X‑ray Emission Spectroscopy of Single Protein Crystals Yields Insights into Heme Enzyme Intermediates
Enzyme reactivity is often enhanced by changes in oxidation state, spin state, and metal–ligand covalency of associated metallocofactors. The development of spectroscopic methods for studying these processes coincidentally with structural rearrangements is essential for elucidating metalloenzyme mec...
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| Veröffentlicht in: | The journal of physical chemistry letters 2023-01, Vol.14 (1), p.41-48 |
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| creator | Emamian, Sahand Ireland, Kendra A. Purohit, Vatsal McWhorter, Kirklin L. Maximova, Olga Allen, Winter Jensen, Scott Casa, Diego M. Pushkar, Yulia Davis, Katherine M. |
| description | Enzyme reactivity is often enhanced by changes in oxidation state, spin state, and metal–ligand covalency of associated metallocofactors. The development of spectroscopic methods for studying these processes coincidentally with structural rearrangements is essential for elucidating metalloenzyme mechanisms. Herein, we demonstrate the feasibility of collecting X-ray emission spectra of metalloenzyme crystals at a third-generation synchrotron source. In particular, we report the development of a von Hamos spectrometer for the collection of Fe Kβ emission optimized for analysis of dilute biological samples. We further showcase its application in crystals of the immunosuppressive heme-dependent enzyme indoleamine 2,3-dioxygenase. Spectra from protein crystals in different states were compared with relevant reference compounds. Complementary density functional calculations assessing covalency support our spectroscopic analysis and identify active site conformations that correlate to high- and low-spin states. These experiments validate the suitability of an X-ray emission approach for determining spin states of previously uncharacterized metalloenzyme reaction intermediates. |
| doi_str_mv | 10.1021/acs.jpclett.2c03018 |
| format | Article |
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The development of spectroscopic methods for studying these processes coincidentally with structural rearrangements is essential for elucidating metalloenzyme mechanisms. Herein, we demonstrate the feasibility of collecting X-ray emission spectra of metalloenzyme crystals at a third-generation synchrotron source. In particular, we report the development of a von Hamos spectrometer for the collection of Fe Kβ emission optimized for analysis of dilute biological samples. We further showcase its application in crystals of the immunosuppressive heme-dependent enzyme indoleamine 2,3-dioxygenase. Spectra from protein crystals in different states were compared with relevant reference compounds. Complementary density functional calculations assessing covalency support our spectroscopic analysis and identify active site conformations that correlate to high- and low-spin states. 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Phys. Chem. Lett</addtitle><description>Enzyme reactivity is often enhanced by changes in oxidation state, spin state, and metal–ligand covalency of associated metallocofactors. The development of spectroscopic methods for studying these processes coincidentally with structural rearrangements is essential for elucidating metalloenzyme mechanisms. Herein, we demonstrate the feasibility of collecting X-ray emission spectra of metalloenzyme crystals at a third-generation synchrotron source. In particular, we report the development of a von Hamos spectrometer for the collection of Fe Kβ emission optimized for analysis of dilute biological samples. We further showcase its application in crystals of the immunosuppressive heme-dependent enzyme indoleamine 2,3-dioxygenase. Spectra from protein crystals in different states were compared with relevant reference compounds. Complementary density functional calculations assessing covalency support our spectroscopic analysis and identify active site conformations that correlate to high- and low-spin states. 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Phys. Chem. Lett</addtitle><date>2023-01-12</date><risdate>2023</risdate><volume>14</volume><issue>1</issue><spage>41</spage><epage>48</epage><pages>41-48</pages><issn>1948-7185</issn><eissn>1948-7185</eissn><abstract>Enzyme reactivity is often enhanced by changes in oxidation state, spin state, and metal–ligand covalency of associated metallocofactors. The development of spectroscopic methods for studying these processes coincidentally with structural rearrangements is essential for elucidating metalloenzyme mechanisms. Herein, we demonstrate the feasibility of collecting X-ray emission spectra of metalloenzyme crystals at a third-generation synchrotron source. In particular, we report the development of a von Hamos spectrometer for the collection of Fe Kβ emission optimized for analysis of dilute biological samples. We further showcase its application in crystals of the immunosuppressive heme-dependent enzyme indoleamine 2,3-dioxygenase. 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| subjects | Catalytic Domain Heme - metabolism INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY Metalloproteins Metals Physical Insights into Light Interacting with Matter Spectrometry, X-Ray Emission |
| title | X‑ray Emission Spectroscopy of Single Protein Crystals Yields Insights into Heme Enzyme Intermediates |
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