Oxidative Uncoupling in Cysteine Dioxygenase Is Gated by a Proton-Sensitive Intermediate

Cysteine dioxygenase (CDO) is a non-heme mononuclear iron enzyme that catalyzes the O₂-dependent oxidation of l-cysteine (Cys) to produce cysteine sulfinic acid (CSA). This enzyme catalyzes the first committed step in Cys catabolism; thus, it is central to mammalian sulfur metabolism and redox homeostasis. Ironically, despite nearly 45 years of continued research on CDO, essentially no information has been reported with respect to its kinetic mechanism. In this work, the timing of chemical steps in the CDO kinetic mechanism is investigated by pH/pD-dependent steady-state kinetics and solvent isotope effects on k_cat, k_cat/K_M, and (O₂/CSA) coupling. Normal solvent kinetic isotope effects of 1.45 ± 0.05 and 2.0 ± 0.1 are observed in k_cat–pL and k_cat/K_M–pL profiles, respectively. Proton inventory experiments within the pL-independent region (pL 8.5) suggest multiple solvent-exchangeable protons in flight for both k_cat and k_cat/K_M data. The influence of solvent viscosity was also investigated to probe non-chemical steps and to verify that the apparent isotope effects were not attributable to increased solvent viscosity of D₂O reactions relative to H₂O. Although solvent viscosity did have a modest influence on k_cat and k_cat/K_M, the response is not sufficient to account for the observed solvent isotope effects. This suggests that product release is only partially rate-limiting for CDO catalysis. Most crucially, proton inventory of (O₂/CSA) coupling indicates that a proton-sensitive transition state directly follows O₂ activation. Thus, protonation of a transient species preceding Cys oxidation is gated by protons in flight. This behavior provides valuable insight into the kinetically masked transients generated during catalysis.