Sunday, November 24, 2024
11/24/2024
Project Summary/Abstract The goal of this proposal is to understand nature’s well-controlled chemistry that occurs in metalloenzymes, by developing the necessary tools and applying them to follow the structural dynamics of the protein and chemical dynamics of the metal-catalyst. For this purpose, we will use X-ray crystallography and X-ray spectroscopy techniques at 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 metalloenzymes at ambient temperature, 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 from XFELs provide an opportunity to overcome the current limitations of room temperature data collection for biological samples at synchrotron X- ray sources. The fs X-ray pulses with shot-by-shot sample replacement make it possible to acquire the signal before damage occurs and the sample is destroyed. We will design and apply a suite of time-resolved X-ray diffraction and X-ray absorption/emission spectroscopy methods, that make use of the unique properties of the XFEL beam. This will provide an unprecedented combination of correlated data between the protein and the co-factors, all of which are necessary to understand the interplay between the geometric structure of the protein and electronic structure of the metal complex, and the functional consequences. Spectroscopy, both emission and absorption spectroscopy, will provide the time-evolution of the electronic structure, while simultaneous room temperature X-ray crystallography will help us visualize the changes in the geometric structure of the overall protein. We will use these methodologies to study some of the most important metalloenzymes in biology to gain insights into the catalytic mechanisms, including mono, and dinuclear systems both with homo- and hetero-metallic centers. A representative example of these systems are Fe enzymes for oxygen and C-H bond activation where the involvement of high-valent Fe are proposed, such as the heme containing Cyt P450 systems, non-heme enzymes ribonucleotide reductase (Mn/Fe and Fe/Fe), and methane mono oxygenase (Fe/Fe). We will also focus on Ni and Ni/Fe enzymes relevant to H2 generation and methane metabolism, and functional analogs of the important class of heme-copper oxidase systems engineered into simpler proteins.
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