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

• Published in: Biochemistry (Easton) 2015-12, Vol.54 (51), p.7477-7490
• Main Authors: Pierce, Brad S, Subedi, Bishnu P, Sardar, Sinjinee, Crowell, Joshua K
• Format: Article
• Language: English
• Subjects: 3-Mercaptopropionic Acid - chemistry; active sites; Azotobacter vinelandii; Azotobacter vinelandii - enzymology; binding capacity; Catalysis; Catalytic Domain; cysteamine; cysteine; cysteinesulfinic acid; Dioxygenases - chemistry; electron paramagnetic resonance spectroscopy; Electron Spin Resonance Spectroscopy; enzyme substrates; enzymes; Glycine - chemistry; Hydrogen-Ion Concentration; Kinetics; nitric oxide; oxidation; oxygen; Protein Conformation; Variovorax paradoxus
• ISSN: 0006-2960; 1520-4995; 1520-4995
• DOI: 10.1021/acs.biochem.5b00636

Cysteine dioxygenase (CDO) is a non-heme iron enzyme that catalyzes the O2-dependent oxidation of l-cysteine to produce cysteinesulfinic acid. Bacterial CDOs have been subdivided as either “Arg-type” or “Gln-type” on the basis of the identity of conserved active site residues. To date, “Gln-type” enzymes remain largely uncharacterized. It was recently noted that the “Gln-type” enzymes are more homologous with another thiol dioxygenase [3-mercaptopropionate dioxygenase (MDO)] identified in Variovorax paradoxus, suggesting that enzymes of the “Gln-type” subclass are in fact MDOs. In this work, a putative “Gln-type” thiol dioxygenase from Azotobacter vinelandii (Av) was purified to homogeneity and characterized. Steady-state assays were performed using three substrates [3-mercaptopropionic acid ( 3mpa ), l-cysteine ( cys ), and cysteamine ( ca )]. Despite comparable maximal velocities, the “Gln-type” Av enzyme exhibited a specificity for 3mpa (k cat/K M = 72000 M–1 s–1) nearly 2 orders of magnitude greater than those for cys (110 M–1 s–1) and ca (11 M–1 s–1). Supporting X-band electron paramagnetic resonance (EPR) studies were performed using nitric oxide (NO) as a surrogate for O2 binding to confirm obligate-ordered addition of substrate prior to NO. Stoichimetric addition of NO to solutions of 3mpa -bound enzyme quantitatively yields an iron-nitrosyl species (Av ES-NO) with EPR features consistent with a mononuclear (S = 3/2) {FeNO}7 site. Conversely, two distinct substrate-bound conformations were observed in Av ES-NO samples prepared with cys and ca , suggesting heterogeneous binding within the enzymatic active site. Analytical EPR simulations are provided to establish the relative binding affinity for each substrate ( 3map > cys > ca ). Both kinetic and spectroscopic results presented here are consistent with 3mpa being the preferred substrate for this enzyme.