Insights into Structure and Function of the Active Site of SoxAX Cytochromes

Abstract

SoxAX cytochromes catalyze the formation of heterodisulfide bonds between inorganic sulfur compounds and a carrier protein, SoxYZ. They contain unusual His/Cys-ligated heme groups with complex spectroscopic signatures. The heme-ligating cysteine has been implicated in SoxAX catalysis, but neither the SoxAX spectroscopic properties nor its catalysis are fully understood at present. We have solved the first crystal structure for a group 2 SoxAX protein (SnSoxAX), where an N-terminal extension of SoxX forms a novel structure that supports dimer formation. Crystal structures of SoxAX with a heme ligand substitution (C236M) uncovered an inherent flexibility of this SoxA heme site, with both bonding distances and relative ligand orientation differing between asymmetric units and the new residue, Met(236), representing an unusual rotamer of methionine. The flexibility of the SnSoxAX(C236M) SoxA heme environment is probably the cause of the four distinct, new EPR signals, including a high spin ferric heme form, that were observed for the enzyme. Despite the removal of the catalytically active cysteine heme ligand and drastic changes in the redox potential of the SoxA heme (WT, -479 mV; C236M, +85 mV), the substituted enzyme was catalytically active in glutathione-based assays although with reduced turnover numbers (WT, 3.7 s(-1); C236M, 2.0 s(-1)). SnSoxAX(C236M) was also active in assays using SoxYZ and thiosulfate as the sulfur substrate, suggesting that Cys(236) aids catalysis but is not crucial for it. The SoxYZ-based SoxAX assay is the first assay for an isolated component of the Sox multienzyme system.