100 µmol l1 sulfide made near 50% air saturation over 260 min showed no change in sulfide consumption rates or S:O2 ratios (data not shown). However, normoxic sulfide consumption rates following bouts of anoxic sulfide consumption were consistently lower than pre-anoxic rates. A bolus injection of 100 µmol l1 sulfide near 50% air saturation after an anoxic sulfide consumption bout resulted in lower oxygen and sulfide consumption rates and a significantly lowered S:O2 ratio of 0.74±0.11 (N=3) compared to the pre-anoxic ratio of 0.88±0.16 (N=8) (P=0.035).
Mitochondrial respiration
Mitochondria from sulfide-maintained mussels
Mitochondria isolated from gills of sulfide-maintained mussels exhibited
RCRs of 3.7±1.3 (N=24) for the substrate malate.
Representative traces of oxygen and sulfide levels over time in the presence
of gill mitochondria are shown in Fig.
6A. The time derivatives, multiplied by 1, of these traces
are the oxygen and sulfide consumption rates, shown in
Fig. 6B. Bolus injections of
12.5 µmol l1 sulfide caused brief increased oxygen
consumption. At PO2 >5 kPa, the increase was
threefold (Fig. 6B), and both
sulfide and oxygen consumption rates exhibited single coincident peaks. As
PO2 declined (42 kPa), the sulfide and
oxygen consumption rate peaks were no longer coincident. Instead, sulfide
consumption rates exhibited single peaks while oxygen consumption rates became
kinetically complex, showing initial sulfide-stimulated high rates that
decreased to sulfide-inhibited rates, then rose again as sulfide levels
declined, but finally dropping as sulfide was exhausted. Interestingly, the
total amount of consumed oxygen, determined from peak integral, remained
constant (Fig. 6B). Multiphasic
oxygen consumption rates also occurred at lower
PO2, although with lower magnitude and for
longer times. As PO2 decreased toward anoxia,
sulfide-stimulated oxygen and sulfide consumption rates were both truncated.
Once anoxia was reached, the sulfide trace followed a two-phase kinetic event
(Fig. 6A) similar to that seen
with whole gills (Fig. 5).
|
The effects of three concentrations of sulfide, each added together with ADP, on the respiration of gill mitochondria from sulfide-maintained mussels are shown as a function of time in Fig. 7. Fig. 7A,B are the parent traces, and Fig. 7C,D are the respective time derivatives (note that B and D have a compressed time scale). These mitochondria had a State 2 respiration rate of 2.1±0.5 nmol O2 min1 mg1 protein (N=24) and exhibited relatively rapid coincident bouts of oxygen and sulfide consumption upon injections of 6.25 and 12.5 µmol l1 sulfide, with matched consumption rates (Fig. 7A,C). At 18.75 µmol l1 sulfide, sulfide consumption rate was proportionately increased (Fig. 7C) but the coincident increased oxygen consumption rate was truncated, indicating a limited sulfide-supported State 3 respiration rate that did not match the sulfide consumption rate (see Fig. 6B, below 4 kPa PO2). Sulfide at 18.75 µmol l1 inhibited the oxygen consumption rate even though the total amount of consumed oxygen remained proportional to the total amount of sulfide, with a S:O2 ratio near unity. The oxygen consumption rate between sulfide injections remained elevated above the initial State 2 rate. Selected rates from respirometry experiments are given in Table 3.
|
|
In the presence of 1 mmol l1 KCN, the oxygen consumption
rate decreased approximately fivefold from 7 to 1.5 nmol O2
min1 mg1 protein
(Fig. 7C), as observed
previously (Parrino et al.,
2000). Following KCN exposure, sulfide at all three concentrations
stimulated oxygen consumption to a single limited level of about 4 nmol
O2 min1 mg1 protein,
approximately 15% of the pre-KCN 12.5 µmol l1
sulfide-stimulated rate (Fig.
7B,D). Under these conditions, sulfide consumption rate also
reached a limit near 15 nmol H2S min1
mg1 protein, approximately 25% of the pre-KCN highest
sulfide consumption rate at 18.75 µmol l1
(Fig. 7B,D). The limited oxygen
and sulfide consumption rates resembled square waves with a duration that
varied proportionally to chamber sulfide concentration
(Fig. 7D). Sulfide consumption
integrals, both pre- and post-KCN exposure, indicated that all sulfide was
consumed at each sulfide concentration. However, the much lower
sulfide-stimulated oxygen consumption rates after KCN resulted in a
stoichiometric shift of the S:O2 ratio to near 2.5, indicating that
sulfide consumption may be uncoupled from oxygen consumption and that the
reducing equivalents generated by sulfide oxidation may not be used for
immediate oxygen reduction.
In previous studies, we provided evidence that an alternative oxidase was
operational within mussel gill mitochondria
(Parrino et al., 2000).
Addition of SHAM, an inhibitor of alternative oxidase activity, subsequent to
KCN inhibition of cytochrome oxidase further decreased the State 2 oxygen
consumption rate to approximately 5% of the pre-KCN rate
(Fig. 7B,D). Exposure to
sulfide at all concentrations after inhibition of both terminal oxidases did
not stimulate oxygen consumption rate while the sulfide consumption rate
increased to a single level of 4 nmol H2S min1
mg1 protein, with a duration again proportional to sulfide
concentration. This sulfide consumption rate was approximately 7% of the
pre-KCN treated, 18.75 µmol l1 sulfide-stimulated rate,
and about 25% of the post-KCN treated, pre-SHAM rate, although it was three-
to fourfold greater than the spontaneous sulfide oxidation rate under similar
PO2 conditions. Again, sulfide consumption
integrals, as with the pre-KCN integrals, indicated that all sulfide was
consumed even though terminal oxidase activity was negligible.
Mitochondria from sulfide-free mussels
In general, gill mitochondria from sulfide-free mussels exhibited State 2
oxygen consumption rates similar to mitochondria from sulfide-maintained
mussels, but exhibited much lower sulfide and oxygen consumption rates
following sulfide exposure (similar to whole gills; see
Lee et al., 1996). These
mitochondria responded to substrates such as malate, succinate and ADP, and
exhibited a malate-supported RCR of 3.8±1.8 (N=6). Typical
traces of mitochondrial respiratory responses to sulfide are provided in
Fig. 8.
Fig. 8A,B are the parent oxygen
and sulfide traces, and Fig.
8C,D are the respective time derivatives (B and D have a
compressed time scale). These mitochondria had a much lower response to
sulfide exposure (note that the ordinate scales of the derivative panels are
about 12% of those in Fig. 7C,D
for mitochondria from sulfide-maintained mussels, SMM). Both sulfide and
oxygen consumption rates increased upon exposure to all three sulfide levels,
but the oxygen consumption rate reached a limit at 7 nmol O2
min1 mg1 protein. Sulfide consumption
rates at 6.25 and 12.5 µmol l1 sulfide were approximately
25% of SMM mitochondrial rates, and at 18.75 µmol l1 the
rate was only 8% of the SMM rate. At 18.75 µmol l1
sulfide, the oxygen consumption rate exhibited multiphasic kinetics similar to
those seen in Fig. 6B for
sulfide exposure of control mitochondria under low oxygen conditions. Although
sulfide and oxygen consumption rates were depressed, the S:O2 ratio
remained near unity, as observed with SMM mitochondria. The addition of
terminal oxidase inhibitors KCN and then SHAM resulted in matched reductions
in oxygen and sulfide consumption rates at all three sulfide concentrations.
The rates appeared to reach limits, with duration proportional to sulfide
concentration, and in general were less than 20% of the comparable SMM
mitochondria rates (Fig. 7D).
Although respiration rates were consistently lower than SMM mitochondria
rates, the S:O2 ratio of mitochondria from sulfide-free mussels
remained near unity under all conditions, indicating that sulfide and oxygen
consumption were not uncoupled under these conditions.
|
Oxygen and sulfide consumption rate apparent P50
It is possible to estimate apparent P50 values of
oxygen and sulfide consumption (partial pressure of oxygen at half maximal
rate) for mitochondria from sulfide-maintained mussels from a plot of initial
sulfide-stimulated oxygen and sulfide consumption rates
(Fig. 6) as a function of
ambient PO2
(Fig. 9). The resultant
apparent P50 values were 2 kPa and 1 kPa for oxygen and
sulfide consumption rates, respectively, indicating that the oxygen
consumption rate was more sensitive to declining
PO2 than the sulfide consumption rate. However,
the S:O2 ratio remained near unity until <0.5 kPa as anoxia was
approached, where sulfide consumption continued at a low rate and oxygen
consumption reached zero. At PO2 above the
oxygen-limiting region, KCN inhibited oxygen and sulfide consumption rates of
sulfide-maintained mitochondria to about 10% and 20% of pre-KCN values,
respectively, and the S:O2 ratio increased to about 2.5. The
addition of SHAM further depressed oxygen consumption to near zero while
sulfide consumption rate decreased to about 10% of control, a response similar
to sulfide consumption under anoxic conditions where oxygen and sulfide
consumption rates were unmatched. Terminal oxidase-inhibited rates from
sulfide-free mitochondria were lower but coincident, indicating that sulfide
and oxygen consumption remained coupled.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sulfide levels in solution are usually measured in discrete samples by
various multi-step wet chemical, colorimetric or gas extraction/gas
chromatography methods (Lawrence et al.,
2000), or with the sulfide electrode. This bare Ag2S
electrode in combination with a reference electrode was developed to detect
S2 in solution at strongly alkaline pH
(Vesely et al., 1972
). Recent
work in which it was used to determine the maximum sulfide consumption rate of
a protozoan over a range of steady state sulfide concentrations suggests that
it may also be sensitive to submicromolar concentrations of
HS (Searcy and Peterson,
2004
). The bare Ag2S coating requires daily
reconditioning to remove interfering deposits that form from constituents
present in biologically relevant solutions. In contrast, the PSS continuously
reports hydrogen sulfide levels in solutions near neutral pH under
physiological conditions in real time. The sulfide-reactive interior
components are protected from other sample components by a polymer membrane.
To use the PSS accurately, solution pH must be known. As hydrogen sulfide is
ionized to the hydrosulfide anion above pH 7, the proportion of hydrogen
sulfide in solution decreases. Although the PSS loses sensitivity at alkaline
pH, it was successfully calibrated at pH 8.5.
The concentration of hydrogen sulfide in biological tissues
(Abe and Kimura, 1996;
Wang, 2002
) or in sulfide-rich
environments where organisms survive (Lee
et al., 1996
; Grieshaber and
Voelkel, 1998
) is usually in the micromolar range, comparable to
oxygen concentration. Spontaneous reaction between hydrogen sulfide and
oxygen, catalyzed by numerous organic and inorganic agents
(Chen and Morris, 1972
), can
rapidly change the solution sulfide concentration. In addition, cellular
enzymatic oxidation of sulfide can also be rapid. To obtain accurate sulfide
measurements under physiological time frames and condition, it is necessary to
use a highly sensitive, continuously recording sulfide sensor such as the PSS.
By using this novel device in a closed chamber respirometer, we have observed
sulfide oxidation kinetics of representative tissues and organelles under
physiological conditions.
Respiratory studies
General response
The influence of oxygen partial pressure PO2
on tissue oxygen consumption rate is well known. Oxygen diffusion rate into
tissue is dictated by PO2, and mitochondrial
cytochrome c oxidase is kinetically dependent on
PO2. Mussel gill oxygen consumption is
independent of PO2 down to about 5 kPa, where
it begins to show conformity (Doeller et
al., 1993). Gill sulfide consumption is also dependent on oxygen,
showing conformity at about 4 kPa following a 50100 µmol
l1 sulfide exposure. The lugworm Arenicola marina,
another inhabitant of sulfide sediments, exhibits oxygen-dependent sulfide
consumption and readily switches to anaerobic metabolic pathways (Völkel
and Grieshaber, 1992
,
1994
). The complex kinetic
responses of oxygen and sulfide consumption rates indicate interdependence of
each process on the other substrate (Fig.
6). At low PO2, sulfide stimulates
oxygen consumption by providing reducing equivalents to cytochrome c
(Doeller et al., 1999
) or to
ubiquinone (Parrino et al.,
2000
), but it simultaneously competes with oxygen for cytochrome
c oxidase and in turn partially inhibits oxygen consumption. As the
sulfide level declines, inhibition is partly removed and oxygen consumption
rises until sulfide is exhausted, when it then falls. The throughput of
electrons from sulfide oxidation to oxygen via mitochondrial electron
transport also controls the rate of sulfide consumption. As
PO2 decreases further, the sulfide consumption
rate also decreases. More evidence of the interdependence of oxygen and
sulfide consumption is seen in the S:O2 ratio being maintained near
unity until anoxia is approached. Evidence from the S:O2 ratio,
rates of ATP and thiosulfate production, and cytochrome redox states supports
the hypothesis that electrons from sulfide oxidation enter the mitochondrial
electron transport chain, coupling sulfide consumption with ATP production
(Doeller et al., 2001
).
However, at low PO2, as sulfide consumption
continues (albeit at a low level) and oxygen consumption drops to zero, it is
unclear where reducing equivalents from sulfide are delivered or sequestered
or if sulfide-stimulated ATP production continues.
Sink for reducing equivalents
The effect of low PO2 on oxygen and sulfide
consumption is also seen at non-limiting PO2 in
the presence of terminal oxidase inhibitors. Previous investigations have
demonstrated that KCN and SHAM limit sulfide-stimulated oxygen consumption and
ATP production (Parrino et al.,
2000). KCN inhibition of cytochrome c oxidase results in
an 80% decreased mitochondrial oxygen consumption rate and a 50% decreased
sulfide consumption rate, compared to the pre-KCN 12.5 µmol
l1 sulfide-stimulated rates. This apparent uncoupling of
oxygen and sulfide consumption, also observed under anoxic conditions,
suggests that the path of reducing equivalents from sulfide to oxygen includes
a component that can either accumulate electrons or pass them onto an
alternative electron acceptor. Sulfide-maintained mussels have much higher
total glutathione and sulfite levels in sulfide-exposed gill tissue compared
to sulfide-free mussels (Doeller et al.,
2001
). Glutathione disulfide (GSSG), which can be reduced to
glutathione (GSH) by glutathione reductase, may represent a competent electron
acceptor if sulfide was the reductant. The total glutathione concentration,
approximately 1 mmol kg1 gill tissue, and the equilibrium
GSH/GSSG redox potential, 240 mV
(Sevier and Kaiser, 2002
),
compared to 270 mV for HS/S
(Kelly, 1982
), lends support
for such consideration. Ascorbate is another agent involved in cellular redox
cycling that represents a potential sink for reducing equivalents. Compared to
GSH, gill tissue ascorbate concentration, approximately 50 µmol
kg1, is low. However, the levels of both ascorbic acid (AA)
and dehydroascorbate (DHAA) are two- to fourfold higher in gills from
sulfide-maintained mussels compared to those from sulfide-free mussels (D.W.K.
and J.E.D., preliminary data). The operation of a cellular sink for electrons
produced from sulfide consumption under oxygen-limiting conditions may include
the subsequent delivery of those electrons to mitochondria upon return to
oxygenated conditions. This is evident in intact gills by the decrease in
S:O2 ratio after a bout of anoxic sulfide consumption. The
S:O2 ratio decline might also suggest that the anoxic event
mediates a persistent partial mitochondria uncoupling. The GSH/GSSG and the
AA/DHAA ratios before and after sulfide exposure must be determined to
evaluate the roles of GSH and AA in sulfide consumption under oxygen-limiting
conditions. Both GSH and AA participate in the plant mitochondrial
HalliwellAsada pathway, which operates to deliver reducing equivalents
and limit oxidative stress (Noctor and
Foyer, 1998
). Perhaps a similar cyclical pathway could operate in
mussel gills to sequester abundant reducing equivalents. Using matrix volume
determination (Walajtys-Rhode et al.,
1992
), total glutathione concentration in rat liver and heart
mitochondria is 34 mmol l1
(Shiva et al., 2004
;
Shu et al., 2003
), and
ascorbate concentration in heart mitochondria is approximately 1.3 mmol
l1 (Shu et al.,
2003
). Another possible sink for molecular sulfide under
oxygen-limiting conditions may be the formation of disulfides, which occurs on
free cysteine residues of sulfide binding proteins in Riftia
pachyptila blood (Zal et al.,
1998
; Bailly et al.,
2002
) and trisulfide glutathione
(Prutz, 1993
;
Moutiez et al., 1994
). Further
sulfide and oxygen respirometric investigations are needed to evaluate the
existence and nature of a reducing equivalent/sulfide store present in
sulfide-maintained mussel gills and lost after the mussels are acclimated to
sulfide-free conditions.
Alternative oxidase pathway
Inhibition by KCN of gill mitochondria suggests that under non-limiting
PO2, cytochrome c oxidase accounts for
about 80% of the sulfide-stimulated oxygen consumption rate. A
cyanide-insensitive alternative oxidase accounts for the remaining 20%, as
seen by SHAM inhibition. Alternative oxidases, previously found in plants and
microorganisms (Vanlerberghe and McIntosh,
1997), have been implicated in sulfide oxidation pathways in the
lugworm (Völkel and Grieshaber,
1997
) and in mussel gill mitochondria
(Parrino et al., 2000
). Mussel
gill mitochondria exhibiting a 200300% increase in oxygen consumption
rate during exposure to 510 µmol l1 sulfide also
exhibit a <50% inhibition at the same sulfide concentration if the
alternative oxidase is first blocked by SHAM
(Parrino et al., 2000
). The
alternative oxidase may serve as a shunt for sulfide oxidation electrons, thus
allowing cytosolic sulfide concentration to be regulated and limiting
inhibitory effects of sulfide on cytochrome c oxidase. Because
alternative oxidases are not involved in proton pumping, sulfide consumption
could continue even during low metabolic demand for ATP generation. The
partitioning of electrons through each terminal oxidase may be adjustable and
depend on metabolic demand and the concentration of available substrates, a
potentially complex matrix. Inhibition of both terminal oxidases under
non-limiting oxygen conditions creates anoxia-like conditions for sulfide and
oxygen consumption, demonstrating that sulfide consumption is not totally
dependent on PO2. With further respirometric
studies, the metabolic role of each terminal oxidase could be defined.
Mitochondria from sulfide-free mussels
Although gill mitochondria isolated from sulfide-free mussels are
stimulated by typical mitochondrial substrates, they exhibit a much weaker
response to sulfide (Lee et al.,
1996), and reach limits for both oxygen and sulfide consumption
rates that are 25% or less of the rates exhibited by mitochondria from
sulfide-maintained mussels under comparable conditions. However, the
S:O2 ratios are maintained near unity at all sulfide concentrations
and under conditions of cytochrome c oxidase and alternative oxidase
inhibition. These results suggest that specific components of sulfide
metabolic pathways may be downregulated during acclimation to sulfide-free
conditions, although the ability of these mitochondria to metabolize organic
substrates and ADP indicates that cytochrome c oxidase activity is
maintained. The limited sulfide consumption rates suggest that the activities
of both sulfide oxidase and alternative oxidase have been substantially
lowered. In addition, the maintenance of coupled oxygen and sulfide
consumption suggests that the reducing equivalent/sulfide store concentration
is also decreased, which is in agreement with lower GSH and AA levels in gills
of sulfide-free mussels. Preliminary data from a 2-D gel electrophoresis
proteomics study of G. demissa gills demonstrated that approximately
36 distinct proteins are differentially expressed in gills from
sulfide-maintained mussels compared to those from sulfide-free mussels. These
results indicate that specific sulfide metabolic pathway components may be
inducible by environmental sulfide in animals adapted to high sulfide
exposure. Identification of inducible components of sulfide metabolic pathways
may help us understand how organisms adapt to the extreme conditions of high
sulfide and low oxygen and also understand the role that sulfide may play in
cell signaling in all biological organisms, including humans
(Wang, 2002
;
Moore et al., 2003
).
![]() |
List of abbreviations |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abe, K. and Kimura, H. (1996). The possible role of hydrogen sulfide as an endogenous neuromodulator. J. Neurosci. 16,1066 -1071.[Abstract]
Arp, A. J., Menon, J. G. and Julian, D. (1995). Multiple mechanisms provide tolerance to environmental sulfide in Urechis caupo. Am. Zool. 35,132 -144.
Atlas, R. M. (1996). Principles of Microbiology. 2nd edition. Dubuque, IA: W. C. Brown Publishers. pp. 1298.
Bailly, X., Jollivet, D., Vanin, S., Deutsch, J., Zal, F.,
Lallier, F. and Toulmond, A. (2002). Evolution of the
sulfide-binding function within the globin multigenic family of the deep-sea
hydrothermal vent tubeworm Riftia pachyptila. Mol. Biol.
Evol. 19,1421
-1433.
Berzofsky, J. A., Peisach, J. and Blumberg, W. E.
(1971). Sulfheme proteins. I. Optical and magnetic properties of
sulfmyoglobin and its derivatives. J. Biol. Chem.
246,3367
-3377.
Brookes, P. S., Kraus, D. W., Shiva, S., Doeller, J. E., Barone,
M. C., Patel, R. P., Lancaster, J. R., Jr and Darley-Usmar., V. M.
(2003). Control of mitochondrial respiration by NO, effects of
low oxygen and respiratory state. J. Biol. Chem.
278,31603
-31609.
Chen, K. Y. and Morris, J. C. (1972). Oxidation of sulfide by O2; catalysis and inhibition. J. Sanit. Eng. Div. Amer. Soc. Civil Eng. 98(SA1),215 -227.
Cline, J. D. (1969). Spectrophotometric determination of hydrogen sulfide in natural waters. Lim. Oceanog. 14,454 -458.
Doeller, J. E., Kraus, D. W., Shick, J. M. and Gnaiger, E. (1993). Heat flux, oxygen flux, and mitochondrial redox state as a function of oxygen availability and ciliary activity in excised gills of Mytilus edulis. J. Exp. Zool. 265, 1-8.[Medline]
Doeller, J. E., Gashen, B. K., Parrino, V. and Kraus, D. W.
(1999). Chemolithoheterotrophy in a metazoan tissue. I. Sulfide
supports cellular work in ciliated mussel gills. J. Exp.
Biol. 202,1953
-1961.
Doeller, J. E., Grieshaber, M. K. and Kraus, D. W.
(2001). Chemolithoheterotrophy in a metazoan tissue: thiosulfate
production matches ATP demand in ciliated mussel gills. J. Exp.
Biol. 204,3755
-3764.
Fahey, R. C., Newton, G. L., Dorian, R. and Kosower, E. M. (1981). Analysis of biological thiols: quantitative determination of thiols at the picomole level based upon derivatization with monobromobimane and separation by cation-exchange chromatography. Anal. Biochem. 111,357 -365.[Medline]
Gnaiger, E. (2001). Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply. Resp. Physiol. 128,277 -297.[CrossRef][Medline]
Grieshaber, M. K. and Voelkel, S. (1998). Animal adaptations for tolerance and exploitation of poisonous sulfide. Ann. Rev. Physiol. 60,30 -53.
Jensen, P. E., Reid, J. D. and Hunter, C. N. (2000). Modification of cysteine residues in the ChlI and ChlH subunits of magnesium chelatase results in enzyme inactivation. Biochem. J. 352,435 -441.[CrossRef][Medline]
Jeroschewski, P., Steuckart, C. and Kuhl, M. (1996). An amperometric microsensor for the determination of H2S in aquatic environments. Anal. Chem. 68,4351 -4357.[CrossRef]
Kelly, D. P. (1982). Biochemistry of the chemolithotrophic oxidation of inorganic sulphur. Phil. Trans. R. Soc. Lond. B 298,499 -528.[Medline]
Kraus, D. W., Doeller, J. E. and Powell, C. S. (1986). Sulfide may directly modify cytoplasmic hemoglobin deoxygenation in Solemya reidi gills. J. Exp. Biol. 199,1343 -1352.
Kuenen, J. G., Robertson, L. A. and Van Gemerden, H. (1985). Microbial interactions among aerobic and anaerobic sulfur-oxidizing bacteria. Adv. Microb. Ecol. 8, 1-59.
Lawrence, N. S., Davis, J. and Compton, R. G. (2000). Analytical strategies for the detection of sulfide: a review. Talanta. 52,771 -784.[CrossRef]
Lee, R. W., Kraus, D. W. and Doeller, J. E.
(1996). Sulfide-stimulation of oxygen consumption rate and
cytochrome reduction in gills of the estuarine mussel Geukensia demissa.Biol. Bull. 191,421
-430.
Millero, F. J. (1986). The thermodynamics and kinetics of the hydrogen sulfide system in natural waters. Mar. Chem. 18,121 -147.[CrossRef]
Millero, F. J. and Hershey, J. P. (1989). Thermodynamics and kinetics of the hydrogen sulfide in natural waters. In Biogenic Sulfur in the Environment (ed. E. S. Saltzman and W. J. Cooper), pp. 282-313. Washington, DC: American Chemical Society.
Moore, P. K., Bhatia, M. and Moochhala, S. (2003). Hydrogen sulfide: from the smell of the past to the mediator of the future? Trends Pharmacol. Sci. 24,609 -611.[CrossRef][Medline]
Moutiez, M., Aumercier, M., Teissier, E., Parmentier, B., Tartar, A. and Sergheraert, C. (1994). Reduction of a trisulfide derivative of glutathione by glutathione reductase. Biochem. Biophys. Res. Commun. 202,1380 -1386.[CrossRef][Medline]
Nicholls, P. (1975). The effect of sulphide on cytochrome aa3. Isoteric and allosteric shifts of the reduced alpha-peak. Biochim. Biophys. Acta 396, 24-35.[Medline]
Noctor, G. and Foyer, C. H. (1998). Ascorbate and glutathione: keeping active oxygen under control. Annu. Rev. Plant. Physiol. Mol. Biol. 49,249 -279.[CrossRef]
Parrino, V., Kraus, D. W. and Doeller, J. E.
(2000). ATP production from the oxidation of sulfide in gill
mitochondria of the ribbed mussel Geukensia demissa. J. Exp.
Biol. 203,2209
-2218.
Powell, M. A. and Somero, G. N. (1986). Hydrogen sulfide oxidation is coupled to oxidative phosphorylation in mitochondria of Solemya reidi. Science 233,563 -566.
Prutz, W. A. (1993). Sulfane-activated reduction of cytochrome c by glutathione. Free Radic. Res. Commun. 18,159 -165.[Medline]
Searcy, D. G. and Peterson, M. A. (2004). Hydrogen sulfide consumption measured at low steady state concentrations using a sulfidostat. Anal. Biochem. 324,269 -275.[CrossRef][Medline]
Sevier, C. S. and Kaiser, C. A. (2002). Formation and transfer of disulfide bonds in living cells. Nature Rev. Mol. Cell. Biol. 3,836 -847.[CrossRef][Medline]
Shiva, S., Crawford, J. H., Ramachandran, A., Ceasar, E. K., Hillson, T., Brookes, P. S., Patel, R. P. and Darley-Usmar, V. M. (2004). Mechanisms of the interaction of nitroxyl with mitochondria. Biochem. J. 379,359 -366.[CrossRef][Medline]
Shu, J. H., Heath, S. and Hagen, T. M. (2003). Two subpopulations of mitochondria in the aging rat heart display heterogenous levels of oxidative stress. Free Rad. Biol. Med. 35,1064 -1072.[CrossRef][Medline]
Somero, G. N., Childress, J. J. and Anderson, A. E. (1989). Transport, metabolism and detoxification of hydrogen sulfide in animals from sulfide-rich marine environments. Aq. Sci. Rev. 1,591 -614.
Svenson, A. (1980). A rapid and sensitive spectrophotometric method for determination of hydrogen sulfide with 2,2'-dipyridyl disulfide. Anal. Chem. 107, 51-55.
Vanlerberghe, G. C. and McIntosh, L. (1997). Alternative oxidase: From gene to function. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48,703 -734.[CrossRef]
Vesely, J., Jensen, O. J. and Nicolaisen, B. (1972). Ion-selective electrodes based on silver sulphide. Anal. Chim. Acta 62,1 -13.[CrossRef]
Völkel, S. and Grieshaber, M. K. (1992). Mechanisms of sulphide tolerance in the peanut worm, Sipunculus nudus (Sipunculidae) and the lugworm, Arenicola marina (Polychaeta). J. Comp. Physiol. 162,469 -477.
Völkel, S. and Grieshaber, M. K. (1994). Oxygen dependent sulfide detoxification in the lugworm Arenicola marina.Mar. Biol. 118,137 -147.
Völkel, S. and Grieshaber, M. K. (1997).
Sulphide oxidation and oxidative phosphorylation in the mitochondria of the
lugworm Arenicola marina. J. Exp. Biol.
200, 83-92.
Walajtys-Rhode, E., Zapatero, J., Moehren, G. and Hoek, J. B. (1992). Mitochondrial pyruvate carboxylation by glucagon pretreatment. J. Biol. Chem. 287,370 -379.
Wang, R. (2002). Two's company, three's a
crowd: can H2S be the third endogenous gaseous transmitter?
FASEB J. 16,1792
-1798.
Zal, F., Leize, E., Lallier, F. H., Toulmond, A., Van
Dorsselaer, A. and Childress, J. J. (1998).
S-Sulfohemoglobin and disulfide exchange: the mechanisms of sulfide binding by
Riftia pachyptila hemoglobins. Proc. Natl. Acad. Sci.
USA 95,8997
-9002.