The reaction mechanism for dioxygen activation in 2-oxoglutarate-dependent enzymes has been studied by means of hybrid density functional theory. The results reported here support a mechanism in which all chemical transformations take place on a quintet potential-energy surface. More specifically, the activated dioxygen species attacks the carbonyl group of the co-substrate producing the Fe(II)-persuccinate-CO(2) complex, which readily releases the carbon dioxide molecule. The step in which the Fe(II)-peracid-CO(2) complex is formed is found to be rate-limiting and irreversible. Subsequent heterolysis of the Obond;O bond in the Fe(II)-persuccinate complex proceeds in two one-electron steps and produces the high-valent iron-oxo species Fe(IV)dbond;O, which is most likely to be responsible for oxidative reactions catalyzed by 2-oxoglutarate-dependent enzymes. The concerted pathway for simultaneous Obond;O and Cbond;C bond cleavage on the septet potential-energy surface is found to be less favorable. The relative stability of different forms of the active iron-oxo species is assessed, and the quintet five-coordinate complex is found to be most stable.

The catalytic mechanism of naphthalene 1,2-dioxygenase has been investigated by means of hybrid density functional theory. This Rieske-type enzyme, which contains an active site hosting a mononuclear non-heme iron(II) complex, uses dioxygen and two electrons provided by NADH to carry out the cis-dihydroxylation of naphthalene. Since a (hydro)peroxo-iron(III) moiety has been proposed to be involved in the catalytic cycle, it was probed whether and how this species is capable of cis-dihydroxylation of the aromatic substrate. Different oxidation and protonation states of the Fe-O2 complex were studied on the basis of the crystal structure of the enzyme with oxygen bound side-on to iron. It was found that feasible reaction pathways require a protonated peroxo ligand, FeIII-OOH; the deprotonated species, the peroxo-iron(III) complex, was found to be inert toward naphthalene. Among the different chemical patterns which have been explored, the most accessible one involves an epoxide intermediate, which may subsequently evolve toward an arene cation, and finally to the cis-diol. The possibility that an iron(V)-oxo species is formed prior to substrate hydroxylation was also examined, but found to implicate a rather high energy barrier. In contrast, a reasonably low barrier might lead to a high-valent iron-oxo species [i.e. iron(IV)-oxo] if a second external electron is supplied to the mononuclear iron center before dioxygenation.

Density functional theory with the B3LYP hybrid functional has been used to study the mechanisms for dioxygen activation by four families of mononuclear non-heme iron enzymes: alpha-ketoacid-dependent dioxygenases, tetrahydrobiopterin-dependent hydroxylases, extradiol dioxygenases, and Rieske dioxygenases. These enzymes have a common active site with a ferrous ion coordinated to two histidines and one carboxylate group (aspartate or glutamate). In contrast to the heme case, this type of weak field environment always leads to a high-spin ground state. With the exception of the Rieske dioxygenases, which have an electron source outside the active site, the dioxygen activation process passes through the formation of a bridging-peroxide species, which then undergoes O-O bond cleavage finally leading to the four electron reduction of O(2). In the case of tetrahydrobiopterin- and alpha-ketoacid-dependent enzymes, the O-O heterolysis yields a high-valent iron-oxo species, which is capable of performing a two-electron oxidation chemistry on various organic substrates. For the other two families of enzymes (extradiol dioxygenases and Rieske dioxygenases) the substrate oxidation and the O-O bond cleavage are found to be coupled. In the extradiol dioxygenases the product of the O-O bond cleavage is a ferric iron with an oxy-substrate with a mixture of radical and anionic character, which is essential for the selectivity of the catechol cleavage.

Density functional theory using the B3LYP hybrid functional has been employed to investigate the reactivity of Fe(TPA) complexes (TPA = tris(2-pyridylmethyl)amine), which are known to catalyze stereospecific hydrocarbon oxidation when H(2)O(2) is used as oxidant. The reaction pathway leading to O-O bond heterolysis in the active catalytic species Fe(III)(TPA)-OOH has been explored, and it is shown that a high-valent iron-oxo intermediate is formed, where an Fe(V) oxidation state is attained, in agreement with previous suggestions based on experiments. In contrast to the analogous intermediate [(Por.)Fe(IV)=O](+1) in P450, the TPA ligand is not oxidized, and the electrons are extracted almost exclusively from the mononuclear iron center. The corresponding homolytic O-O bond cleavage, yielding the two oxidants Fe(IV)=O and the OH. radical, has also been considered, and it is shown that this pathway is inaccessible in the hydrocarbon oxidation reaction with Fe(TPA) and hydrogen peroxide. Investigations have also been performed for the O-O cleavage in the Fe(III)(TPA)-alkylperoxide species. In this case, the barrier for O-O homolysis is found to be slightly lower, leading to loss of stereospecificity and supporting the experimental conclusion that this is the preferred pathway for alkylperoxide oxidants. The difference between hydroperoxide and alkylperoxide as oxidant derives from the higher O-O bond strength for hydrogen peroxide (by 8.0 kcal/mol).

The chemical pathways leading to the hydroxylated aromatic amino acids in phenylalanine and tryptophan hydroxylases have been investigated by means of hybrid density functional theory. In the catalytic core of these non-heme iron enzymes, dioxygen reacts with the pterin cofactor and is likely to be activated by forming an iron(IV)=O complex. The capability of this species to act as a hydroxylating intermediate has been explored. Depending on the protonation state of the ligands of the metal, two different mechanisms are found to be energetically possible for the hydroxylation of phenylalanine and tryptophan by the high-valent iron-oxo species. With a hydroxo ligand the two-electron oxidation of the aromatic ring passes through a radical, while an arenium cation is involved when a water replaces the hydroxide. After the attack of the activated oxygen on the substrate, it is also found that a 1,2-hydride shift (known as an NIH shift) generates a keto intermediate, which can decay to the true product through an intermolecular keto-enol tautomerization. The benzylic hydroxylation of 4-methylphenylalanine by the Fe(IV)=O species has also been investigated according to the rebound mechanism. The computed energetics lead to the conclusion that Fe(IV)=O is capable not only of aromatic hydroxylation, but also of benzylic hydroxylation.