Crystallographic and cryogenic electron microscopic structures and enzymatic characterization of sulfur oxygenase reductase from Sulfurisphaera tokodaii

Sulfur oxygenase reductases (SORs) are present in thermophilic and mesophilic archaea and bacteria, and catalyze oxygen-dependent oxygenation and disproportionation of elemental sulfur. SOR has a hollow, spherical homo-24-mer structure and reactions take place at active sites inside the chamber. The crystal structures of SORs from two Acidianus species have been reported. However, the states of the active site components (mononuclear iron and cysteines) and the entry and exit paths of the substrate and products are still in dispute. Here, we report the biochemical and structural characterizations of SORs from the thermoacidophilic archaeon Sulfurisphaera tokodaii (StSOR) and present high-resolution structures determined by X-ray crystallography and cryogenic electron microscopy (cryo-EM). The crystal structure of StSOR was determined at 1.73 Å resolution. At the catalytic center, iron is ligated to His86, His90, Glu114, and two water molecules. Three conserved cysteines in the cavity are located 9.5∼13 Å from the iron and were observed as free thiol forms. A mutational analysis indicated that the iron and one of the cysteines (Cys31) were essential for both activities and the other two cysteines (Cys101 and Cys104) had a supportive role. The cryo-EM structure was determined at 2.24 Å resolution using an instrument operating at 200 kV. The two structures determined by different methodologies showed similar main chain traces, but the maps exhibited different features at catalytically important components. Given the high resolution achieved in this study, StSOR was shown to be a good benchmark sample for cryo-EM measurements.HighlightsSulfur oxygenase reductase (SOR) was biochemically and structurally characterized.High resolution structures of SOR were determined by crystallography and cryo-EM.Twenty-four identical subunits of SOR form a hollow sphere.Catalytic components exhibited different features in the crystal and cryo-EM structures.

Insight into the Sulfur Metabolism by Thermoacidophilic Archaeon Metallosphaera cuprina with Genomic, Proteomic and Biochemical Tools

The thermoacidophilic archaeon Metallosphaeracuprina was isolated from a sulfuric hot spring. M. cuprina is able to oxidize elemental sulfur, tetrathionate (S4O62+) pyrite, and a range of low-grade ores, thus is attractive to biomining industry. Dissimilatory sulfur metabolism with a sulfur oxygenase reductase (SOR) system has been reported for members of Sulfolobus and Acidianus. But SOR system was not identified in the genome of M. cuprina. Recently, we have explored the sulfur metabolism of M. cuprina with genomic, proteomic, and biochemical tools. A hypothetical model of sulfur metabolism in M. cuprina was proposed on proteomic and genomic data, and proteins that involved in sulfur metabolism have been identified in our following studies. Specifically, DsrE/TusA homologs were biochemically characterized, and a novel thiosulfate transfer reaction was found during sulfur oxidation with M. cuprina. More recently, we cloned and identified a CoA-dependent NAD(P)H sulfur oxidoreductase from M.cuprina. The study will cover new understandings of the sulfur metabolism with M. cuprina.

Theoretical Model of the Structure and the Reaction Mechanisms of Sulfur Oxygenase Reductase in Acidithiobacillus thiooxidans

Sulfur oxygenase reductase (SOR), which is thought to be an important enzyme involved in sulfur oxidation in many microorganisms, may play a key role in sulfur oxidation in Acidithiobacillusthiooxidans. Draft genome sequence of A. thiooxidans A01 indicated the presence of sulfur oxygenase reductase gene (sor). The complementary DNA fragment was speculated to encode a putative 311-aa full-length protein SOR. Structural analysis of SOR revealed that three cysteines located in the two conserved domains, C32 at V-G-P-K-V-C32 as well as C102 and C105 at C102-X-X-C105, might form the substrate activation and binding site. It was proposed that conserved motif H87-X3-H91-X23-E115 acted as ligands might combine with iron atom to constitute a mononuclear non-heme iron center, catalyzing the oxidation reaction of substrate.