Site-directed mutagenesis reveals new and essential elements for iron-coordination of the sulfur oxygenase reductase from the acidothermophilic Acidianus tengchongensis

Previous study on refolding of sulfur oxygenase reductase (SOR) inclusion bodies from recombinant Escherichia coli showed that iron was critical to the activity of the SOR from Acidianus ambivalens. In this study, enzymatic assays showed that 2,2′-Dipyridyl, Tiron and 8-hydroxyquinoline, which are specific for chelating ferrous or ferric ions, strongly inhibited the activity of SOR from A. tengchongensis, suggesting that iron atom is essential for SOR activity. Alignment of several functionally identified SORs and SOR-like sequences from genome database revealed a conserved, putative iron binding motif, H⁸⁶-X₃-H⁹⁰-X _n -E¹¹⁴-X _n -E¹²⁹ (numbering according to the Acidianus tengchongensis SOR sequence). Three mutants of SOR were generated by site-directed mutagenesis of H₈₆, H₉₀ and E₁₂₉ into phenylalanine or alanine residue in this study. Circular dichroism spectrum determination indicated that there was no change of the secondary structures of mutant SORs, H⁸⁶F, H⁹⁰F and E¹²⁹A, but all mutants were completely inactive. Through determination of iron contents we found that SOR mutants of H⁸⁶F, H⁹⁰F and E¹²⁹A completely or partially lost iron, while mutants of C³¹S, C¹⁰¹S, and C¹⁰⁴S (generated in a previous study) did not. This result indicated that H⁸⁶, H⁹⁰ and E¹²⁹ but not C³¹, C¹⁰¹, and C¹⁰⁴ were involved in binding to iron atom. Based on this and previous studies, it is proposed that the conserved motifs, C³¹-X _n -C¹⁰¹-X₂-C¹⁰⁴ and H⁸⁶-X₃-H⁹⁰-X₂₃-E¹¹⁴-X₁₄-(E/D)¹²⁹, are respectively for sulfur and molecular oxygen binding and activation. These two conserved motifs are essential elements for the SOR activity. Supported by National Natural Science Foundation of China (Grant Nos. 30670018, 30621005) and State Key Basic Research and Development Program of China (Grant No. 2004CB719602)