The broad objective of this proposal is a better understanding of how metal-containing enzymes work. The more specific aims involve characterization of three classes of Fe-based metalloenzymes with 'unusual'active sites -- nitrogenase (N2ase), hydrogenase (H2ase), and methane monooxygenase (MMO). We also seek to understand how special Fe-S cluster ('Wbl') proteins from Mycobacteria and Streptomyces bacteria sense NO and O2 in their environment. The questions that we hope to answer revolve around molecular structure (what atoms are where) and dynamics (how the atoms move). To achieve this knowledge, we have arranged close collaborations between chemists, biochemists, and spectroscopists. We will develop or enhance spectroscopic 'probes'that allow us to answer questions about the structure of enzyme intermediates. These include x-ray and nuclear techniques at synchrotron radiation labs -- XAFS and NRVS. We will balance these 'large facility'methods with campus-based spectroscopies including EPR, FT-IR, and M"ssbauer, both as direct probes and combined with UV-visible photolysis methods. For N2ase, H2ase, and MMO, the questions that we plan to address are: How does structure change during the course of the catalytic cycle? Where do substrates and inhibitors bind? What are the undefined light atoms? The same questions apply to the interactions of NO and O2 with Wbl proteins, except that the reactions involve sensing small molecules as opposed to catalysis. Our spectroscopic techniques will allow us to monitor the enzyme active sites. We will use this capability to focus on structural and dynamic issues that are beyond the reach of protein crystallography.

Public Health Relevance

How is research on these proteins related to public health? Quite simply: N2ase is responsible for the first step in the production of more than half of the protein we eat;a better understanding of the H2ase catalytic mechanism could help progress toward a pollution-free 'hydrogen economy';MMO and related enzymes are the key agents in biological remediation of oil spills such as the recent BP disaster;while better understanding of Wbl protein interactions with NO and O2 would help discover better ways to treat tuberculosis. Apart from specific knowledge about these metalloproteins, the techniques developed in the course of this study we be applicable to the hundreds of metalloproteins involved in human metabolism.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM065440-09A1
Application #
8240263
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Smith, Ward
Project Start
2002-03-01
Project End
2016-01-31
Budget Start
2012-05-01
Budget End
2013-01-31
Support Year
9
Fiscal Year
2012
Total Cost
$449,147
Indirect Cost
$52,580
Name
University of California Davis
Department
Miscellaneous
Type
Schools of Engineering
DUNS #
047120084
City
Davis
State
CA
Country
United States
Zip Code
95618
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