There are three primary goals in this proposal. The first is to understand the molecular details of how the CO sensing transcription factor CooA functions. The binding of CO to the heme iron results in very large structural changes some 20? from the CO. These structural changes switch CooA to the "on-state" such that it specifically recognizes 5'DNA regulatory sequences where CooA promotes specific gene transcription involved in CO oxidation. CooA is a paradigm for how small gaseous ligands lead to large allosteric transitions coupled to important signal transduction processes. In addition, CooA exhibits exquisite ligand selectivity such that only CO results in the allosteric transition and no other biological diatomic gases such as NO and O2. Given that gas sensing now is recognized as a critically important biological process throughout the biosphere, uncovering the structural details of ligand selectivity in CooA is especially important. A second goal is to understand the structural details of heme transport in pathogenic bacteria. Such pathogens must acquire nutrients from the host and one such nutrient is iron. Pathogenic bacteria have developed a complex system where host heme is transported into the cytosol where it is degraded thus releasing iron. The goal of this aim is to determine the structures of all protein components including specific protein complexes. Given that these proteins are unique to bacterial pathogens, they can potentially provide useful therapeutic targets. The third goal is to study structure-function relationships in peroxidases. Recent advances in coupling x-ray data collection, single crystal spectroscopy, and novel composite data collection methods have made it possible to determine the crystal structure of unstable enzyme intermediates. This goal now is to focus on these intermediates in redox protein-protein complexes. A new crystal structure of a peroxidase-cytochrome c complex has been solved which has provided a new system for probing inter-protein electron transfer.

Public Health Relevance

This proposal centers on structure-function relationship in 3 biological systems: carbon monoxide sensing, heme transport in pathogenic bacteria, and peroxidases. Peroxidases are a paradigm for a wide range of metalloenzymes and have and continue to provide fundamental molecular level details on enzyme structure and function.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM042614-24
Application #
8643234
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Barski, Oleg
Project Start
1989-07-01
Project End
2017-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
24
Fiscal Year
2014
Total Cost
$301,031
Indirect Cost
$106,031
Name
University of California Irvine
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
046705849
City
Irvine
State
CA
Country
United States
Zip Code
92697
Poulos, Thomas L (2014) Heme enzyme structure and function. Chem Rev 114:3919-62
Tripathi, Sarvind; O'Neill, Maura J; Wilks, Angela et al. (2013) Crystal structure of the Pseudomonas aeruginosa cytoplasmic heme binding protein, Apo-PhuS. J Inorg Biochem 128:131-6
Benabbas, Abdelkrim; Karunakaran, Venugopal; Youn, Hwan et al. (2012) Effect of DNA binding on geminate CO recombination kinetics in CO-sensing transcription factor CooA. J Biol Chem 287:21729-40
Jasion, Victoria S; Poulos, Thomas L (2012) Leishmania major peroxidase is a cytochrome c peroxidase. Biochemistry 51:2453-60
Jasion, Victoria S; Polanco, Julio A; Meharenna, Yergalem T et al. (2011) Crystal structure of Leishmania major peroxidase and characterization of the compound i tryptophan radical. J Biol Chem 286:24608-15
Meharenna, Yergalem T; Doukov, Tzanko; Li, Huiying et al. (2010) Crystallographic and single-crystal spectral analysis of the peroxidase ferryl intermediate. Biochemistry 49:2984-6
Sundaramoorthy, Munirathinam; Gold, Michael H; Poulos, Thomas L (2010) Ultrahigh (0.93A) resolution structure of manganese peroxidase from Phanerochaete chrysosporium: implications for the catalytic mechanism. J Inorg Biochem 104:683-90
Poulos, Thomas L (2010) Thirty years of heme peroxidase structural biology. Arch Biochem Biophys 500:3-12
Ioanoviciu, Alexandra; Meharenna, Yergalem T; Poulos, Thomas L et al. (2009) DevS oxy complex stability identifies this heme protein as a gas sensor in Mycobacterium tuberculosis dormancy. Biochemistry 48:5839-48
Meharenna, Yergalem T; Oertel, Patricia; Bhaskar, B et al. (2008) Engineering ascorbate peroxidase activity into cytochrome c peroxidase. Biochemistry 47:10324-32

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