We propose to study the mechanisms of dioxygen activation by non-heme Fe-containing dioxygenases and related biosynthetic oxidases. The most important oxygen activation strategies used in nature are represented within the group of enzymes selected for study, allowing the problem to be approached on a broad front. Three of the classes of enzymes we will study are the aromatic ring cleaving, intra- and extradiol catecholic dioxygenases and the Rieske type cis-diol forming aromatic dioxygenases. These enzymes perform the key steps in the biodegradation of aromatic compounds in the environment from both natural and man-made sources; thus, they are of substantial significance for health issues related to the environment. Similar enzymes catalyze essential steps in mammalian biosynthetic pathways using the same oxygen activation strategies. The dioxygenase enzymes proposed for study include: (intradiol) protocatechuate 3,4-dioxygenase, (extradiol) homoprotocatechuate 2,3-dioxygenase, (Rieske) naphthalene 1,2- dioxygenase, and (Rieske) benzoate 1,2-dioxygenase. The oxidase enzymes that will be investigated include isopenicillin N-synthase (IPNS) and fosfomycin synthase (HppE). IPNS catalyzes the essential formation of both rings of the penicillin and cephalosporin class antibiotics while HppE forms the unusual epoxide ring of fosfomycin. These enzymes are representative of a large group of bacterial and mammalian oxidases that use oxygen to promote a specific reaction but do not incorporate O atoms into the products. We have developed a set of hypotheses for the mechanisms by which active site iron in both the dioxygenases and the biosynthetic oxidases is used to promote catalysis. These mechanisms are related by the known use of multiple sites in the metal ligand sphere to bind O2 and, in most cases, simultaneously organic substrates, thereby allowing precise juxtaposition of reactants as well as electron transfer between reactants to promote catalysis. Investigations of each enzyme class will employ a wide variety of active site mutants in conjunction with transient kinetics, spectroscopy (optical, EPR, ENDOR, rRaman, NMR, EXAFS, NIR CD, MCD, and M""""""""ssbauer), and crystallography to trap and characterize intermediates in the oxygen activation, O-O bond cleavage, insertion, and substrate oxidation processes. Past studies have led to the development of single turnover and peroxide shunt systems for several of the enzyme classes that will be used in the investigation. Application of these techniques allowed the key reactive oxygen species to be identified in both the extradiol and Rieske classes in recent months. Many mutations have been characterized for each dioxygenase and oxidase class that will allow the specific intermediates and the unique mechanistic features of the enzymes to be explored. Finally, a new approach for the initiation of reactions in enzyme crystals has been developed that allows reaction cycle intermediates to be trapped and their 3 dimensional structures determined. This work will yield fundamental information about the chemistry of oxygenases, oxidases, oxygen, and metals in biological systems. The basic concepts that emerge will be useful in such areas as the design of drugs to modulate the reactivity of oxygen activating enzymes, the synthesis of new antibiotics, and interdiction in the production of deleterious diffusible reactive oxygen species in humans.
Dioxygen is both the single most important molecule for human existence and a powerful agent for destruction of that existence. The great oxidizing potential of dioxygen is held in check by a quirk in the physics of its molecular structure that prevents facile reaction with other molecules at ambient temperature. Nature utilizes oxygenase and oxidase enzymes to unlock this potential specifically when and where it is needed to build biological structures, tap energy from metabolism, and detoxify molecules in our organs and the environment. When these enzymes go awry, nonspecific oxidation, aberrant oxygen insertion and random oxidation by powerful reactive oxygen species occurs, leading to some of the most devastating of human diseases. It is proposed here to study the mechanism of dioxygen activation at a fundamental level using a group of non- heme iron containing dioxygenase and oxidase enzymes that are representative of their respective classes. ? ? ?
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