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Aspartate or arginine? Validated redox state X-ray structures elucidate mechanistic subtleties of FeIV = O formation in bacterial dye-decolorizing peroxidases

Lučić, Marina and Wilson, Michael T and Svistunenko, Dimitri A and Owen, Robin L and Hough, Michael A and Worrall, Jonathan AR (2021) 'Aspartate or arginine? Validated redox state X-ray structures elucidate mechanistic subtleties of FeIV = O formation in bacterial dye-decolorizing peroxidases.' Journal of Biological Inorganic Chemistry, 26 (7). pp. 743-761. ISSN 0949-8257

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Structure determination of proteins and enzymes by X-ray crystallography remains the most widely used approach to complement functional and mechanistic studies. Capturing the structures of intact redox states in metalloenzymes is critical for assigning the chemistry carried out by the metal in the catalytic cycle. Unfortunately, X-rays interact with protein crystals to generate solvated photoelectrons that can reduce redox active metals and hence change the coordination geometry and the coupled protein structure. Approaches to mitigate such site-specific radiation damage continue to be developed, but nevertheless application of such approaches to metalloenzymes in combination with mechanistic studies are often overlooked. In this review, we summarize our recent structural and kinetic studies on a set of three heme peroxidases found in the bacterium Streptomyces lividans that each belong to the dye decolourizing peroxidase (DyP) superfamily. Kinetically, each of these DyPs has a distinct reactivity with hydrogen peroxide. Through a combination of low dose synchrotron X-ray crystallography and zero dose serial femtosecond X-ray crystallography using an X-ray free electron laser (XFEL), high-resolution structures with unambiguous redox state assignment of the ferric and ferryl (Fe<sup>IV</sup> = O) heme species have been obtained. Experiments using stopped-flow kinetics, solvent-isotope exchange and site-directed mutagenesis with this set of redox state validated DyP structures have provided the first comprehensive kinetic and structural framework for how DyPs can modulate their distal heme pocket Asp/Arg dyad to use either the Asp or the Arg to facilitate proton transfer and rate enhancement of peroxide heterolysis.

Item Type: Article
Uncontrolled Keywords: Heme peroxidase; Ferryl; X-ray free electron laser; Kinetic isotope effect; Serial crystallography
Divisions: Faculty of Science and Health
Faculty of Science and Health > Life Sciences, School of
SWORD Depositor: Elements
Depositing User: Elements
Date Deposited: 21 Sep 2021 08:53
Last Modified: 06 Jan 2022 14:26

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