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

Abstract

ABSTRACT Sequence comparisons showed that the sulfur oxygenase reductase (SOR) of the haloalkaliphilic bacterium Thioalkalivibrio paradoxus Arh 1 ( Tp SOR) is branching deeply within dendrograms of these proteins (29 to 34% identity). A synthetic gene encoding Tp SOR 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. Tp SOR 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 Tp SOR 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 Cys 44 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.