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

Abstract

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 kcat, kcat/KM, and (O2/CSA) coupling. Normal solvent kinetic isotope effects of 1.45 ± 0.05 and 2.0 ± 0.1 are observed in kcat-pL and kcat/KM-pL profiles, respectively. Proton inventory experiments within the pL-independent region (pL 8.5) suggest multiple solvent-exchangeable protons in flight for both kcat and kcat/KM 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 kcat and kcat/KM, 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.

Non-chemical proton-dependent steps prior to O2-activation limit Azotobacter vinelandii 3-mercaptopropionic acid dioxygenase (MDO) catalysis

“…Among thiol dioxygenase enzymes, the mammalian ‘Arg-type’ CDO has been extensively characterized spectroscopically [32–35], crystallographically [36–38] and kinetically [16,22,23]. However, almost no information is available regarding the relative timing of chemical and non-chemical steps in ‘Gln-type’ MDO catalyzed reactions.…”

“…The rate of dioxygen consumption in activity assays was determined polarographically using a standard Clark electrode (Hansatech Instruments, Norfolk, England) in a jacketed 2.5 mL cell. Calibration of O 2 -electrode is described in detail elsewhere [22,23]. All reactions were initiated by addition of 1.0 μM Av MDO under identical buffer conditions as described for HPLC assays.…”

The “Gln-Type” Thiol Dioxygenase from Azotobacter vinelandii Is a 3-Mercaptopropionic Acid Dioxygenase

“…Calibration of the O 2 electrode is described in detail elsewhere. 27,28 As with HPLC assays, all reactions were initiated by addition of 1.0 µM Av MDO under identical buffer conditions as described for HPLC assays. Reaction temperatures were maintained at 25 ± 2 °C by a circulating water bath (ThermoFlex 900, Thermo Scientific).…”

Thiol dioxygenase turnover yields benzothiazole products from 2-mercaptoaniline and O2-dependent oxidation of primary alcohols

“…Recombinant Mus musculus cysteine dioxygenase (Mm CDO) and Azotobacter vinelandii 3-mercaptopropionic acid dioxygenase ( Av MDO) were expressed, purified, and activity verified as described previously [29, 30]. For all batches of enzyme used in these experiments, spectrophotometric determination of ferrous and ferric iron content was measured using 2, 4, 6-tripyridyl-s-triazine (TPTZ).…”

“…Calibration of O 2 -electrode is described in detail elsewhere [30, 33]. All reactions were initiated by addition of 2.0 μM enzyme ( Mm CDO or Av MDO) under identical buffer conditions as described for UV-visible experiments.…”