Altered inositol metabolism is implicated in a number of diabetic complications. The first committed step in mammalian inositol catabolism is performed by myo-inositol oxygenase (MIOX), which catalyzes a unique four-electron dioxygen-dependent ring cleavage of myo-inositol to D-glucuronate. Here, we present the crystal structure of human MIOX in complex with myo-inosose-1 bound in a terminal mode to the MIOX diiron cluster site. Furthermore, from biochemical and biophysical results from N-terminal deletion mutagenesis we show that the N terminus is important, through coordination of a set of loops covering the active site, in shielding the active site during catalysis. EPR spectroscopy of the unliganded enzyme displays a two-component spectrum that we can relate to an open and a closed active site conformation. Furthermore, based on site-directed mutagenesis in combination with biochemical and biophysical data, we propose a novel role for Lys 127 in governing access to the diiron cluster.myo-Inositol is a cyclitol that plays a crucial role in all eukaryotic cells by serving as a backbone for the most important second messengers: inositol phosphates and their lipid derivatives, phosphoinositides. Although inositol phosphates and phosphoinositides have been and continue to be the focus of intense study, the mechanisms for maintenance of total cellular inositol levels and the medical implications of deranged intracellular inositol levels are less studied. It is known that inositol homeostasis is often disturbed in diabetes (1) and that intracellular depletion of myo-inositol is associated with common diabetic complications, such as cataracts, nephropathies, retinopathies, and neuropathies (2). In mammalian cells, the only pathway for inositol breakdown utilizes myo-inositol oxygenase (MIOX) 3 to catalyze the first committed step by a dioxygen-dependent cleavage between C1 and C6 of the inositol ring to form D-glucuronate, which can then enter the glucuronate-xylulose pathway (2). Therapeutic intervention aimed at inhibiting MIOX activity may be a future cure for diabetic complications caused by inositol depletion.18 O labeling studies have shown that MIOX incorporates only a single oxygen into the product and that oxygen is found exclusively in the D-glucuronate carboxylate group. MIOX has a pH optimum of 9.5 (3) with myo-inositol (K m ϭ 5.9 mM; k cat ϭ 11 min Ϫ1 (2)) and its epimer D-chiroinositol (K m ϭ 33 mM; k cat ϭ 2.3 min Ϫ1 (2)) as its only known substrates. Moreover, the only known reasonably potent inhibitor is myo-inosose-1 (K i ϭ 62 M (3)). It has been noted that an N-terminally truncated fragment starting at Thr 32 is formed upon storage of recombinant protein (4). Interestingly, this uncharacterized N-terminal fragment is also observed in vivo (5).It was recently shown, using EPR and Mössbauer spectroscopy, that MIOX is a new member of the nonheme diiron-dependent dioxygen-activating family (6). Interestingly, the sequence indicated strong dissimilarity to other members of this family (6) that all fe...