We propose to detect and characterize intermediates in the reaction cycles of a varied and versatile set of non-heme Fe- containing dioxygenases as a means to study the mechanisms of biological dioxygen activation. The most important oxygen activation strategies used in nature are represented within this group of enzymes, 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" from the active site causes damage of 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 bacterial classes of enzymes we will study all use aromatic substrates and represent the major means by which natural and man-made aromatics are biodegraded in the environment. This provides the first line of defense against the toxic and carcinogenic effects of many 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. We have developed a set of hypotheses for the mechanisms by which the active site iron in each type of dioxygenase is used to promote catalysis, which 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. Also, novel in crystallo techniques will be used in which the reactions are slowed (and often stopped at intermediates) by carrying them out in single enzyme crystals. The intermediates trapped by RFQ will be characterized by a range of spectroscopies including optical, EPR, rRaman, NRVS, EXAFS, NIR CD, VTVH MCD, and M?ssbauer. The 3D structures of intermediate trapped in crystallo will be solved by X-ray crystallography and through the use of newly developed single crystal optical and (r)Raman spectroscopies. In preliminary studies, we have detected and trapped several widely postulated, but unobserved, intermediates in each of the enzyme classes proposed for study. These will be now be characterized as methods to detect and trap additional intermediates are developed. In parallel studies, methods to replace the Fe(II) in extradiol dioxygenases with Co(II) and Mn(II) without loss of activity have been developed. The new spectroscopic and kinetic properties of these metal-substituted enzymes promise new insight into the dioxygenase mechanism as well as the rationale for metal choice in enzymes. 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 reactive oxygen species in humans.

Public Health Relevance

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 step by step at a fundamental level using a diverse group of non-heme iron containing dioxygenase enzymes.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Barski, Oleg
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Minnesota Twin Cities
Schools of Medicine
United States
Zip Code
Lipscomb, John D (2014) Life in a sea of oxygen. J Biol Chem 289:15141-53
Fielding, Andrew J; Lipscomb, John D; Que Jr, Lawrence (2014) A two-electron-shell game: intermediates of the extradiol-cleaving catechol dioxygenases. J Biol Inorg Chem 19:491-504
Yin, DeLu Tyler; Purpero, Vince M; Fujii, Ryota et al. (2013) New structural motif for carboxylic acid perhydrolases. Chemistry 19:3037-46
Hayden, Joshua A; Farquhar, Erik R; Que, Lawrence et al. (2013) NO binding to Mn-substituted homoprotocatechuate 2,3-dioxygenase: relationship to Oýýý reactivity. J Biol Inorg Chem 18:717-28
Fielding, Andrew J; Lipscomb, John D; Que Jr, Lawrence (2012) Characterization of an O2 adduct of an active cobalt-substituted extradiol-cleaving catechol dioxygenase. J Am Chem Soc 134:796-9
Mbughuni, Michael M; Chakrabarti, Mrinmoy; Hayden, Joshua A et al. (2011) Oxy intermediates of homoprotocatechuate 2,3-dioxygenase: facile electron transfer between substrates. Biochemistry 50:10262-74
Fielding, Andrew J; Kovaleva, Elena G; Farquhar, Erik R et al. (2011) A hyperactive cobalt-substituted extradiol-cleaving catechol dioxygenase. J Biol Inorg Chem 16:341-55
Mbughuni, Michael M; Chakrabarti, Mrinmoy; Hayden, Joshua A et al. (2010) Trapping and spectroscopic characterization of an FeIII-superoxo intermediate from a nonheme mononuclear iron-containing enzyme. Proc Natl Acad Sci U S A 107:16788-93
Yin, De Lu Tyler; Bernhardt, Peter; Morley, Krista L et al. (2010) Switching catalysis from hydrolysis to perhydrolysis in Pseudomonas fluorescens esterase. Biochemistry 49:1931-42
(2009) Abstracts of the 14th International Conference on Biological Inorganic Chemistry. July 25-30, 2009. Nagoya, Japan. J Biol Inorg Chem 14 Suppl 1:S3-254

Showing the most recent 10 out of 77 publications