Persulfide Dioxygenase From Acidithiobacillus caldus: Variable Roles of Cysteine Residues and Hydrogen Bond Networks of the Active Site.

Persulfide dioxygenases (PDOs) are abundant in Bacteria and also crucial for H2S detoxification in mitochondria. One of the two pdo-genes of the acidophilic bacterium Acidithiobacillus caldus was expressed in Escherichia coli. The protein (AcPDO) had 0.77 ± 0.1 Fe/subunit and an average specific sulfite formation activity of 111.5 U/mg protein (Vmax) at 40°C and pH 7.5 with sulfur and GSH following Michaelis-Menten kinetics. KM for GSH and Kcat were 0.5 mM and 181 s-1, respectively. Glutathione persulfide (GSSH) as substrate gave a sigmoidal curve with a Vmax of 122.3 U/mg protein, a Kcat of 198 s-1 and a Hill coefficient of 2.3 ± 0.22 suggesting positive cooperativity. Gel permeation chromatography and non-denaturing gels showed mostly tetramers. The temperature optimum was 40-45°C, the melting point 63 ± 1.3°C in thermal unfolding experiments, whereas low activity was measurable up to 95°C. Site-directed mutagenesis showed that residues located in the predicted GSH/GSSH binding site and in the central hydrogen bond networks including the iron ligands are essential for activity. Among these, the R139A, D141A, and H171A variants were inactive concomitant to a decrease of their melting points by 3-8 K. Other variants were inactivated without significant melting point change. Two out of five cysteines are likewise essential, both of which lie presumably in close proximity at the surface of the protein (C87 and C224). MalPEG labeling experiments suggests that they form a disulfide bridge. The reducing agent Tris(2-carboxyethyl)phosphine was inhibitory besides N-ethylmaleimide and iodoacetamide suggesting an involvement of cysteines and the disulfide in catalysis and/or protein stabilization. Mass spectrometry revealed modification of C87, C137, and C224 by 305 mass units equivalent to GSH after incubation with GSSH and with GSH in case of the C87A and C224A variants. The results of this study suggest that disulfide formation between the two essential surface-exposed cysteines and Cys-S-glutathionylation serve as a protective mechanism against uncontrolled thiol oxidation and the associated loss of enzyme activity.

The Sulfur Oxygenase Reductase Activity Assay: Catalyzing a Reaction with Elemental Sulfur as Substrate at High Temperatures.

The sulfur oxygenase reductase (SOR) reaction is a dioxygen-dependent disproportionation of elemental sulfur (S0), catalyzed at optimal temperatures between 65 °C and 85 °C. Thiosulfate and sulfite are formed as oxidized products as well hydrogen sulfide as reduced product. External co-factors are not required. Usually, the SOR assay is performed in a milliliter scale in S0-containing Tris-buffer at high temperatures followed by colorimetric product quantification. In order to make the SOR assay more sensitive and better reproducible, several modifications were implemented compared to the original SOR assay (Kletzin, 1989). Here we present the modified SOR assay and the following quantification of the reaction products.

A Sulfur Oxygenase from the Haloalkaliphilic Bacterium Thioalkalivibrio paradoxus with Atypically Low Reductase Activity.

Sequence comparisons showed that the sulfur oxygenase reductase (SOR) of the haloalkaliphilic bacterium Thioalkalivibrio paradoxus Arh 1 (TpSOR) is branching deeply within dendrograms of these proteins (29 to 34% identity). A synthetic gene encoding TpSOR expressed in Escherichia coli resulted in a protein 14.7 ± 0.9 nm in diameter and an apparent molecular mass of 556 kDa. Sulfite and thiosulfate were formed from elemental sulfur in a temperature range of 10 to 98°C (optimum temperature ≈ 80°C) and a pH range of 6 to 11.5 (optimum pH ≈ 9; 308 ± 78 U/mg of protein). Sulfide formation had a maximum specific activity of 0.03 U/mg, or <1% of the corresponding activity of other SORs. Hence, reductase activity seems not to be an integral part of the reaction mechanism. TpSOR was most active at NaCl or glycine betaine concentrations of 0 to 1 M, although 0.2% of the maximal activity was detected even at 5 M NaCl and 4 M betaine. The melting point of TpSOR was close to 80°C, when monitored by circular dichroism spectroscopy or differential scanning fluorimetry; however, the denaturation kinetics were slow: 55% of the residual activity remained after 25 min of incubation at 80°C. Site-directed mutagenesis showed that the active-site residue Cys44 is essential for activity, whereas alanine mutants of the two other conserved cysteines retained about 0.5% residual activity. A model of the sulfur metabolism in T. paradoxus is discussed. IMPORTANCE: Sulfur oxygenase reductases (SORs) are the only enzymes catalyzing an oxygen-dependent disproportionation of elemental sulfur and/or polysulfides to sulfite, thiosulfate, and hydrogen sulfide. SORs are known from mesophilic and extremophilic archaea and bacteria. All SORs seem to form highly thermostable 24-subunit hollow spheres. They carry a low-potential mononuclear nonheme iron in the active site and an indispensable cysteine; however, their exact reaction mechanisms are unknown. Typically, the reductase activity of SORs is in the range of 5 to 50% of the oxygenase activity, but mutagenesis studies had so far failed to identify residues crucial for the reductase reaction. We describe here the first SOR, which is almost devoid of the reductase reaction and which comes from a haloalkaliphilic bacterium.