The objective of the proposed research is to elucidate the mechanism of the integral membrane metalloenzyme particulate methane monooxygenase (pMMO). pMMO efficiently catalyzes the selective oxidation of methane to methanol under ambient conditions. Our central hypothesis is that pMMO catalyzes the selective oxidation of methane using a novel mechanistic pathway. Central to the catalytic pathway is an oxo-bridged dicopper species somewhat similar to that in previously characterized dicopper enzymes and model compounds. However, the coordination environment of the pMMO dicopper center is significantly different from that in all other known dicopper enzymes, and likely defines a completely new class of enzymes. A novel active site and new O2 activation chemistry will likely emerge, impacting both bioinorganic chemistry and catalysis. The pMMO mechanism will be defined by three approaches. Initial characterization will investigate the O2 binding at the dicopper site of pMMO and a recombinant construct of the soluble pmoB domain (spmoB) using various spectroscopic techniques. spmoB will be used in the studies as a functional model for pMMO and site-specific variants will be made to further probe the properties of the active site. Once the O2 binding has been characterized, enzyme kinetics using gas chromatography and stopped-flow spectroscopy will be determined. These data will define the role of the membrane in pMMO and trap fast timescale intermediates on the reaction pathway. In parallel to the biochemical studies, the high-resolution crystal structures of oxidized and reduced pMMO and spmoB will be employed. All the proposed studies will be run in the presence and absence of a suitable substrate to investigate the site of methane entry and oxidation. This proposal is relevant to the mission of the NIH by developing new strategies to diminish both cancer causing environmental contaminates and diseases induced by climate change. pMMO breaks down the most inert hydrocarbon, methane, under ambient conditions and therefore represents an attractive target in the development of green catalysts to target bioremediation and minimize greenhouse gas emissions. Halogenated hydrocarbon pollutants, such as trichloroethylene (TCE) and vinylchloride (VC) that pose a threat to human health are effectively degraded by pMMO. According to the Centers of Disease Control, chlorinated hydrocarbons are implicated in endocrine disorders and many forms of cancer. Additionally, pMMO represents a target for minimizing greenhouse gas emissions that pose a threat to human health by increasing the earth

Public Health Relevance

The studies proposed in this work are significant because they address the growing concern in our society on the effect environmental contamination and the global climate change has on human health. The development of greener, safer catalysis is essential in diminishing these environmental health concerns. The strategies proposed here are the first steps in developing these catalysts and minimizing the effects environmental stressors pose on human health and wellbeing.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
1F32GM097049-01
Application #
8061095
Study Section
Special Emphasis Panel (ZRG1-F04A-B (20))
Program Officer
Gerratana, Barbara
Project Start
2011-04-01
Project End
2013-03-31
Budget Start
2011-04-01
Budget End
2012-03-31
Support Year
1
Fiscal Year
2011
Total Cost
$48,398
Indirect Cost
Name
Northwestern University at Chicago
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
160079455
City
Evanston
State
IL
Country
United States
Zip Code
60201
Culpepper, Megen A; Cutsail 3rd, George E; Gunderson, William A et al. (2014) Identification of the valence and coordination environment of the particulate methane monooxygenase copper centers by advanced EPR characterization. J Am Chem Soc 136:11767-75
Culpepper, Megen A; Cutsail 3rd, George E; Hoffman, Brian M et al. (2012) Evidence for oxygen binding at the active site of particulate methane monooxygenase. J Am Chem Soc 134:7640-3
Culpepper, Megen A; Rosenzweig, Amy C (2012) Architecture and active site of particulate methane monooxygenase. Crit Rev Biochem Mol Biol 47:483-92