An [Fe IV 2(μ-O) 2] diamond core structure has been postulated for intermediate Q of soluble methane monooxygenase (sMMO-Q), the oxidant responsible for cleaving the strong C-H bond of methane and its hydroxylation. By extension, analogous species may be involved in the mechanisms of related diiron hydroxylases and desaturases. Because of the paucity of well-defined synthetic examples, there are few, if any, mechanistic studies on the oxidation of hydrocarbon substrates by complexes with high-valent [Fe 2(μ-O) 2] cores. We report here that water or alcohol substrates can activate synthetic [Fe IIIFe IV(μ-O) 2] complexes supported by tetradentate tris(pyridyl-2-methyl)amine ligands (1 and 2) by several orders of magnitude for C-H bond oxidation. On the basis of detailed kinetic studies, it is postulated that the activation results from Lewis base attack on the [Fe IIIFe IV(μ-O) 2] core, resulting in the formation of a more reactive species with a [X-Fe III-O-Fe IV=O] ring-opened structure (1-X, 2-X, X = OH - or OR -). Treatment of 2 with methoxide at -80 °C forms the 2-methoxide adduct in high yield, which is characterized by an S = 1/2 EPR signal indicative of an antiferromagnetically coupled [S = 5/2 Fe III/S = 2 Fe IV] pair. Even at this low temperature, the complex undergoes facile intramolecular C-H bond cleavage to generate formaldehyde, showing that the terminal high-spin Fe IV=O unit is capable of oxidizing a C-H bond as strong as 96 kcal mol -1. This intramolecular oxidation of the methoxide ligand can in fact be competitive with intermolecular oxidation of triphenylmethane, which has a much weaker C-H bond (D C-H 81 kcal mol -1). The activation of the [Fe IIIFe IV(μ- O) 2] core is dramatically illustrated by the oxidation of 9,10-dihydroanthracene by 2-methoxide, which has a second-order rate constant that is 3.6 × 10 7-fold larger than that for the parent diamond core complex 2. These observations provide strong support for the DFT-based notion that an S = 2 Fe IV=O unit is much more reactive at H-atom abstraction than its S = 1 counterpart and suggest that core isomerization could be a viable strategy for the [Fe IV 2(μ-O) 2] diamond core of sMMO-Q to selectively attack the strong C-H bond of methane in the presence of weaker C-H bonds of amino acid residues that define the diiron active site pocket.