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
| Autor: | Brad S. Pierce, Andrew C. Weitz, Sinjinee Sardar, Michael P. Hendrich |
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| Rok vydání: | 2019 |
| Předmět: |
Models
Molecular Denticity Stereochemistry Iron Amino Acid Motifs Sulfinic acid Biochemistry Article Conserved sequence Dioxygenases 03 medical and health sciences Dioxygenase Catalytic Domain 3-Mercaptopropionic Acid chemistry.chemical_classification 0303 health sciences biology Chemistry Hydrogen bond 030302 biochemistry & molecular biology Active site biology.organism_classification Amino acid Azotobacter vinelandii Azotobacter Mutation biology.protein Tyrosine |
| Zdroj: | Biochemistry |
| ISSN: | 1520-4995 |
| Popis: | Thiol dioxygenases are non-heme mononuclear iron enzymes that catalyze the O(2)-dependent oxidation of free thiols (-SH) to produce the corresponding sulfinic acid (-SO(2)(–)). 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 [Formula: see text] 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. |
| Databáze: | OpenAIRE |
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