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First published online September 15, 2004
Journal of Experimental Biology 207, 3667-3679 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01212

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Sulfide consumption by mussel gill mitochondria is not strictly tied to oxygen reduction: measurements using a novel polarographic sulfide sensor

David W. Kraus^* and Jeannette E. Doeller

Department of Biology, University of Alabama at Birmingham, 1300 University Boulevard, Birmingham AL 35294-1170, USA

View larger version (50K): [in a new window]   Fig. 1. Diagram of the polarographic sulfide sensor, PSS. A side view and expanded tip view of the PSS illustrates the component parts. The overall dimensions are 25 mm long and 12 mm diameter, with individual components proportionally sized. The sawtooth lines indicate threaded pieces. The platinum anode and cathode are cemented into the polyether ether ketone (PEEK) housing with epoxy resin. The membranes are cemented together with silicone cement. The 0.5 mm hole in the H2S-impermeable membrane is concentric with the anode and provides a small reservoir of electrolyte between the anode and the H2S permeable membrane. Lateral diffusion of H2S from this region at the tip into the bulk electrolyte is limited and greatly shortens response time as the impermeable membrane serves as a virtual guard ring.

View larger version (18K): [in a new window]   Fig. 2. Calibration of the polarographic sulfide sensor, PSS. A typical calibration curve is derived from sequential injections (inset) of Na2S stock into anoxic 20 mmol l–1 Tris at pH 7.0. A linear regression fits the PSS signals at <200 µmol l–1 sulfide, whereas a second order polynomial fits the PSS signals above this concentration.

View larger version (17K): [in a new window]   Fig. 3. The polarographic sulfide sensor (PSS) signal compared to the standard chemical analysis of sulfide with 2,2 dipyridyl disulfide (2-PDS). A single injection of anoxic Na2S stock brought the concentration to 100 µmol l–1 in 3 ml air-saturated 20 mmol l–1 Tris at pH 7.0. 50 µl aliquot samples of solution were removed at timed intervals and immediately added to the 2-PDS reaction mixture. Both methods reported the same change in sulfide concentration over time due to spontaneous sulfide oxidation and volatilization. Signals were not influenced by the presence of a polarographic oxygen sensor (POS) or PSS in the chamber.

View larger version (26K): [in a new window]   Fig. 4. The polarographic sulfide sensor (PSS) signal is dependent on solution pH (5.5–8.5) with increased sensitivity of the PSS at lower pH. The titration at a specific sulfide concentrations (10–200 µmol l–1) over a pH range (inset) illustrates the pK for H2S /HS– to be near 6.75 at 20°C and that the PSS is detecting only H2S.

View larger version (25K): [in a new window]   Fig. 5. Consumption of oxygen and sulfide by intact gill tissue. Traces of oxygen partial pressure (black line) and sulfide concentration (red line) over time demonstrate a rapid decline in oxygen and a coincident rapid removal of sulfide following bolus injections of 100 µmol l–1 Na2S (arrows along abscissa). Once anoxia is reached at 70 s with no further oxygen consumption, consumption of sulfide injected at 100 s continues more slowly than under normoxic conditions. Following an anoxic bout, a subsequent addition of sulfide results in decreased rates of oxygen and sulfide consumption and a decreased S:O2 ratio. Spontaneous sulfide oxidation under oxygenated conditions (open circles) or anoxic conditions (open squares) is much slower than biological sulfide consumption. Sulfide oxidation rates in chambers with heat-killed gills (microwave oven for 1 min, see Materials and methods; data not shown) are comparable to rates without gills, indicating that biological sulfide consumption is catalyzed enzymatically.

View larger version (32K): [in a new window]   Fig. 6. Mitochondrial oxygen and sulfide consumption from normoxic to anoxic conditions. (A) Oxygen partial pressure PO2 (black line) and sulfide concentration (red line) as isolated mitochondria were exposed to repeated additions of 12.5 µmol l–1 sulfide (arrows). (B) Time derivatives (x –1) of oxygen (black line) and sulfide (red line) lines, giving oxygen and sulfide consumption rates (O2 and H2S, respectively). At higher PO2, both oxygen and sulfide consumption events are coincident. As oxygen levels decline, the events become uncoupled, with oxygen consumption being limited first. The multiphasic oxygen consumption kinetics may result from transient sulfide stimulation and inhibition of cytochrome c oxidase. Under anoxia, sulfide consumption continues at a low level.

View larger version (34K): [in a new window]   Fig. 7. Respiration of gill mitochondria from sulfide-maintained mussels with sequential inhibition of terminal oxidases. (A,B) Traces of oxygen partial pressure PO2 (black line) and sulfide concentration (red line) over time. Sulfide additions at 6.25 µmol l–1 (a), 12.5 µmol l–1 (b) and 18.75 µmol l–1 (c) were repeated under non-limiting oxygen conditions and in the presence of 1 mmol l–1 potassium cyanide (KCN) and 1 mmol l–1 salicylhydroxamic acid (SHAM) to inhibit cytochrome c oxidase and the alternative oxidase, respectively. (C) Derivatives of oxygen (black line) and sulfide (red line) traces showing consumption rates. Sulfide consumption rate (H2S) proportionally increases while oxygen consumption rate (O2) reaches a limit and is inhibited at the highest sulfide concentration. (D) KCN-inhibition of cytochrome c oxidase results in truncated and unmatched rates of oxygen and sulfide consumption. SHAM-inhibition of alternative oxidase results in complete oxygen consumption inhibition while sulfide consumption continues at a low level. Note the compressed time scale in B and D.

View larger version (37K): [in a new window]   Fig. 8. Respiration of gill mitochondria from sulfide-free mussels with sequential inhibition of terminal oxidases. (A,B) Traces of oxygen partial pressure PO2 (black line) and sulfide concentration (red line) over time. Sulfide additions at 6.25 µmol l–1 (a), 12.5 µmol l–1 (b) and 18.75 µmol l–1 (c) were repeated under non-limiting oxygen conditions and in the presence 1 mmol l–1 potassium cyanide (KCN) and 1 mmol l–1 salicylhydroxamic acid (SHAM) to inhibit cytochrome c oxidase and the alternative oxidase, respectively. (C) Derivatives of oxygen (black line) and sulfide (red line) traces showing consumption rates (O2 and H2S, respectively). Rates are lower than those from sulfide-maintained mussels (Fig. 7) but remain coupled, although limits are reached at all sulfide concentrations. (D) KCN-inhibition of cytochrome c oxidase and then alternative oxidase with SHAM results in further decreases in both oxygen and sulfide consumption rates, however the two events remain coupled. Note the compressed time scale in B and D.

View larger version (17K): [in a new window]   Fig. 9. Sulfide-stimulated oxygen and sulfide consumption rates of mussel gill mitochondria as a function of oxygen partial pressure. Three experiments (differentiated by symbols) of repeated (5–8) additions of 10–13 µmol l–1 sulfide, as shown in Fig. 6, demonstrate a conformity of oxygen (black symbols) and sulfide (red symbols) consumption rates as PO2 declines. The rate of oxygen consumption shows limitation at higher PO2 with an apparent P50 near 2 kPa whereas the apparent P50 for sulfide consumption is approximately 1 kPa; data fitted by Michaelis–Menten equation.

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