We propose to determine the molecular and regulatory mechanisms of a varied and versatile set of non-heme Fe- containing mono- and dioxygenases as a means to study the mechanisms of biological dioxygen activation. A major focus will be on the detection and characterization of reaction cycle intermediates. The most important O2 activation strategies used in nature are represented within the group of enzymes we have chosen, allowing the problem to be approached on a broad front. The activation of O2 by Fe-containing enzymes is highly regulated because escape of reactive oxygen species (ROS) causes damage to genetic material and other biomolecules, leading to cell death in bacteria and diseases in humans. Consequently, our study will delineate the stabilizing and destabilizing forces for the reactive intermediates in the O2 activation process. The mononuclear Fe-containing dioxygenase enzymes we study all use aromatic substrates and represent the major means by which natural and man-made aromatics are biodegraded. This provides the first line of defense against the toxic and carcinogenic effects of these compounds, and thus these enzymes have another substantial impact on human health. The dioxygenase enzymes proposed for study include: Fe(II)-containing extradiol dioxygenases, Fe(III)-containing intradiol dioxygenases, and redox cycling Rieske dioxygenases. The dinuclear Fe cluster-containing monooxygenases being studied are soluble methane monooxygenase (MMO) and two enzymes from the biosynthetic pathway for chloramphenicol, CmlA and CmlI. MMO is the principal barrier to release of the potent greenhouse gas methane into our atmosphere. CmlA and CmlI have numerous homologs in the natural product biosynthetic pathways for some of our most effective antibiotic and chemotherapy drugs, but remain mechanistically uncharacterized. We have developed hypotheses for the mechanisms by which the active site iron(s) in each type oxygenase we study is used to promote catalysis. These will now be tested by exploiting methods we have perfected to observe their reactions step by step as they occur. Past studies have led to the development of single turnover systems for each of the enzyme classes. Intermediates will be detected and trapped by coordinated use of stopped-flow transient kinetics, rapid mixing freeze quench (RFQ), site directed mutagenesis, and use of slow substrate analogs. The trapped intermediates will be characterized by a range of spectroscopies including optical, EPR, rRaman, NRVS, EXAFS, NIR CD, VTVH MCD, Mssbauer and a novel time resolved rRaman technique (TR3). Also, novel in crystallo techniques will be used in which the reactions are slowed (and often stopped at intermediates) by carrying them out in enzyme crystals. The 3D structures of intermediates trapped in crystallo will be solved crystallographically and through the use of newly developed time resolved techniques that employ the Linac Coherent Light Source. In ongoing studies, we have detected and trapped several widely postulated, but previously unobserved, intermediates in each of the enzyme classes. This work will yield fundamental information about the chemistry of oxygenases, oxygen, and metals in biological systems. The basic concepts that emerge will be useful in such areas as the mechanisms of similar enzymes in mammals and methods to interdict the production of deleterious diffusible ROS in humans.
Dioxygen is both the single most important molecule for human existence and the cause of some of the most devastating human diseases when it is activated in an uncontrolled fashion to form reactive oxygen species. Nature utilizes oxygenase and oxidase enzymes to unlock the oxidizing power of dioxygen safely when and where it is needed to build biological structures, tap energy from metabolism, and detoxify molecules in our organs and the environment. It is proposed here to study the mechanism of dioxygen activation step by step at a fundamental level using a diverse group of non-heme iron containing oxygenase enzymes.
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