Outer-Sphere Tyrosine 159 within the 3-Mercaptopropionic Acid Dioxygenase S-H-Y Motif Gates Substrate-Coordination Denticity at the Non-Heme Iron Active Site

Biochemistry. 2019 Dec 24;58(51):5135-5150. doi: 10.1021/acs.biochem.9b00674. Epub 2019 Dec 11.


Thiol dioxygenases are non-heme mononuclear iron enzymes that catalyze the O2-dependent oxidation of free thiols (-SH) to produce the corresponding sulfinic acid (-SO2-). Regardless of the phylogenic domain, the active site for this enzyme class is typically comprised of two major features: (1) a mononuclear ferrous iron coordinated by three protein-derived histidines and (2) a conserved sequence of outer Fe-coordination-sphere amino acids (Ser-His-Tyr) spatially adjacent to the iron site (∼3 Å). Here, we utilize a promiscuous 3-mercaptopropionic acid dioxygenase cloned from Azotobacter vinelandii (Av MDO) to explore the function of the conserved S-H-Y motif. This enzyme exhibits activity with 3-mercaptopropionic acid (3mpa), l-cysteine (cys), as well as several other thiol-bearing substrates, thus making it an ideal system to study the influence of residues within the highly conserved S-H-Y motif (H157 and Y159) on substrate specificity and reactivity. The pKa values for these residues were determined by pH-dependent steady-state kinetics, and their assignments verified by comparison to H157N and Y159F variants. Complementary electron paramagnetic resonance and Mössbauer studies demonstrate a network of hydrogen bonds connecting H157-Y159 and Fe-bound ligands within the enzymatic Fe site. Crucially, these experiments suggest that the hydroxyl group of Y159 hydrogen bonds to Fe-bound NO and, by extension, Fe-bound oxygen during native catalysis. This interaction alters both the NO binding affinity and rhombicity of the 3mpa-bound iron-nitrosyl site. In addition, Fe coordination of cys is switched from thiolate only to bidentate (thiolate/amine) for the Y159F variant, indicating that perturbations within the S-H-Y proton relay network also influence cys Fe binding denticity.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • 3-Mercaptopropionic Acid / metabolism*
  • Amino Acid Motifs
  • Azotobacter / enzymology
  • Catalytic Domain*
  • Dioxygenases / chemistry*
  • Dioxygenases / genetics
  • Dioxygenases / metabolism*
  • Iron*
  • Models, Molecular
  • Mutation
  • Tyrosine*


  • Tyrosine
  • 3-Mercaptopropionic Acid
  • Iron
  • Dioxygenases