POTENTIAL ROLE OF A TYROSINE LIGAND IN STABILIZING THE MIXED-VALENT DIIRON (II/III) COFACTOR OF MYO-INOSITOL OXYGENASE

Open Access
Author:
Cioffi, Katherine Joyann
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • J. Martin Bollinger, Thesis Supervisor
  • Craig Eugene Cameron, Honors Advisor
Keywords:
  • myo-inositol oxygenase
  • metalloenzymes
  • mixed-valent diiron oxygenase
  • enzymology
Abstract:
myo-inositol (MI) oxygenase (MIOX) uses a non-heme diiron cofactor in its mixed-valent Fe2(II/III) oxidation state to activate dioxygen for cleavage of MI to D-gluconorate. MIOX is the founding member of a growing class of mixed-valent diiron oxygenase (MVDO) enzymes. PhnZ and TmpB, the second and third MVDO enzymes to be recognized, were recently shown to have stabilizing tyrosine ligands to the Fe(II) sites of their respective cofactors. The possibility that MIOX also has such a stabilizing tyrosinate is probed in this paper by comparison of the ultraviolet-visible absorption, EPR, and Raman spectra of the wild-type (WT) and variant MIOX proteins. The WT enzyme exhibits a change in its absorption spectrum from a peak at 600 nm to a feature at 495 nm upon reduction from the diferric to mixed-valent state; the Y13G variant protein failed to exhibit this shift. Upon addition of substrate, however, WT diferric and mixed-valent MIOX display quite similar spectra. Likewise, EPR spectra of mixed-valent WT, Y13A, and Y13G MIOX were similar in the presence of substrate, exhibiting axial symmetry with effective g-values of ~1.96, 1.81, and 1.81. In the absence of substrate, the EPR signal was much weaker and broader in form for the WT and Y13A enzyme, though the variant form was shifted further downfield. Stopped-flow experiments demonstrated that turnover does occur in the Y13A variant, but it has a decreased binding affinity for substrate (KD of ~0.8 mM as found in this paper), as compared to the WT form (literature value of KD is ~0.4 mM). Resonance Raman spectroscopy was also attempted on diferric WT MIOX and PhnZ at 637.8 nm. Though fluorescence was observed, signals possibly attributable to resonance-enhanced tyrosine vibrations appeared in both PhnZ (866 and 1276 cm-1) and MIOX (1288 and 853 cm-1). The results suggest the presence of a tyrosinate ligand in MIOX, but additional evidence is required.