Many enzymes containing redox-active metal centers play significant roles in cellular function, and are often involved in a variety of physiologically important processes. In particular, several Mn-containing metalloproteins have emerged with functional roles in O{2} metabolism since the identification of Mn as an essential metal in biological redox catalysis. These include a mitochondrial Mn-superoxide dismutase (SOD2) that detoxifies superoxide radicals into O{2} and peroxide;a non-heme Mn-containing pseudocatalase that catalyzes the decomposition of peroxide into H{2}O and O{2};and the oxygen-evolving complex (OEC) in photosystem II (PSII), which is possibly the most important due to its key role in the oxidation of H{2}O to O{2} during photosynthesis. Nearly all of the atmospheric O{2} that supports aerobic life is produced and replenished by the OEC through H{2}O oxidation;hence, this light-induced reaction is one of the most important biological redox processes found in nature. Although it is known that the OEC is composed of a heteronuclear Mn4CaOx cluster where four electrons are extracted in a stepwise manner from two H{2}O molecules to produce one O{2} molecule, the detailed structure and mechanism of how this process occurs are not well understood. Furthermore, conventional X-ray crystallography and spectroscopy approaches are limited by the sensitivity of the redox-active metal complex to radiation damage by photoreduction. However, the recent development of the powerfully intense X-ray free electron laser (X-FEL) and application of the "collect before destroy" approach provide a viable option for overcoming this obstacle. Thus, a key objective of this proposal is to determine the structure of the intact OEC and elucidate the catalytic mechanism by which H{2}O is oxidized to O{2} by mapping the time evolution of the Mn{4}CaO{x} cluster using this new X-FEL technology. Specifically, X-ray diffraction (XRD) and X-ray emission spectra (XES) will be simultaneously measured from a continuous stream of PSII microcrystals with femtosecond X-FEL pulses in order to determine not only the electronic and geometric structure of the Mn{4}CaO{x} cluster, but also the integrity of the metal complex. Two fundamental points that are central to understanding photosynthetic water oxidation include: (i) the temporal evolution of the OEC electronic structure, and (ii) the structural dynamics in the ligand environment and Mn{4}CaO{x} cluster as it cycles through the catalytic steps. To address these points and map the light-induced chemical steps in real time, a combined laser excitation 'pump'and X-FEL 'probe'with variable time delays will be incorporated into the experimental setup. Not only will this study lead to an understanding of the mechanism of H{2}O oxidation to form O{2}, but the methodology developed here should also have broad applications as a model study for using X-FELs to determine structure and dynamics in other physiologically important membrane proteins and redox- active metalloenzymes that are prone to X-ray radiation damage.

Public Health Relevance

Many enzymes containing metal centers play significant roles in cellular function, and are involved in a variety of physiologically important processes. In particular, the metal-centered oxygen-evolving complex catalyzes the generation of O{2} from H{2}O as part of the O{2} metabolism cycle, which is critical for sustaining all aerobic life on Earth. These proposed studies aim to elucidate the mechanism of how O{2} is formed from H{2}O by the oxygen-evolving complex using novel methodology that will have broad applications in biomedical research for determining the chemistry and structural dynamics in other physiologically important metal-centered enzymes.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Postdoctoral Individual National Research Service Award (F32)
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Special Emphasis Panel (ZRG1-F04B-D (20))
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Flicker, Paula F
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Lawrence Berkeley National Laboratory
Other Basic Sciences
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United States
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Kern, Jan; Hattne, Johan; Tran, Rosalie et al. (2014) Methods development for diffraction and spectroscopy studies of metalloenzymes at X-ray free-electron lasers. Philos Trans R Soc Lond B Biol Sci 369:20130590
Kanady, Jacob S; Tran, Rosalie; Stull, Jamie A et al. (2013) Role of Oxido Incorporation and Ligand Lability in Expanding Redox Accessibility of Structurally Related Mn4 Clusters. Chem Sci 4:3986-3996
Tsui, Emily Y; Tran, Rosalie; Yano, Junko et al. (2013) Redox-inactive metals modulate the reduction potential in heterometallic manganese-oxido clusters. Nat Chem 5:293-9