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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function A Study Section (MSFA)
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Smith, Ward
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University of California Davis
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Scott, Aubrey D; Pelmenschikov, Vladimir; Guo, Yisong et al. (2014) Structural characterization of CO-inhibited Mo-nitrogenase by combined application of nuclear resonance vibrational spectroscopy, extended X-ray absorption fine structure, and density functional theory: new insights into the effects of CO binding and the J Am Chem Soc 136:15942-54
Wang, Hongxin; Alp, Esen Ercan; Yoda, Yoshitaka et al. (2014) A practical guide for nuclear resonance vibrational spectroscopy (NRVS) of biochemical samples and model compounds. Methods Mol Biol 1122:125-37
Gu, Weiwei; Wang, Hongxin; Wang, Kun (2014) Nickel L-edge and K-edge X-ray absorption spectroscopy of non-innocent Ni[S?C?(CF?)?]?(n) series (n = -2, -1, 0): direct probe of nickel fractional oxidation state changes. Dalton Trans 43:6406-13
Kuchenreuther, Jon M; Myers, William K; Suess, Daniel L M et al. (2014) The HydG enzyme generates an Fe(CO)2(CN) synthon in assembly of the FeFe hydrogenase H-cluster. Science 343:424-7
Schilter, David; Pelmenschikov, Vladimir; Wang, Hongxin et al. (2014) Synthesis and vibrational spectroscopy of (57)Fe-labeled models of [NiFe] hydrogenase: first direct observation of a nickel-iron interaction. Chem Commun (Camb) 50:13469-72
Mitra, Devrani; George, Simon J; Guo, Yisong et al. (2013) Characterization of [4Fe-4S] cluster vibrations and structure in nitrogenase Fe protein at three oxidation levels via combined NRVS, EXAFS, and DFT analyses. J Am Chem Soc 135:2530-43
Dong, Weibing; Wang, Hongxin; Olmstead, Marilyn M et al. (2013) Inelastic X-ray scattering of a transition-metal complex (FeCl4(-)): vibrational spectroscopy for all normal modes. Inorg Chem 52:6767-9
Wang, Hongxin; Young, Anthony T; Guo, Jinghua et al. (2013) Soft X-ray absorption spectroscopy and resonant inelastic X-ray scattering spectroscopy below 100 eV: probing first-row transition-metal M-edges in chemical complexes. J Synchrotron Radiat 20:614-9
Kuchenreuther, Jon M; Guo, Yisong; Wang, Hongxin et al. (2013) Nuclear resonance vibrational spectroscopy and electron paramagnetic resonance spectroscopy of 57Fe-enriched [FeFe] hydrogenase indicate stepwise assembly of the H-cluster. Biochemistry 52:818-26
Zhou, Zhao-Hui; Wang, Hongxin; Yu, Ping et al. (2013) Structure and spectroscopy of a bidentate bis-homocitrate dioxo-molybdenum(VI) complex: insights relevant to the structure and properties of the FeMo-cofactor in nitrogenase. J Inorg Biochem 118:100-6

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