Chinese Journal of Oceanology and Limnology Vol. 28 No. 3, P. 521-526, 2010 DOI: 10.1007/s00343-010-9104-4

Sulfide-based ATP production in Urechis unicinctus*

MA Zhuojun (马卓君)†,††, BAO Zhenmin (包振民) †, WANG Sifeng (王思锋) †,†††, ZHANG Zhifeng (张志峰) †,** † Key Laboratory of Marine Genetics and Breeding of Ministry of Education, Ocean University of China, Qingdao 266003, China †† The Centre for Applied Aquatic Genomics, Chinese Academy of Fishery Sciences, Beijing 100141, China ††† Biology Institute of Shandong Academy of Sciences, Jinan 250014, China

Received June 17, 2009; revision accepted Aug. 25, 2009

© Chinese Society for Oceanology and Limnology, Science Press, and Springer-Verlag Berlin Heidelberg 2010

Abstract We measured sulfide-based ATP production by isolated mitochondria from four tissues of Urechis unicinctus and the effects of inhibitors of respiratory complexes on ATP production were evaluated. The results show that these mitochondria could oxidize sulfide to produce ATP. The yield of sulfide-stimulated ATP varied from 5 nmol ATP/min/mg to 90 nmol ATP/min/mg according to the sulfide concentration and the source of the mitochondria. The maximum ATP synthesis occurred in hindgut mitochondria using 5 μmol/L sulfide as a substrate. The effects of inhibitors (Rotenone, Antimycin A, Cyanide, and Salicylhydroxamic acid) on mitochondrial ATP production varied with the source of the mitochondria. Our results indicate that sulfide-based ATP production and the associated electron transport pathway are tissue-specific in U. unicinctus.

Keyword: Urechis unicinctus; sulfide oxidation; mitochondria; ATP production; inhibitor

1 INTRODUCTION

Sulfide, which is commonly viewed as a toxic substance, exists in three forms in natural waters (H2S, HS-, S2-) and is distributed extensively in deep sea hydrothermal vents, cold seeps, muddy sediment, and marshes (Childress, 1995; Somero et al., 1989). The harmful effects of sulfide are mainly due to its reversible inhibition of cytochrome c oxidase in the mitochondrial electron transport chain and respiration of aerobic animals. It has been reported that the inhibition by sulfide of cytochrome c oxidase is even higher than that of cyanide; cytochrome c oxidase may be inhibited by up to 50% in the presence of 1 μmol/L sulfide (Nicholls, 1975).

Intertidal muddy coasts present sulfide-rich habitats for burrowing organisms. At low tide, in the burrows of muddy coasts, the CO2 concentration increases remarkably while the O2 content of declines to 05 mmHg (1 mmHg=0.133 kPa) because of respiration, redox, and shortage of fresh seawater (Nates et al., 1998; Felder, 1979). Extreme hypoxia or anoxia might lead to accumulation of reduced compounds, such as sulfide and ammonia, in burrow waters and sediment. During that time, burrowing animals might be exposed to oxygen deficiency and

high concentrations of sulfide for several hours. Many of these burrowing organisms were found to be able to tolerate sulfide, and the mechanism of their sulfide tolerance has been investigated (Fenchel et al., 1970; Atkinson et al., 1988; Arp et al., 1995; Völkel et al., 1995; Nates et al., 1998).

Among these organisms, the sulfide toleration mechanism of the marine echiuran (Urechis caupo) has been extensively studied. These studies involved physiology, biochemistry, and molecular biology, by which U. caupo became a model organism for research on sulfide-tolerating organism (Powell et al., 1989; Arp et al., 1992, 1995; Menon et al., 1998; Julian et al., 1999; Boore, 2004).

U. unicinctus is mainly found in China, Korea, Japan, and Russia and inhabits a U-shaped burrow in the intertidal and subtidal sediment. Studies on U. unicinctus have involved many fields, such as taxology, chorology, morphology, anatomy, reproductive biology, and nutriology (Li et al., 1994; Li et al., 1998; Yang et al., 1999). Recently, due to the high sulfide concentration in its habitat, many

Supported by the National Natural Science Foundation of China (No.

40776074) Corresponding author: zzfp107@ouc.edu.cn

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studies of the sulfide tolerance of U. unicinctus have been published (Shao et al., 2003; Ma et al., 2005; Zhang et al., 2006; Huo et al., 2007; Zhang et al., 2008).

Research has shown that the mitochondria of several kinds of invertebrates can use sulfide as substrate to produce ATP (Powell et al., 1986a; Völkel et al., 1997; Parrino et al., 2000; Bourgeois et al., 2001). In the present study, sulfide-based ATP production of isolated mitochondria from four tissues of U. unicinctus was measured and the effects of inhibitors of respiratory complexes on ATP production were determined to investigate sulfide oxidation and electron transport in U. unicinctus.

2 MATERIALS AND METHODS

2.1 Animals

Experimental animals were collected from Yantai, China. Healthy worms ranging from 15 to 20 cm in length were selected and maintained in filtered seawater for a week. The temperature was 20–25°C and the stocking density was five or six worms per square meter. The seawater was aerated and changed once a day. Experimental worms were not fed during the experiment.

2.2 Reagents and stock solutions

Rotenone, Antimycin A, Salicylhydroxamic acid (SHAM), glucose-6-phophate dehydrogenase (GPD), hexokinase, and Bovine Serum Albumin (BSA fat free) were purchased from Sigma. Stock solutions of rotenone (0.3 mmol/L) and salicylhydroxamic acid (100 mmol/L) were dissolved in dimethylsulphoxide (DMSO). Antimycin A (10 mmol/L) was dissolved in ethanol. KCN (100 mmol/L, Merck) was dissolved in buffer solution (pH 7.5) for mitochondrial ATP production.

2.3 Isolation of mitochondria

The body wall, hindgut, midgut, and anal sac were sampled from the experimental worms and mitochondria from the above tissues were isolated according to the method of Parrino et al. (2000) with slight modifications, in isolation buffer (58.4 mmol/L sucrose, 140.2 mmol/L glycine, 40 mmol/L Tris, 2 mmol/L EGTA, 0.2% BSA, pH7.5). The mitochondrial content was assayed as protein according to Bradford (1976).

2.4 Measurement of ATP production

Mitochondrial ATP production was measured by the procedure of Powell et al. (1986b). A coupled enzyme assay (see schematic below) was used to

convert ATP to NADPH. The production of NADPH was monitored at 340 nm on a SHIMADIU UV-730 micro-flow spectrophotometer.

The buffer solution for this reaction contained 58.4 mmol/L sucrose, 140.2 mmol/L glycine, 40 mmol/L Tris, 2 mmol/L EGTA, 0.2%BSA (fat free), 0.1 mol/L KCl, 10 mmol/L K2HPO4, 3 mmol/L MgCl2, 10 mmol/L glucose, hexokinase 7 U/ml, GPD 2 U/ml, and corresponding substrates, 0.5 mmol/L NADH, 0.1 mmol/L ADP, pH7.5.

The isolated mitochondria were added to the buffer solution and the reaction was initiated by adding succinate, malate, or sulfide, respectively. The activity of the mitochondria was confirmed by measurement of ATP production with succinate, and only the active mitochondria were used. Boiled mitochondria were used as a negative control.

Sulfide, at concentrations ranging from 5 to 100 μmol/L, was used as a substrate in the absence of succinate or malate to analyze the effect of sulfide on mitochondrial ATP production. Sulfide stock solutions (20 mmol/L) were diluted to obtain serial concentrations of sulfide, which were prepared daily by adding washed crystals of Na2S·9H2O to nitrogen-saturated distilled water. For inhibitor studies, the respective inhibitor stock solution was added to the buffer solution and then substrate (succinate, malate or sulfide) was added to start the reaction.

2.5 Statistical analysis

The statistical package SPSS11.5 was used for data analysis. Means and 95% confidence intervals (95% CI) were calculated for parallel treatments. Comparisons between means of different treatments were made using a two-sample t-test.

3 RESULTS

3.1 ATP production with sulfide as substrate

The mitochondria of U. unicinctus could synthesize ATP with sulfide as the only substrate (Fig.1). However, the ATP yields differed depending on the tissue source and sulfide concentration. The maximal ATP yield of mitochondria from body wall and hindgut was reached at a sulfide concentration of 5 μmol/L and their unit ATP synthesis was similar. Moreover, these tissue’s mitochondrial ATP

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synthesis rates in different sulfide concentrations showed similar changes, i.e. decreasing gradually as the sulfide concentration increased after reaching maximum (Fig.1a). However, a significant decrease of ATP synthesis rate was observed at sulfide concentrations higher than 10 μmol/L and again at 35 μmol/L in body wall mitochondria, whereas a significant decrease did not take place until concentrations above 50 μmol/L sulfide was reached for hindgut mitochondria. The ATP synthesis rate of mitochondria from the midgut reached its maximum at 5–10 μmol/L sulfide, but only reached half of that in mitochondria from body wall or hindgut, and then decreased significantly to almost zero at 100 μmol/L sulfide (Fig.1b). For crissal bursa mitochondria, the ATP synthesis rate increased gradually with sulfide concentration until a maximum at 20 μmol/L sulfide was reached before decreasing gradually.

Fig.1 ATP synthesis rate of mitochondria from four tissues with sulfide as a substrate

Note: n=8

▲ Significantly different from the maximum rate of ATP synthesis with

sulfide as substrate in the same tissue (two sample t-test, P<0.05)

* No ATP synthesis was detected in the negative control

3.2 Effects of inhibitors on mitochondrial electron transport chain

Mitochondrial ATP synthesis rates in the four tissues were different when using the same substrate, including succinate, malate, or sulfide, and were highest in the hindgut and lowest in crissal bursa (Table 1). For the same mitochondria, the ATP synthesis rate was highest when using succinate as substrate and lowest when using sulfide.

To investigate effects of different inhibitors, the mitochondrial ATP synthesis rate was measured in

the presence of rotenone, antimycin A, cyanide, and SHAM. When rotenone was used as an inhibitor and malate as a substrate, the ATP synthesis rate in all tissues was inhibited by more than 70%. With succinate as the substrate, there was no significant inhibition. With sulfide as substrate, rotenone only inhibited ATP synthesis of midgut mitochondria. With succinate or malate as a substrate, antimycin A and cyanide inhibited mitochondrial ATP production by more than 50% in all tissues. By contrast, inhibition by antimycin A on mitochondria from the hindgut was not observed with sulfide as the substrate. As a specific inhibitor of alternative oxidation enzymes, SHAM showed no significant inhibition of ATP synthesis with succinic acid or malate as the substrate. With sulfide as the substrate, mitochondrial ATP synthesis in body wall and crissal bursa were inhibited by more than 55% by SHAM. However, this inhibition was not observed in mitochondria isolated from the hindgut and midgut.

4 DISCUSSION

4.1 Mitochondrial oxidation of sulfide in U. unicinctus tissues

Some animals are able to oxidize sulfide to less toxic or nontoxic sulfur compounds, such as thiosulfate, sulfite, sulfate, or elementary sulfur. When sulfide is oxidized to sulfate a Gibbs free energy (ΔG) of -796.6 kJ/mol plus 1 proton is produced and when oxidized to thiosulfate a ΔG =-818.4 kJ/mol plus two protons is produced. The energy is enough to combine ADP and Pi to make ATP (ΔG=-30.5 kJ/mol). In addition, protons released from the oxidation reaction can be used to produce a proton gradient to make more ATP. The sulfide-based ATP synthesis has been found in several marine animals, such as Solemya reidi, Arenicola marina, and Geukensia demissa (Powell et al., 1986b; Völkel et al., 1997; Parrino et al., 2000). In this study, we showed that ATP could also be produced using sulfide as the sole substrate in the mitochondria of U. unicinctus but that ATP synthesis rates vary in different tissues.

Like U. caupo, U. unicinctus inhabits U-shaped burrows and pumps seawater for O2 by peristaltic contraction and relaxation of its body wall (Julian et al, 1996, Li et al., 1998). Its hindgut is a water lung and is responsible to O2 uptake (Menon et al. 1992). Therefore, when sulfide is present in the water, the body wall and hindgut might be directly exposed to it. Thus, we could hypothesize that the mitochondria of body wall and hindgut would have greater ability to

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Table 1 Effects of inhibitors on mitochondrial ATP synthesis rate of four U. unicinctus tissues mitochondria (n=8)

ATP synthesis rate Inhibition rate of ATP synthesis (%)

Mitochondria Source

Substrate (nmol/ (min g)) Rotenone

(1.5 μmol/L) Antimycin A (100 μmol/L)

Cyanide (100 μmol/L)

SHAM (100 μmol/L)

Body wall 2.5 mmol/L succinate 111.76±49.42 21.95±14.99* 81.76±15.66 69.68±23.88 38.85±21.48*

2.5 mmol/L malate 89.92±43.35 71.83±26.10 93.94±6.28 60.71±23.35 17.15±17.08*

10 μmol/L sulfide 79.25±36.09 22.93±19.71* 53.19±11.56 72.30±23.73 56.85±25.79

Hindgut 2.5 mmol/L succinate 123.90±29.01 11.93±7.53* 52.72±22.90 76.26±18.65 21.16±13.11*

2.5 mmol/L malate 111.44±25.15 76.52±17.15 62.69±34.58 81.60±15.83 5.32±4.75*

10 μmol/L sulfide 86.16±20.00 25.38±20.59* 31.98±28.08* 51.41±14.65 26.12±16.02*

Midgut 2.5 mmol/L succinate 61.70±28.08 35.88±21.58* 95.31±4.77 84.81±10.85 25.08±17.21*

2.5 mmol/L malate 51.67±28.50 84.05±15.14 98.63±1.17 80.22±18.78 15.93±12.88*

10 μmol/L sulfide 34.32±19.57 84.00±15.81 77.74±21.02 100.00±0.00 24.20±18.99*

Cressal bursa 2.5 mmol/L succinate 39.74±11.33 26.36±13.33* 86.26±12.69 65.00±34.37 23.65±15.80*

2.5 mmol/L malate 33.87±16.03 72.02±24.02 100.00±0.00 61.88±37.54 21.75±20.88*

10 μmol/L sulfide 31.11±18.09 29.10±20.06* 64.24±34.92 72.75±26.68 69.09±30.83

Note: * No significant difference between in the presence or absence of the inhibitor when using the same substrate (two sample t-test, P>0.05). Values are means

± S.D. ATP synthesis rate of negative control was 0

oxidize sulfide for respiratory metabolism and to synthesize ATP at low concentrations of sulfide. In contrast, the midgut is a digestive organ with little or no opportunity to come into contact with high concentration sulfide. When 5 µmol/L sulfide was used as the substrate, the ATP synthesis rates of the body wall and hindgut mitochondria were much higher than that of the midgut mitochondria. This phenomenon indicated that sulfide tolerance might be related to the differential abilities of the mitochondria from different tissues to oxidize sulfide for ATP synthesis.

The crissal bursa is a special organ in U. unicinctus of unclear function. It was suspected to be an excretion organ functioning as a physiological filter (Harris et al., 1981). In our experiments, even at sulfide concentrations up to 100 mmol/L, the ATP synthesis rate of crissal bursa was significant. Considering that the crissal bursa is located near the spiracle, this phenomenon indicates that the crissal bursa cells, as well as their mitochondria might also be in contact with sulfide.

Another interesting phenomenon is that with increasing sulfide concentration, the decrease of ATP synthesis rates in body wall mitochondria was more significant than that of the hindgut. This phenomenon might be explained by the different structures of those two organs. There are thick mucus layers and horny layers outside the body wall that are able to prevent or delay the invasion of sulfide. In contrast, the hindgut wall is thin and lacks these

protective structures. Therefore, the hindgut mitochondria might encounter higher concentrations of sulfide and be more tolerant to sulfide compared to the body wall.

4.2 The mechanism of sulfide oxidation in U. unicinctus mitochondria

The alternative oxidase has been found in the mitochondria of plants, as well as in some fungi and protists (McDonald et al., 2004). It catalyzes a cyanide-resistant reduction of oxygen to water without translocation of protons across the inner mitochondrial membrane (Berthold et al., 2000). Studies on mitochondrial sulfide oxidation in certain animals indicate that the alternative oxidase might act as a secondary pathway of electron transport responding to cyanide, high sulfide, or other extreme conditions (Völkel et al., 1997; Parrino et al., 2000; Hahlbeck et al, 2000; Kraus et al., 2004).

In the lugworm (Arenicola marina) research on the mechanism of mitochondrial sulfide oxidation and sulfide-based ATP synthesis have been rigorously pursued by Prof. Grieshaber’s group, and a model for the electron transport pathway of sulfide-based ATP synthesis has been proposed (Völkel et al., 1996, 1997). They showed that sulfide is oxidized by sulfide oxidase, and electrons from sulfide oxidation enter the mitochondrial electron transport chain at the level of the ubiquinone cycle or directly via complex III (Fig.2). At low sulfide concentrations, these electrons are mainly transferred to oxygen by

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Fig.2 Proposed model of electron transport during sulfide oxidation in Arenicola marina (from Völkel et al., 1997)

complex IV (cytochrome c oxidase). At high sulfide concentrations complex IV is inhibited and electrons from sulfide are transferred to oxygen by an alternative oxidase that is sulfide insensitive.

Our results showed that the sensitivity of mitochondria isolated from the body wall and the crissal bursa to inhibitors was similar. ATP synthesis was inhibited by antimycin A and cyanide, but not rotenone. This indicated that the electrons produced by sulfide oxidation mainly entered the electron transport chain after complex I. ATP production with sulfide as the substrate was inhibited by more than 55% after the addition of SHAM, the inhibitor of the alternative oxidase (Parrino et al., 2000). This indicated that the alternative oxidase was involved the mitochondrial electron transport pathway in these two tissues and contributed to the tolerance to sulfide.

Sulfide-based ATP production in midgut mitochondria was strongly inhibited by rotenone, antimycin A, and cyanide, indicating that electrons from sulfide enter the electron transport chain via Complex I. In addition, the midgut mitochondria were insensitive to SHAM, which means it may lack the alternative oxidase.

However, sulfide oxidation in hindgut mitochondria was insensitive to all inhibitors; even cyanide had an inhibition rate of only about 50%. Recently, in the lugworm (A. Marina), besides the sulfide-based ATP synthesis pathway, a redox detoxifying sulfide mechanism was revealed (Völkel et al., 1996, 1997; Hidebrandt et al., 2008). This specific redox mechanism, which oxidizes sulfide but does not produce any ATP, is believed to contribute to protecting mitochondrial sulfide oxidation from the inhibition by SHAM and high concentrations of sulfide (Hildebrandt et al., 2008). The low sensitivity of the hindgut mitochondria to inhibitors, especially to SHAM, indicates that a

similar protection mechanism might exist in the hindgut mitochondria of U. unicinctus. This mechanism might help the worm to maintain sulfide oxidation and ATP production to provide energy to perform physiological functions during low tide sulfide exposure. Under the electron microscope, many mitochondria were observed in the cells of the hindgut (Shao et al, 2003). Numerous mitochondria able to oxidize, and be tolerant to, sulfide might represent the first line of defense against sulfide.

In conclusion, ATP production based on sulfide oxidation occurs in the mitochondria of several tissues of U. unicinctus. The ATP production rates and electron transport pathways are significantly different in the different tissues when sulfide is used as the sole substrate. However, which genes contribute to the specificity of these mitochondria? Does the redox mechanism exist in the hindgut mitochondria of U. unicinctus as in A. marina? These interesting questions are currently under investigation.

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