A Stepwise Thiol Dioxygenation Mechanism in Mercaptosuccinate Dioxygenase Revealed by A Combined Experimental and...

Thiol dioxygenases (TDOs) catalyze the incorporation of molecular oxygen into thiol metabolites and N -terminal cysteine residues of regulatory proteins, thereby playing critical roles in sulfur metabolism and oxygen sensing. Despite extensive study over the past two decades, the molecular basis for substrate recognition and the catalytic mechanism of TDOs remains controversial, owing to the scarcity of substrate-bound structures and direct evidence for catalytic intermediates. Herein, we present a comprehensive study of mercaptosuccinate dioxygenase (MSDO), a TDO originally identified in Variovorax paradoxus B4, using a combination of structural, biochemical, spectroscopic, and computational approaches. MSDO oxidizes both ( S )- and ( R )-mercaptosuccinate (MS) with similar K _m values but exhibits approximately 2.5-fold higher turnover for the ( S )-enantiomer. Crystal structures of MSDO reveal that both ( S )- and ( R )-MS coordinate the iron in a bidentate mode via their thiolate and proximal carboxylate groups, with the distal carboxylate adopting distinct orientations. Two active-site Arg residues recognize the substrate carboxylate groups and thereby stabilize a flexible C -terminal loop, underpinning a catalytic site gating mechanism in MSDO. EPR spectroscopy corroborates bidentate coordination, showing conversion of a high-spin {FeNO} ⁷ complex to a low-spin species upon substrate binding. Time-resolved in crystallo reactions capture two key iron-bound intermediates, namely an unprecedented monooxygenated sulfenate and a dioxygenated sulfinate product. These structural snapshots are supported by DFT calculations that point to a stepwise oxygen atom transfer pathway. Computational analysis further accounts for the kinetic differences between the substrate enantiomers, as rationalized by structural comparisons, active-site geometry, and second coordination sphere interactions. Together, these results elucidate fundamental principles of TDO catalysis and advance our understanding of nonheme iron-dependent oxygen activation.