Free radicals and chemiluminescence as products of the spontaneous oxidation of sulfide in seawater, and their biological implications

Abstract

The discovery of symbioses between marine invertebrates and sulfide-oxidizing bacteria at deep-sea hydrothermal vents and in other high-sulfide marine environments has stimulated research into the adaptations of metazoans to potentially toxic concentrations of sulfide. Most of these studies have focused on a particular action of sulfide--its disruption of aerobic metabolism by the inhibition of mitochondrial respiration--and on the adaptations of sulfide-tolerant animals to avoid this toxic effect (1). We propose that sulfidic environments impose another, hitherto over-looked type of toxicity: exposure to free radicals of oxygen, which may be produced during the spontaneous oxidation of sulfide, thus imposing an oxidative stress. Here we present evidence that oxygen- and sulfur-centered free radicals are produced during the oxidation of sulfide in seawater, and we propose a reaction pathway for sulfide oxidation that is consistent with our observations. We also show that chemiluminescence at visible wavelengths occurs during sulfide oxidation, providing a possible mechanism for the unexplained light emission from hydrothermal vents (2, 3).

Figure 1

Electron paramagnetic resonance (EPR) spectra of DMPO adducts formed during sulfide oxidation in air-saturated artificial seawater (ASW) (27) at pH 7.4 and room temperature (≈20°C). (A) Control spectrum of DMPO in ASW with no sulfide added. (B) Spectrum obtained when sulfide (1 mM) is added. (C) Computer simulation of the spectrum in B, a composite of DMPO/HO^• (30%) and DMPO/^•SO₃⁻ (70%). (D) Control spectrum of DMPO and dimethyl sulfoxide (DMSO) in ASW with no sulfide added. (E) Spectrum obtained when 1 mM sulfide is added to DMPO and DMSO in ASW. Computer simulation of this spectrum indicates these relative abundances: DMPO/HO^• (20%), DMPO/^•SO₃⁻ (60%), and DMPO/^•CH₃ (20%). Horizontal scale = 10 gauss; since in this type of spectrometry the relative positions of the lines are more important than their absolute positions, spectra do not usually include an absolute scale. The vertical axis is in arbitrary units. The vertical ticks in (E) mark the positions of DMPO/^•CH₃ peaks. Reaction components, when present, were in the following final concentrations: DMPO, 50 mM; sulfide, 1 mM; DMSO, 0.7 M. Sulfide was added after all the other reactants, and immediately before transferring the sample to the EPR cell. Spectra were obtained with a Bruker ESP 300 EPR spectrometer equipped with a TM₁₁₀ cavity and an aqueous flat cell. Computer simulations of spectra were carried out using SIMEPR software (28).

Figure 2

Time course of the disappearance of sulfide from ASW (pH 7.4) continuously sparged with air or nitrogen gas. Sulfide rapidly disappeared from solution in the aerated treatment (■). When DTPA was present, the loss of sulfide from aerated solution (▲) was reduced to control rates (●: nitrogen-sparged; ◆: nitrogen-sparged, DTPA present). In the anoxic control experiments, as well as those with DTPA, the loss of sulfide was owing to the degassing of H₂S by the nitrogen or air stream, not to the chemical oxidation of sulfide. In experiments where the pH was more acidic (and the proportion of sulfide as H₂S was therefore greater), the loss of sulfide under otherwise identical conditions was greatly increased (data not shown). ASW (50 ml) was placed in a glass Erlenmeyer flask and sparged with air or nitrogen for 1 h before the experiments were begun and continuously thereafter. The experiments were started by addition of sufficient sulfide stock, pH 7.4, to result in an initial sulfide concentration of 500 μM. Sulfide concentration was determined at 10-min intervals using the diamine method (29).

Figure 3

Time course of chemiluminescence, measured as counts per second (cps), during sulfide oxidation at various initial sulfide concentrations, with and without chelators. Each point is the average of three determinations; error bars are ± one standard deviation. The amount of light emitted was positively correlated with initial sulfide concentration (□: 0 μM; ◆: 200 μM; ▲: 500 μM). Luminescence was reduced substantially, and there was a 30 min induction period, when the chelator EDTA was included in 500 μM sulfide (▼). No chemiluminescence was observed in the presence of DTPA and 500 μM sulfide (+) or with 500 μM sulfide under anoxic conditions (data not shown). Reaction mixtures were prepared in ASW adjusted to pH 7.4 after the addition of chelators, when they were used. Chemiluminescence was measured in a Packard Tri-Carb model 3255 liquid scintillation counter set on the tritium channel in the out-of-coincidence mode (30). The photomultiplier in this counter is sensitive to wavelengths between 380 and 620 nm. Background counts from the empty chamber were 172 ± 4 cps (mean ± SD) and of an empty vial were 186 ± 11 cps. For each determination 20 ml of aerated ASW were placed into a vial, and a sufficient amount of sulfide stock was added to obtain the desired concentration. Counts were accumulated over 10-min intervals; for the experiments employing 500 μM sulfide, counts were accumulated over 1-min intervals for the first 10 min.