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Taking Snapshots of Enzymatic Reactions Using X-ray Crystallography and Spectroscopy

Award Number: R35GM149528
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 05/01/2023
PERIOD OF PERFORMANCE END DATE: 04/30/2028

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Taking Snapshots of Enzymatic Reactions Using X-ray Crystallography and Spectroscopy - Project Summary/Abstract Metalloproteins containing Mn and Fe in a redox-active role are involved in a variety of physiologically important reactions of dioxygen metabolism and activation. Perhaps the most complex is the Mn4CaO5 cluster that is involved in the oxidation of water to dioxygen in photosystem II (PS II), a multi-subunit membrane protein complex. The water-oxidation reaction in PS II involves removal of four electrons from two water molecules, in a stepwise manner by light-induced oxidation, to produce a molecule of oxygen. PS II and the Mn4CaO5 cluster generate almost all of the dioxygen that supports aerobic life, and it is abundant in the atmosphere because of its constant regeneration by the oxidation of water. The light-induced oxidation of water to dioxygen is one of the most important chemical processes occurring on such a large scale in the biosphere. Although the structure of PS II and the chemistry at the catalytic site have been studied intensively, understanding the sequence in the chemistry at atomic-scale from light absorption to water-oxidation 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 structure of PS II and the Mn4CaO5 cluster at ambient conditions at physiological temperatures, while overcoming the severe X-ray damage to the redox active center is key for deriving the mechanism. The very intense, ultra-short femtosecond (fs) X-ray pulses from a X-ray free electron laser (XFEL) provide an opportunity to overcome the current limitations in room temperature data collection for biological samples at traditional X-ray sources. The fs X-ray pulses allow us to acquire the signal before the sample is destroyed, thus making the light-induced snapshot study possible. The objective of this proposal is to study the protein structure and dynamics of PS II with X-ray diffraction, as well as the chemical structure and changes in the Mn4CaO5 cluster (charge and spin density, and covalency) with X-ray spectroscopy during the light-driven process of PS II. We will use the XFEL facilities at Stanford and elsewhere to collect X-ray diffraction and emission spectra simultaneously, and X-ray absorption spectra of the Mn cluster in its native and intermediates states at room temperature in a time-resolved manner, to capture short-lived intermediates and the step that includes the O-O bond formation. We have also started studying the chemistry of other Mn/Fe/Ni containing metalloenzymes of importance such as methane monooxgygenase, ribonucleotide reductase, isopenicillin N synthase and other related enzymes. These studies have the potential to provide an unprecedented combination of correlated data between the proteins and the metal co-factors, providing the geometric and electronic structure and the changes that occur during the catalytic cycle, all of which are necessary for a complete understanding of the mechanism of the enzymatic reactions.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2025 ( Subtotal = $603,913 )
2025	2025	THE REGENTS OF THE UNIVERSITY OF CALIFORNIA	1 CYCLOTRON RD	BERKELEY	CA	94720	ALAMEDA	USA	Biomedical Research and Research Training	001	3	4/8/2025	NON-COMPETING CONTINUATION	$603,913
2025	2024	THE REGENTS OF THE UNIVERSITY OF CALIFORNIA	1 CYCLOTRON RD	BERKELEY	CA	94720	ALAMEDA	USA	Biomedical Research and Research Training	000	2	11/5/2024	NON-COMPETING CONTINUATION	$0
														Subtotal = $603,913

Issue Date FY: 2024 ( Subtotal = $771,536 )
2024	2024	THE REGENTS OF THE UNIVERSITY OF CALIFORNIA	1 CYCLOTRON RD	BERKELEY	CA	94720	ALAMEDA	USA	Biomedical Research and Research Training	000	2	4/4/2024	NON-COMPETING CONTINUATION	$543,522
2024	2024	THE REGENTS OF THE UNIVERSITY OF CALIFORNIA	1 CYCLOTRON RD	BERKELEY	CA	94720	ALAMEDA	USA	Biomedical Research and Research Training	002	2	9/17/2024	SUPPLEMENT FOR EXPANSION	$167,623
2024	2024	THE REGENTS OF THE UNIVERSITY OF CALIFORNIA	1 CYCLOTRON RD	BERKELEY	CA	94720	ALAMEDA	USA	Biomedical Research and Research Training	001	2	6/17/2024	NON-COMPETING CONTINUATION	$60,391
														Subtotal = $771,536

Issue Date FY: 2023 ( Subtotal = $674,268 )
2023	2023	REGENTS OF THE UNIVERSITY OF CALIFORNIA, THE	1 CYCLOTRON RD	BERKELEY	CA	94720	ALAMEDA	USA	Biomedical Research and Research Training	000	1	3/27/2023	NEW	$674,268
														Subtotal = $674,268

Grand Total All Awards = $2,049,717

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Taking Snapshots of Enzymatic Reactions Using X-ray Crystallography and Spectroscopy

Award Number: R35GM149528

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 05/01/2023

PERIOD OF PERFORMANCE END DATE: 04/30/2028

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