Oxidation of Sulfur and Inorganic Sulfur Compounds in the Thermoacidophilic Archaeon Acidianus ambivalens²
Zusammenfassung der Projektergebnisse
The microbial oxidation of sulfur and inorganic sulfur compounds (RISCs) to sulfuric acid is an important reaction in the global sulfur cycle. It provides the energy for growth of those archaea and bacteria that can fix atmospheric CO2 without light. Sulfur oxidation also plays a role in the formation of acidic drainage from mines and slag heaps, which leads to environmental problems and which is also used for biological recovery of precious metals from slag heaps and low-grade ores. Our model organism, the archaeon Acidianus ambivalens grows by oxidation of sulfur and RISCs at 80˚C and pH 1-3.5 producing copious amounts of sulfuric acid. Our goal is to understand the biochemistry behind the sulfur oxidation in Acidianus and the mechanisms how the enzymes work. We had purified several of the sulfur and RISC-oxidizing enzymes and had established the role of A. ambivalens as a model organism for sulfur oxidation in archaea. One of the most interesting and most important proteins is the sulfur oxygenase reductase (SOR), one of the few proteins that can actually metabolize the almost water-insoluble elemental sulfur (≈120 µg/l at 80˚C). The SOR catalyzes a sulfur disproportionation reaction depending on the presence of dioxygen with sulfite, thiosulfate and H2S as products. We had solved the 3D structure of the A. ambivalens SOR, which showed that the iron-containing protein forms a large, hollow, ball-shaped complex consisting of 24 identical subunits. We also found that one out of three conserved cysteine residues is essential for catalysis, and we had inactivated the enzyme by mutagenesis of the Fe-coordinating residues. Our main focus for this project is the reaction mechanism of the SOR, meaning to resolve how the enzyme works. We constructed mutants affecting active site residues. Most of them diminished specific activities and only a few had more drastic effects (mutants of the amino acids Glu87, Trp80 and Gln76): They diminishing the activity of the enzyme to <15 % by disrupting hydrogen bonds between active site residues. We also exchanged the iron in the active site by other (transition) metals. Activity was obtained not only with FeII, but also with CoII (even with higher reductase activites than FeII), NiII, MnII, and GaIII. The results suggested that a valence change is not necessarily required for catalysis. We also showed that mercury and iodoacetamide inhibit enzyme activity by binding to active site cysteines, whereas zinc binds far from the active site and probably inhibits the enzyme by restricting protein flexibility. In order to reach the 24 separate active sites of the SOR, substrate and products must first pass the outer shell of the enzyme and then another pore into the active site pocket. Two types of pores provide entrance/exit to the interior hollow cavity, six each at the fourfold symmetry axes and six each at the threefold symmetry axes. We used mutagenesis to open the pores and to change the properties of the amino acids involved. It showed that opening of the pores increases the activity of the protein up to sevenfold, while the stoichiometry of the reaction products changes towards more reduced products. We mutagenized the active site pore getting a decrease in enzyme activity showing that its integrity is important. SORs were mostly found in thermophilic microorganisms, both archaea and bacteria. Recently, sor genes were also identified in mesophilic bacteria. Since the solubility of sulfur is low in water (5 µg/l at 25˚C), we also chose one of the low-temperature SORs from the bacterium Halothiobacillus neapolitanus for study (optimal growth temperature ≈30˚C). The enzyme proved to be highly thermostable (temperature optimum 80˚C) in spite of the low growth temperature. It had an up to 10-fold higher specific activity compared to the A. ambivalens SOR and a very broad range of activity (10-99˚C). X-ray crystallography showed the same ballshaped structure as the A. ambivalens enzyme with several modifications in the pore and in the active site, which could contribute to the activity at low temperature. The reaction mechanism of the SOR remains enigmatic. It seems to be a combination of polysulfide hydrolysis (for H2S production) by Lewis acid chemistry, hydroxyl-catalyzed sulfur disproportionation and metal- or substrate-catalyzed dioxygen activation.
