myo-Inositol oxygenase: a radical new pathway for O(2) and C-H activation at a nonheme diiron cluster

Abstract

The enzyme myo-inositol oxygenase (MIOX) catalyzes conversion of myo-inositol (cyclohexan-1,2,3,5/4,6-hexa-ol or MI) to d-glucuronate (DG), initiating the only known pathway in humans for catabolism of the carbon skeleton of cell-signaling inositol (poly)phosphates and phosphoinositides. Recent kinetic, spectroscopic and crystallographic studies have shown that the enzyme activates its substrates, MI and O(2), at a carboxylate-bridged nonheme diiron(ii/iii) cluster, making it the first of many known nonheme diiron oxygenases to employ the mixed-valent form of its cofactor. Evidence suggests that: (1) the Fe(iii) site coordinates MI via its C1 and C6 hydroxyl groups; (2) the Fe(ii) site reversibly coordinates O(2) to produce a superoxo-diiron(iii/iii) intermediate; and (3) the pendant oxygen atom of the superoxide ligand abstracts hydrogen from C1 to initiate the unique C-C-bond-cleaving, four-electron oxidation reaction. This review recounts the studies leading to the recognition of the novel cofactor requirement and catalytic mechanism of MIOX and forecasts how remaining gaps in our understanding might be filled by additional experiments.

Figure 1

(A) Ribbon diagram of the mouse MIOX fold showing helices 4, 5 and 8 (in pink) comprising the conserved HD domain structure that contributes four of the six Fe ligands and helices 7 and 8 (in blue) containing the remaining two unique histidine ligands that complete the Fe2 site. (B) Active site of mouse MIOX adapted from the structure solved by Baker and coworkers with superoxide modeled into the Fe1 site representing the superoxo-diiron(III/III) intermediate, G. The distance from the terminal oxygen atom of superoxide to C1 of MI is 1.92 Å. (C and D) Superposition of the structures of mouse and human enzymes (in grey and blue, respectively). Note that in the structure of human MIOX by Thorsell et al. the protein has a known inhibitor, myo-inosose-1, rather than MI bound and the active site and one molecule of L-cysteine, a known activator that reduces MIOX(III/III) state to MIOX(II/III), bound to the periphery of the enzyme in a disulfide with Cys235.

Scheme 1

Reaction catalyzed by MIOX.

Scheme 2

Interconversion of three different oxidation states of the MIOX diiron cluster [(II/II), (II/III), and (III/III)] in the absence and presence of substrate, MI. Selected X-band EPR and 4.2K/53-mT Mossbauer reference spectra are shown next to the respective structures.

Scheme 3

Proposed mechanism of conversion of MI to DG initiated by the formally (superoxo)diiron(III/III) intermediate, G, via the abstraction of hydrogen atom, from C1. X-band EPR of the various states and the optical spectroscopy change upon addition of MI to II/III are shown underneath the structures., The mechanism of decay of the (hydroperoxo)diiron(III/III) intermediate and subsequent steps, as well as the nature of intermediate H are not well understood at present.

Scheme 4

Two possible reaction pathways for breakdown of the (hydroperoxo)diiron(III/III) intermediate. The upper pathway involves O-O homolysis and rebound of the hydroxyl radical equivalent with the C1-centered substrate radical. The lower pathway involves Fe-O homolysis and rebound of the hydroperoxyl radical with the substrate radical.