Background

Sulfur oxygenase reductases (SOR) catalyze a dioxygen-dependent disproportionation of elemental sulfur with sulfite, thiosulfate and hydrogen sulfide as detectable products. The initially found SORs were derived from hyperthermophilic, sulfur-oxidizing Archaea and Bacteria. The reported temperature optima were between 65 °C and 85 °C and the pH optima between pH 5 and pH 7.4 (Emmel et al., 1986; Kletzin, 1989; Sun et al., 2003; Pelletier et al., 2008). Surprisingly, SORs derived from the mesophilic bacterium Halothiobacillus neapolitanus (Veith et al., 2012) and the alkalihalophilic Thioalkalivibrio paradoxus (Rühl et al., 2017) also had temperature optima of around 80 °C at pH 8.4 and pH 9.0, respectively. Most SORs can be produced in E. coli by heterologous gene expression and only the first descriptions of the enzyme were derived from proteins purified from their native sources (Emmel et al., 1986; Kletzin, 1989; Pelletier et al., 2008). Today, sor genes are found in approximately 35 different Bacteria and Archaea (Rühl et al., 2017).

Usually, SORs have a stoichiometry between 4:1 and 10:1 of oxidized (thiosulfate and sulfite) and reduced products (hydrogen sulfide). The oxidation and reduction reactions could not be separated by site-directed mutagenesis, although the product stoichiometries may vary (Veith et al., 2012). So far, the only exception is the Thioalkalivibrio paradoxus SOR with stoichiometries of 100-1,000:1, which makes the enzyme an oxygenase with almost no reductase activity (Rühl et al., 2017). The thiosulfate:sulfite ratio increases with temperature and pH (Kletzin, 1989; Veith et al., 2012; Rühl et al., 2017). Therefore, the bulk of the thiosulfate is most likely formed rapidly by a non-enzymatic reaction between sulfur and sulfite at pH values above 6 and temperatures exceeding 70 °C.

The original SOR activity assay (Kletzin, 1989; Urich et al., 2004) involves shaking of an aliquot of the enzyme in a buffer containing elemental sulfur at the given reaction temperature coupled to the colorimetric determination of the amount of products at different time points. Product determination is also possible using HPLC (e.g., Rethmeier et al., 1997). However the number of samples that can be processed per day is higher using the colorimetric assays because of the longer time required for each HPLC run. Specific SOR activities were in the range of 10 U/mg of protein for the Acidianus ambivalens SOR and 40 U/mg for the Halothiobacillus enzyme, both determined with the original enzyme assay.

After developing several modifications, the activity assay became more sensitive, resulting in higher product formation and lower amount of the required enzyme together with a reduced incubation time of the enzyme assay (Table 1). The original assay (Kletzin, 1989) was performed in stoppered glass vials with the enzyme being added at room temperature prior to incubation. All vials were transferred simultaneously to a shaking bath with preheated heating liquid. In order to stop the reaction, the vials were transferred sequentially into an ice bath. Urich et al. (2004) modified the procedure for the use of 1.5 ml plastic reaction vials and thermomixers but kept the order of the steps. In the modified procedure, the enzyme solution is added last to the 0 min time point immediately before transfer of the entire set of vials to an ice bath. Thus, all vials remain at the assay temperature for exactly the same time minimizing background effects. Here we describe the modified SOR activity assay and the quantification of its three reaction products.

Table 1. Incubation conditions for the original and modified SOR enzyme assays