Abstract
Thiosulfate dehydrogenases are bacterial cytochromes that contribute to the oxidation of inorganic sulfur. The active sites of these enzymes contain low-spin c-type heme with Cys(-)/His axial ligation. However, the reduction potentials of these hemes are several hundred mV more negative than that of the thiosulfate/tetrathionate couple (E(m), +198 mV), making it difficult to rationalize the thiosulfate oxidizing capability. Here, we describe the reaction of Campylobacter jejuni thiosulfate dehydrogenase (TsdA) with sulfite, an analogue of thiosulfate. The reaction leads to stoichiometric conversion of the active site Cys to cysteinyl sulfonate (C(α)-CH(2)-S-SO(3)(-)) such that the protein exists in a form closely resembling a proposed intermediate in the pathway for thiosulfate oxidation that carries a cysteinyl thiosulfate (C(α)-CH(2)-S-SSO(3)(-)). The active site heme in the stable sulfonated protein displays an E(m) approximately 200 mV more positive than the Cys(-)/His-ligated state. This can explain the thiosulfate oxidizing activity of the enzyme and allows us to propose a catalytic mechanism for thiosulfate oxidation. Substrate-driven release of the Cys heme ligand allows that side chain to provide the site of substrate binding and redox transformation; the neighboring heme then simply provides a site for electron relay to an appropriate partner. This chemistry is distinct from that displayed by the Cys-ligated hemes found in gas-sensing hemoproteins and in enzymes such as the cytochromes P450. Thus, a further class of thiolate-ligated hemes is proposed, as exemplified by the TsdA centers that have evolved to catalyze the controlled redox transformations of inorganic oxo anions of sulfur.