A Coupled Dinuclear Iron Cluster that Is Perturbed by Substrate Binding in myo-Inositol Oxygenase

Abstract

myo-Inositol oxygenase (MIOX) uses iron as its cofactor and dioxygen as its cosubstrate to effect the unique, ring-cleaving, four-electron oxidation of its cyclohexan-(1,2,3,4,5,6-hexa)-ol substrate to d-glucuronate. The nature of the iron cofactor and its interaction with the substrate, myo-inositol (MI), have been probed by electron paramagnetic resonance (EPR) and Mössbauer spectroscopies. The data demonstrate the formation of an antiferromagnetically coupled, high-spin diiron(III/III) cluster upon treatment of solutions of Fe(II) and MIOX with excess O(2) or H(2)O(2) and the formation of an antiferromagnetically coupled, valence-localized, high-spin diiron(II/III) cluster upon treatment with either limiting O(2) or excess O(2) in the presence of a mild reductant (e.g., ascorbate). Marked changes to the spectra of both redox forms upon addition of MI and analogy to changes induced by binding of phosphate to the diiron(II/III) cluster of the protein phosphatase, uteroferrin, suggest that MI coordinates directly to the diiron cluster, most likely in a bridging mode. The addition of MIOX to the growing family of non-heme diiron oxygenases expands the catalytic range of the family beyond the two-electron oxidation (hydroxylation and dehydrogenation) reactions catalyzed by its more extensively studied members such as methane monooxygenase and stearoyl acyl carrier protein Delta(9)-desaturase.

Organophosphonate-degrading PhnZ reveals an emerging family of HD domain mixed-valent diiron oxygenases

“…The signals are broad and suggest the presence of two or more Fe II complexes (SI Appendix, Fig. S2), as has been seen in MIOX and other nonheme diiron proteins (16,(27)(28)(29).…”

“…. .D motif binds two iron ions, and the enzyme uses an unprecedented mixed-valent Fe II /Fe III cofactor form to catalyze the four-electron (4e -) oxidative C-C bond cleavage converting myo-inositol (MI; cyclohexan-1,2,3,5/4, 6-hexa-ol) to D-glucuronate (13,16,17). The Fe III site coordinates MI, serving as Lewis acid to activate the substrate, and the Fe II site activates O 2 to produce a superoxo-Fe III /Fe III intermediate that initiates the reaction by abstracting a hydrogen atom from C1 of MI (6,18).…”

tRNA-modifying MiaE protein fromSalmonella typhimuriumis a nonheme diiron monooxygenase

“…The large quadrupole splittings of theses ions are the signature of a -oxo-diferric system (24). Finally, a broad magnetic contribution, reminiscent of those observed for enzymes with a localized mixedvalent [Fe II -Fe III ] center (MMOH, uteroferrin and myo-inositol oxygenase), was observed (20,21,25), this contribution could be fitted as well. However, because the limited contribution of this species [30 (5)%] and the spread of its spectrum over a wide velocity range, the Mössbauer parameters cannot be reliably determined with a high enough accuracy.…”

“…The simulated curves shown in Fig. 4 have been obtained from the spin Hamiltonian parameters reported for the mixed-valent center of myo-inositol oxygenase complexed with myo-inositol (MIOX-MI, see Table 2) (25). The excellent agreement between the experimental and simulated spectra is further shown in the difference spectrum obtained by subtracting the parallel from the perpendicular spectrum (Fig.…”

“…This is further confirmed by in vitro assays showing that ms 2 i 6 A-to-ms 2 io 6 A conversion can be achieved by incubating pure MiaE2H and the tRNA substrate with E. coli cell-free extracts under air. More recently, a different mechanism was discovered in the case of myo-inositol oxygenase (44), another diiron enzyme with a slightly different active site, which activates dioxygen through its mixed-valent Fe II -Fe III state (25,45). The sequence homology of MiaE with the hydroxylase component of MMO and the fact that it can function in vitro with hydrogen peroxide as the oxidant, in the absence of a source of electrons, strongly suggests that MiaE operates in a similar way to MMOH through a two-electron O 2 reductive activation mechanism.…”