Finding Intermediates in the O₂ Activation Pathways of Non-Heme Iron Oxygenases

Abstract

Intermediates in the reaction cycle of an oxygenase are usually very informative of the chemical mechanism of O 2 activation and insertion. However, detection of these intermediates is often complicated by their short lifetime and the regulatory mechanism of the enzyme designed to ensure specificity. Here, the methods used to detect the intermediates in an extradiol dioxygenase, a Rieske cis-dihydrodiol dioxygenase, and soluble methane monooxygenase are discussed. The methods include the use of alternative, chromophoric substrates, mutagenesis of active site catalytic residues, forced changes in substrate binding order, control of reaction rates using regulatory proteins, and initialization of catalysis in crystallo.

DFT Study on the Catalytic Reactivity of a Functional Model Complex for Intradiol-Cleaving Dioxygenases

“…The calculated activation free energy of 23.3 kcal/mol is in a good agreement with the experimental barrier of 21 kcal/mol. The involvement of a peroxo intermediate in both intra-and extradiol cleavage reactions is supported by a number of computational studies [5,7,8,32] and by recent experimental studies of Kovaleva et al [3,4], where such an intermediate was actually detected. The peroxide O-O bond cleaves homolytically to give a radical Fe(IV)-oxo intermediate RAD.…”

“…One oxygen atom of the substrate has to dissociate from the metal ion upon oxygen binding to provide the necessary coordination site, see Fig.3 b. Two spin states for the R2 complex with quite close energies were identified, a quartet 4 [R2] 4α1β and a sextet 6 [R2] 4α1α , where the unpaired electrons are residing on iron and oxygen, respectively (no spin on the substrate). The quartet state lies at 18.0 and the sextet state at 18.2 kcal/mol relative to the reaction zero level.…”

“…Thus, in total oxygen binding requires an activation barrier of 16.7 kcal/mol in the presence of the base. This shows that most of the energy needed to form the oxygen bound adduct 4 [R2] 4α1β is due to the dissociation of the semiquinonate. Since the total O 2 binding energy given by the test model was essentially the same as without the base, the investigation was continued with the initial model.…”

Synthetic iron-oxo “diamond core” mimics structure of key intermediate in methane monooxygenase catalytic cycle

“…The enzymes involved in these processes use a rich variety of different active sites to activate O 2 , such as heme cofactors, mono-and binuclear nonheme iron centers, mono-and binuclear copper complexes, and heteronuclear heme iron/copper clusters (1). Typically, oxygen is activated in a tightly controlled manner to ensure that the formation of the key reactive species in the corresponding catalytic cycle occurs only when the target substrate is present, thereby suppressing potentially harmful side reactions in the enzyme active site (2). A particularly well studied example of an enzyme that follows this general strategy is provided by cytochrome P450 (3).…”

Ferric ion (hydr)oxo clusters in the “Venus flytrap” cleft of FbpA: Mössbauer, calorimetric and mass spectrometric studies

“…In general, proteins having non-heme polynuclear iron sites play important roles in a plethora of important physiological reactions including oxygen transport, oxygenases and hydrogenases [11][12][13][14][15][16][17]. Recently it has been shown that a phosphate uptake and transfer protein (Pho U)…”