The scientific aim of this proposal is to understand nature's well-controlled chemistry that occurs in enzymes, by following the structural dynamics of the protein and chemical dynamics of the catalyst simultaneously. It is our goal to understand these design concepts from nature with X-ray crystallography and X-ray spectroscopy techniques using X-ray Free Electron Lasers (XFELs). Although the structure of enzymes and the chemistry at the catalytic sites have been studied intensively, an understanding of the atomic-scale chemistry requires a new approach beyond the conventional steady state X-ray crystallography and X-ray spectroscopy at cryogenic temperatures. Following the dynamic changes in the geometric and electronic structure of metallo-enzymes at ambient conditions, while overcoming the severe X-ray damage to the redox active catalytic center, is key for deriving the reaction mechanism. The intense and ultra-short femtosecond (fs) X-ray pulses of the LCLS (Linac Coherent Light Source) X-ray free electron laser provide an opportunity to overcome the current limitations of room temperature data collection for biological samples at regular synchrotron X-ray sources. The fs X-ray pulses make it possible to acquire the signal before the sample is destroyed. The objective of this proposal is to study the protein structure and dynamics of metallo-enzymes using crystallography, as well as the chemical structure and dynamics of the catalytic complexes (charge, spin, and covalency) using spectroscopy during the reaction to understand the electron-transfer processes and elucidate the mechanism. We will design and apply a full suite of time-resolved X-ray diffraction and X-ray absorption/emission spectroscopy methods to follow the reaction at room temperature, that will provide an unprecedented combination of correlated data between the protein, the co-factors, all of which are necessary for a complete understanding of structure and mechanism. Spectroscopy will include both K-edge-emission and L-edge absorption spectroscopy to get a complete understanding of the time-evolution of the electronic structure, while simultaneous room temperature time-resolved X-ray crystallography would provide the changes in the geometric structure of the overall protein complex. The proposal will also focus on simultaneously following the chemistry that occurs at multiple sites in biological systems. This will allow us to follow the electron transfer between the multiple sites in metalloproteins at various time-scales and levels;within a site, between two sites in a molecule or in two different molecules. The systems that will be used for developing these methodologies are some of the most important metallo-enzymes in biology;cytochrome c oxidase (Fe, Cu), ribonucleotide reductase (Mn, Fe), nitrogenase (Mo, Fe) and heme enzymes (Fe), cyctochrome c peroxidase and nitric oxide synthase.

Public Health Relevance

Biological crystallography and spectroscopy are complementary methods that contribute to understanding the structure and function of enzymes. The newly available free electron laser facility, the Linac Coherent Light Source, the LCLS at Stanford, has made possible the study of biological molecules at room temperature, in real time. Development of both crystallography and spectroscopy methods that make use of this novel national facility is important for understanding how complex biological molecules, such as metal containing proteins, interact and function under, ambient, physiological conditions.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM110501-01
Application #
8673988
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
2014-09-15
Project End
2018-08-31
Budget Start
2014-09-15
Budget End
2015-08-31
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Lawrence Berkeley National Laboratory
Department
Type
DUNS #
City
Berkeley
State
CA
Country
United States
Zip Code
94720
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Kubin, Markus; Kern, Jan; Guo, Meiyuan et al. (2018) X-ray-induced sample damage at the Mn L-edge: a case study for soft X-ray spectroscopy of transition metal complexes in solution. Phys Chem Chem Phys 20:16817-16827
Kern, Jan; Chatterjee, Ruchira; Young, Iris D et al. (2018) Structures of the intermediates of Kok's photosynthetic water oxidation clock. Nature 563:421-425
Koralek, Jake D; Kim, Jongjin B; Br?┼ża, Petr et al. (2018) Generation and characterization of ultrathin free-flowing liquid sheets. Nat Commun 9:1353
Kubin, Markus; Guo, Meiyuan; Ekimova, Maria et al. (2018) Direct Determination of Absolute Absorption Cross Sections at the L-Edge of Dilute Mn Complexes in Solution Using a Transmission Flatjet. Inorg Chem 57:5449-5462
Brewster, Aaron S; Waterman, David G; Parkhurst, James M et al. (2018) Improving signal strength in serial crystallography with DIALS geometry refinement. Acta Crystallogr D Struct Biol 74:877-894
Kroll, Thomas; Weninger, Clemens; Alonso-Mori, Roberto et al. (2018) Stimulated X-Ray Emission Spectroscopy in Transition Metal Complexes. Phys Rev Lett 120:133203
Kubin, Markus; Guo, Meiyuan; Ekimova, Maria et al. (2018) Cr L-Edge X-ray Absorption Spectroscopy of CrIII(acac)3 in Solution with Measured and Calculated Absolute Absorption Cross Sections. J Phys Chem B 122:7375-7384
Kubin, Markus; Guo, Meiyuan; Kroll, Thomas et al. (2018) Probing the oxidation state of transition metal complexes: a case study on how charge and spin densities determine Mn L-edge X-ray absorption energies. Chem Sci 9:6813-6829
Fuller, Franklin D; Gul, Sheraz; Chatterjee, Ruchira et al. (2017) Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers. Nat Methods 14:443-449

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