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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
journal contribution
posted on 2019-12-11, 04:13 authored by Sinjinee Sardar, Andrew Weitz, Michael P. Hendrich, Brad S. PierceThiol 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.