First published online August 8, 2008
Journal of Experimental Biology 211, 2617-2623 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.019729
Redox regulation of mitochondrial sulfide oxidation in the lugworm, Arenicola marina
Tatjana M. Hildebrandt* and
Manfred K. Grieshaber
Institut für Zoophysiologie, Heinrich-Heine-Universität, 40225
Düsseldorf, Germany
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Fig. 1. Regulation of mitochondrial sulfide oxidation by glutathione (GSH),
ascorbate and dehydroascorbate (DHA). (A–C) Initial rates of ATP
production (gray circles) and oxygen consumption in state 3 (black circles)
and state 4 (black squares) with sulfide (5–100
µmoll–1) added as a substrate. (A) Sulfide oxidation in
the absence of redox-competent molecules. (B) Sulfide oxidation in the
presence of 2.5 mmoll–1 GSH and 2.5 mmoll–1
ascorbate. Oxygen consumption rates were identical in the presence and in the
absence of ADP. (C) Sulfide oxidation in the presence of 1
mmoll–1 DHA. *Significantly different
(P>0.05) from corresponding data points without redox competent
molecules. (D–F) Inhibition (%) of mitochondrial respiration (black
bars) and ATP production (grey bars) with sulfide added as a substrate by 5
µmoll–1 myxothiazole, 0.05 mmoll–1 KCN, 1
mmoll–1 NaN3 or 0.5 mmoll–1 SHAM.
(D) Inhibition of oxygen consumption (state 3) and ATP production with
5–10 µmoll–1 sulfide added as a substrate in the
absence of redox-competent molecules. (E) Inhibition of oxygen consumption
with 10–100 µmoll–1 sulfide added as a substrate in
the presence of 2.5 mmoll–1 GSH and 2.5
mmoll–1 ascorbate. ATP production rates were too low to study
the effects of inhibitors. (F) Inhibition of oxygen consumption (state 3) and
ATP production with 5–50 µmoll–1 sulfide added as a
substrate in the presence of 1 mmoll–1 DHA. Data are given as
means ± s.d. of the results from 3 to 12 different preparations, each
comprising approximately 10–15 animals.
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Fig. 2. Original recordings of (A) oxygen concentrations
(µmoll–1) and (B) ATP concentration
(µmoll–1) calculated from the NADPH absorbance at 340 nm
in mitochondrial suspensions from body wall tissue of Arenicola
marina. (A) Effect of redox-competent molecules on oxygen consumption
with sulfide as a substrate. The assays contained 0.45 mg
ml–1 mitochondrial protein and either 1
mmoll–1 dehydroascorbate (DHA) (trace 2) or 2.5
mmoll–1 GSH plus 2.5 mmoll–1 ascorbate
(trace 3). The reactions were started by the addition of 50
µmoll–1 sulfide. In the presence of DHA, the oxygen
consumption rate could be stimulated by ADP. (B) Activation of ATP production
with 50 µmoll–1 sulfide as a substrate by DHA. The amount
of mitochondrial protein present was 0.16 mg ml–1. Additions
are indicated by arrows.
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Fig. 3. Dehydroascorbate (DHA) protects mitochondrial respiration with malate as a
substrate from inhibition by sulfide. Oxygen consumption rates of mitochondria
from the body wall tissue of Arenicola marina as a function of time.
Additions (indicated by arrows) were 8 mmoll–1 malate and 1
mmoll–1 ADP. Subsequently 1 mmoll–1 DHA was
added only to the assay shown by the black line at the time marked with X. The
injection of 50 µmoll–1 sulfide inhibited malate
respiration in the absence of DHA (gray line) and, by contrast, stimulated the
oxygen consumption rate in the presence of DHA (black line).
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Fig. 4. Original recordings of oxygen concentrations (µmoll–1)
in mitochondrial suspensions from body wall tissue of Arenicola
marina. Additions of 4 mmoll–1 succinate, 1
mmoll–1 ADP, 5 mmoll–1 glutathione (GSH), 1
mmoll–1 NaN3 and 50 µmoll–1
sulfide are marked with arrows. Mitochondrial protein: (A) 0.53·mg
ml–1, (B) 0.48 mg ml–1, (C) 0.35 mg
ml–1.
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Fig. 5. Mitochondrial oxygen consumption (% of oxygen consumption rate at air
saturation) as a function of oxygen concentration
(µmoll–1). State 3 respiration was recorded with 8
mmoll–1 malate added as a substrate in the presence of 0.5
mmoll–1 salicylhydroxamic acid (SHAM) (black dots) and with
50 µmoll–1 sulfide added as a substrate in the presence of
1 mmoll–1 DHA (open squares) or in the presence of 2.5
mmoll–1 GSH + 2.5 mmoll–1 ascorbate (gray
circles). Mitochondria were allowed to completely consume the oxygen contained
in the respiration medium, thus providing respiration rates at variable oxygen
concentrations.
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Fig. 6. Proposed model of the two different pathways for sulfide oxidation in the
mitochondria of Arenicola marina. A membrane-bound sulfide:quinone
oxidoreductase (SQR) oxidizes sulfide (H2S) to persulfides (R-SSH;
R=cysteine residue of the SQR) and transfers the electrons to the ubiquinone
pool (Theissen and Martin,
2008 ). In the presence of GSH and ascorbate, representing reducing
cellular conditions, a pathway of sulfide detoxification is activated (A).
Electrons are transferred to oxygen via an alternative oxidase (AOX)
without proton translocation. The energy-conserving sulfide oxidation pathway
can be activated by dehydroascorbate (DHA) in vitro (B). Electrons
are channelled to oxygen through the classical respiratory chain complexes III
and IV, which transport protons across the membrane, thus allowing ATP
production by complex V (ATP synthase). Inhibitors used in this study are
specified above their target enzymes.
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© The Company of Biologists Ltd 2008
