Studies of the di-iron(VI) Intermediate in ferrate-dependent oxygen evolution from water.

Molecular oxygen is produced from water via the following reaction of potassium ferrate (K(2)FeO(4)) in acidic solution: 4[H(3)Fe(VI)O(4)](+) + 8H(3)O(+) → 4Fe(3+) + 3O(2) + 18H(2)O. This study focuses upon the mechanism by which the O-O bond is formed. Stopped-flow kinetics at variable acidities in H(2)O and D(2)O are used to complement the analysis of competitive oxygen-18 kinetic isotope effects ((18)O KIEs) upon consumption of natural abundance water. The derived (18)O KIEs provide insights concerning the identity of the transition state. Water attack (WA) and oxo-coupling (OC) transition states were evaluated for various reactions of monomeric and dimeric ferrates using a calibrated density functional theory protocol. Vibrational frequencies from optimized isotopic structures are used here to predict (18)O KIEs for comparison to experimental values determined using an established competitive isotope-fractionation method. The high level of agreement between experimental and theoretic isotope effects points to an intramolecular OC mechanism within a di-iron(VI) intermediate, consistent with the analysis of the reaction kinetics. Alternative mechanisms are excluded based on insurmountably high free energy barriers and disagreement with calculated (18)O KIEs.

Experimental and computational investigations of oxygen reactivity in a heme and tyrosyl radical-containing fatty acid α-(di)oxygenase.

Rice α-(di)oxygenase mediates the regio- and stereospecific oxidation of fatty acids using a persistent catalytic tyrosyl radical. Experiments conducted in the physiological O(2) concentration range, where initial hydrogen atom abstraction from the fatty acid occurs in a kinetically reversible manner, are described. Our findings indicate that O(2)-trapping of an α-carbon radical is likely to reversibly precede reduction of a 2-(R)-peroxyl radical intermediate in the first irreversible step. A mechanism of concerted proton-coupled electron transfer is proposed on the basis of natural abundance oxygen-18 kinetic isotope effects, deuterium kinetic isotope effects, and calculations at the density functional level of theory, which predict a polarized transition state in which electron transfer is advanced to a greater extent than proton transfer. The approach outlined should be useful for identifying mechanisms of concerted proton-coupled electron transfer in a variety of oxygen-utilizing enzymes.

Oxygen isotope effects as structural and mechanistic probes in inorganic oxidation chemistry.

Oxidative transformations using molecular oxygen are widespread in nature but remain a major challenge in chemical synthesis. Limited mechanistic understanding presents the main obstacle to exploiting O(2) in "bioinspired" industrial processes. Isotopic methods are presently being applied to characterize reactions of natural abundance O(2) including its coordination to reduced transition metals and cleavage of the O-O bond. This review describes the application of competitive oxygen-18 isotope effects, together with Density Functional Theory, to examine O(2) reductive activation under catalytically relevant conditions. The approach should be generally useful for probing small-molecule activation by transition-metal complexes.

Molecular oxygen dependent steps in fatty acid oxidation by cyclooxygenase-1.

The mechanism by which cyclooxygenase-1 (COX-1), a heme- and tyrosyl radical-containing enzyme, catalyzes the regio- and stereospecific oxygenation of polyunsaturated fatty acids to prostaglandin or hydroperoxide products has not been understood. Steady-state kinetic studies conducted with the native substrate arachidonic acid and the slower substrate linoleic acid are described here. Second-order rate constants, kcat/KM for fatty acid and O2, are found to depend upon the concentration of the other cosubstrate. Competitive oxygen kinetic isotope effects (18O KIEs) kcat/KM(16,16O2)/kcat/KM(18,16O2) reveal that a peroxyl radical is formed in or before the first kinetically irreversible step. Together, the results indicate that the oxygenase reaction occurs by a sequential mechanism which most likely involves reversible abstraction of a hydrogen atom from the fatty acid prior to the trapping of the delocalized substrate radical by O2. The identity of the first kinetically irreversible step, subsequent to forming the peroxyl radical, is also discussed in the context of the magnitude of the oxygen kinetic isotope effects as well as the behavior of kcat/KM(O2) in response to changing solvent pH, pD, and viscosity.

Determination of a large reorganization energy barrier for hydride abstraction by glucose oxidase.

Glucose oxidase mediates the aerobic oxidation of simple sugars to lactones using a noncovalently bound flavin cofactor. The chemical mechanism of this reaction has been uncertain for many years. Here it is shown, using enzymes reconstituted with chemically modified cofactors, that sugar oxidation most likely occurs by concerted hydride (H-) abstraction. Studies of the kinetics and thermodynamics together with the application of Marcus theory reveal a large reorganization energy barrier. The magnitude of this intrinsic contribution appears characteristic of H- transfer in proteins and in solution. The observation that neither the thermodynamics nor reorganization energy is significantly altered in the glucose oxidase active site raises questions concerning how the redox reaction may be catalyzed.