The sulfur oxygenase reductase fromAcidianus ambivalens

Abstract

The microbial oxidation of reduced inorganic sulfur compounds and elemental sulfur to sulfate is one of the major reactions in the global sulfur cycle. Despite its importance, only limited information is available about molecular details of the enzymes involved. The present work was aimed to contribute to the understanding of the underlying molecular mechanisms by investigating the function and structure of the sulfur oxygenase reductase (SOR) from the thermoacidophilic crenarchaeote Acidianus ambivalens. The expression of the sor gene in Escherichia coli resulted in active, soluble SOR and in inclusion bodies from which active SOR could be refolded as long as ferrous ions were present in the refolding solution. The wild type and recombinant SOR preparations possessed indistinguishable properties when analyzed for activity and by gel permeation chromatography, CD spectroscopy and electron microscopy. The analysis of the quaternary structure showed a multi-subunit shell-like assembly with a central hollow core. The subunits formed homodimers as the building blocks of the holoenzyme, as shown by denaturation experiments. Conformational stability studies showed that the apparent unfolding free energy in water was ~5 kcal mol-1, at pH 7. Iron was found in the wild type enzyme at a stoichiometry of one iron per subunit. EPR spectroscopy of the colorless SOR resulted in a single isotropic signal at g = 4.3 characteristic of high-spin ferric iron. The signal disappeared upon reduction with dithionite or incubation with sulfur at elevated temperature. The iron center had a reduction potential of E0´ = -268 mV at pH 6.5. Protein database inspection identified five SOR protein homologues which allowed the prediction of amino acids putatively involved in catalysis. The recombinant SOR was crystallized by the sitting drop vapor diffusion method. The crystal structure was determined at 1.7 A resolution. The homo-icosatetrameric holoenzyme was a highly symmetrical hollow protein particle with 432 point group symmetry and a molecular mass of 871 kDa. The subunits were αβ-proteins and comprised a central β-barrel surrounded by α-helices. Each monomer contained one mononuclear non-heme iron site with the ligands H85, H89, E113 and two water molecules in an octahedral arrangement. The protein ligands formed a 2-His-1-carboxylate facial triad for iron binding. The cysteines C30, C100 and C103 were in the vicinity of the iron site and located along the same cavity within the interior of the subunit, therefore defining the enzyme´s active site. C30 was persulfurated. The 24 active sites were spacially separated from each other, making an electronic interaction during catalysis unlikely. They were accessible solely via the inner compartment. Access of substrate to the inner compartment is most probably provided by six hydrophobic channels along the four-fold symmetry axes of the particle. Furthermore, the structure suggested that a linear polysufide species and not the cyclic α-S8 is the substrate of the SOR. Crystal structures of the SOR in complex with the inhibitors p-hydroxy-mercury-benzoic acid and iodoacetamide identified the cysteines as the inhibitor binding sites. The iron-binding residues H85, H89 and E113 and the three cysteines C30, C100 and C103 were altered by site-directed mutagenesis and the mutant proteins were analyzed for activity and iron content. Mutations of the iron ligands and C30 resulted in inactive enzyme, whereas mutations of C100 and C103 resulted in a reduced activity. All mutations affected the oxygenase and reductase partial reactions to a similar degree. These analyses allowed the first detailed insight into the mode of action of this self-compartmentalizing metalloenzyme. C30 is most probably the sulfur binding residue which aligns the substrate for the initial oxygenation catalyzed by the Fe site. The role of C100 and C103 is not clear, but they might act in the subsequent sulfur disproportionation reaction. The comparison of the SOR with SoxAX, the only other sulfur compound oxidizing enzyme from prokaryotes for which a high resolution structure is available, showed no structural similarity. SoxAX and other sulfur oxidizing enzymes contain different cofactors, demonstrating the diversity of mechanistic approaches utilized for sulfur compound oxidation. In contrast, a basic functional principle seems to be the central role of cysteine residues, acting as covalent binding sites for the substrate.